相关申请Related Applications
本申请根据35U.S.C.§119(e)要求于2020年9月3日提交的标题为“METHODS OFPREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES”的美国临时申请序列No.63/074439以及于2020年9月3日提交的标题为“METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDECOMPLEXES”的美国临时申请序列No.63/074436的优先权；其各自的内容通过引用整体并入本文。This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63/074439, filed on September 3, 2020, entitled “METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES,” and U.S. Provisional Application Serial No. 63/074436, filed on September 3, 2020, entitled “METHODS OF PREPARING PROTEIN-OLIGONUCLEOTIDE COMPLEXES,” the contents of each of which are incorporated herein by reference in their entirety.
技术领域Technical Field
本申请涉及纯化复合物(例如，蛋白质-寡核苷酸缀合物和抗体-药物缀合物)的方法。The present application relates to methods of purifying complexes (eg, protein-oligonucleotide conjugates and antibody-drug conjugates).
参考作为文本文件通过EFS-WEB提交的序列表Reference to a sequence listing submitted as a text file via EFS-WEB
本申请包含已通过EFS-Web以ASCII格式提交并且在此通过引用整体并入的序列表。在2021年9月3日创建的所述ASCII副本被命名为D082470043WO00-SEQ-ZJG并且大小为57,060字节。This application contains a sequence listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on September 3, 2021 is named D082470043WO00-SEQ-ZJG and is 57,060 bytes in size.
背景技术Background Art
近年来，已经开发了数种寡核苷酸(例如，反义寡核苷酸)来对抗组织或细胞特异性疾病(例如，肌肉特异性疾病，例如多种形式的肌营养不良)。然而，已证明有效地将这些寡核苷酸递送至其期望的组织或细胞具有挑战性。In recent years, several oligonucleotides (e.g., antisense oligonucleotides) have been developed to combat tissue or cell-specific diseases (e.g., muscle-specific diseases such as various forms of muscular dystrophy). However, it has proven challenging to effectively deliver these oligonucleotides to their desired tissues or cells.
发明内容Summary of the invention
包含与治疗性寡核苷酸共价连接的组织或细胞特异性蛋白质(例如，抗体)的复合物为递送所述治疗性寡核苷酸提供了极好的机会。这样的治疗性寡核苷酸包含，例如，电荷中性寡核苷酸(例如，PMO、PNA等)以及带电荷寡核苷酸(例如，间隔聚体(gapmer)、混合物、siRNA等)。然而，纯化和分离所述复合物使其远离过量蛋白质和寡核苷酸具有挑战性。在一些方面中，本公开内容提供了处理包含与寡核苷酸共价连接的蛋白质的复合物的方法，其使未缀合的寡核苷酸和蛋白质(例如，抗体)与复合物分离。还提供了产生复合物的方法。在一些实施方案中，产生本文中所述复合物的方法降低了包含炔基的未连接抗体的水平。在一些实施方案中，产生本文中所述复合物的方法在缀合反应之后仅产生痕量的包含炔基的未连接抗体。The complex comprising tissue or cell-specific proteins (e.g., antibodies) covalently linked to therapeutic oligonucleotides provides an excellent opportunity for delivering the therapeutic oligonucleotides. Such therapeutic oligonucleotides include, for example, charge-neutral oligonucleotides (e.g., PMO, PNA, etc.) and charged oligonucleotides (e.g., gapmers, mixtures, siRNA, etc.). However, it is challenging to purify and separate the complex away from excessive proteins and oligonucleotides. In some aspects, the disclosure provides a method for processing a complex comprising a protein covalently linked to an oligonucleotide, which separates unconjugated oligonucleotides and proteins (e.g., antibodies) from the complex. A method for producing a complex is also provided. In some embodiments, the method for producing the complex described herein reduces the level of unconnected antibodies comprising alkynyl groups. In some embodiments, the method for producing the complex described herein only produces trace amounts of unconnected antibodies comprising alkynyl groups after the conjugation reaction.
本公开内容的一个方面涉及处理复合物的方法，所述复合物各自包含与一个或更多个电荷中性寡核苷酸共价连接的抗体，所述方法包括：One aspect of the present disclosure relates to a method of treating complexes, each of which comprises an antibody covalently linked to one or more charge-neutral oligonucleotides, the method comprising:
(i)使包含有机溶剂、所述复合物和未连接电荷中性寡核苷酸的混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂在使所述复合物吸附至所述混合模式树脂的条件下接触，以及(i) contacting a mixture comprising an organic solvent, the complex and unattached neutrally charged oligonucleotides with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions such that the complex is adsorbed to the mixed mode resin, and
(ii)在所述复合物从所述混合模式树脂上解离的条件下从所述混合模式树脂洗脱所述复合物。在一些实施方案中，所述有机溶剂是二甲基乙酰胺(Dimethylacetamide，DMA)、异丙醇(isopropyl alcoho，IPA)、二甲基亚砜(dimethyl sulfoxide，DMSO)、乙腈(acetonitrile，ACN)或丙二醇(propylene glycol，PG)。在一些实施方案中，所述有机溶剂在步骤(i)所述混合物中为5％至30％(v/v)，任选地其中所述有机溶剂在步骤(i)所述混合物中为15％(v/v)。在一些实施方案中，所述有机溶剂在步骤(i)所述混合物中为30％(v/v)。(ii) eluting the complex from the mixed mode resin under conditions where the complex is dissociated from the mixed mode resin. In some embodiments, the organic solvent is dimethylacetamide (DMA), isopropyl alcoho (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG). In some embodiments, the organic solvent is 5% to 30% (v/v) in the mixture of step (i), optionally wherein the organic solvent is 15% (v/v) in the mixture of step (i). In some embodiments, the organic solvent is 30% (v/v) in the mixture of step (i).
在一些实施方案中，步骤(i)中的混合物还包含最高为10mM的磷酸根离子和/或最高为20mM的氯离子。在一些实施方案中，所述方法还包括在步骤(i)与步骤(ii)之间用包含有机溶剂的洗涤溶液来洗涤所述混合模式树脂，任选地其中所述有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。在一些实施方案中，所述有机溶剂在所述洗涤溶液中为5％至30％(v/v)，任选地其中所述有机溶剂在所述洗涤溶液中为15％(v/v)。在一些实施方案中，所述有机溶剂在所述洗涤溶液中为30％(v/v)。在一些实施方案中，所述洗涤溶液还包含最高为10mM的磷酸根离子和/或最高为20mM的氯离子。In some embodiments, the mixture in step (i) also contains up to 10 mM phosphate ions and/or up to 20 mM chloride ions. In some embodiments, the method further comprises washing the mixed mode resin with a washing solution comprising an organic solvent between step (i) and step (ii), optionally wherein the organic solvent is dimethylacetamide (DMA), isopropanol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG). In some embodiments, the organic solvent is 5% to 30% (v/v) in the washing solution, optionally wherein the organic solvent is 15% (v/v) in the washing solution. In some embodiments, the organic solvent is 30% (v/v) in the washing solution. In some embodiments, the washing solution also contains up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
在一些实施方案中，步骤(ii)包括向所述混合模式树脂施加洗脱溶液以洗脱所述复合物，其中所述洗脱溶液包含有机溶剂，任选地其中所述有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。在一些实施方案中，所述有机溶剂在所述洗脱溶液中为10％至30％(v/v)，任选地其中所述有机溶剂在所述洗脱溶液中为10％(v/v)。在一些实施方案中，所述洗脱溶液包含至少30mM的磷酸根离子，任选地其中所述洗脱溶液包含至少100mM的磷酸根离子。在一些实施方案中，所述洗脱溶液包含逐渐提高浓度的磷酸根离子，任选地其中所述磷酸根离子的浓度从至少10mM提高至至少100mM。在一些实施方案中，所述洗脱溶液的pH为7.6至8.5。In some embodiments, step (ii) includes applying an elution solution to the mixed mode resin to elute the complex, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is dimethylacetamide (DMA), isopropanol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG). In some embodiments, the organic solvent is 10% to 30% (v/v) in the elution solution, optionally wherein the organic solvent is 10% (v/v) in the elution solution. In some embodiments, the elution solution comprises at least 30mM of phosphate ions, optionally wherein the elution solution comprises at least 100mM of phosphate ions. In some embodiments, the elution solution comprises a gradually increasing concentration of phosphate ions, optionally wherein the concentration of the phosphate ions is increased from at least 10mM to at least 100mM. In some embodiments, the pH of the elution solution is 7.6 to 8.5.
本公开内容的另一方面涉及产生包含与一个或更多个寡核苷酸共价连接的抗体的复合物的方法，所述方法包括：Another aspect of the present disclosure relates to a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i)获得包含以下结构的寡核苷酸：(i) obtaining an oligonucleotide comprising the following structure:
(B)，其中n为3；(B), where n is 3;
(ii)获得包含以下结构的抗体：(ii) obtaining an antibody comprising the following structure:
(F)，其中m为4；以及(F), where m is 4; and
(iii)使步骤(i)中的所述寡核苷酸与步骤(ii)中所获得的抗体反应以获得所述复合物。(iii) reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
本公开内容的另一方面涉及产生包含与一个或更多个寡核苷酸共价连接的抗体的复合物的方法，所述方法包括：Another aspect of the disclosure relates to a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i)获得包含以下结构的寡核苷酸：(i) obtaining an oligonucleotide comprising the following structure:
(D)；其中n为3并且其中m为4；(D); wherein n is 3 and wherein m is 4;
(ii)获得抗体；以及(ii) obtaining antibodies; and
(iii)使步骤(i)中的所述寡核苷酸与步骤(ii)中所获得的抗体反应以获得所述复合物。(iii) reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
在一些实施方案中，所述复合物包含以下结构：In some embodiments, the complex comprises the following structure:
(E)(E)
其中n为3并且m为4，并且其中所述抗体通过赖氨酸连接。wherein n is 3 and m is 4, and wherein the antibody is linked via a lysine.
本公开内容的另一方面涉及混合物，其包含复合物和未连接寡核苷酸，所述复合物各自包含与一个或更多个寡核苷酸共价连接的抗体，其中所述混合物通过包括以下的方法来产生：Another aspect of the disclosure relates to a mixture comprising complexes and unattached oligonucleotides, wherein the complexes each comprise an antibody covalently attached to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i)获得包含与含有缬氨酸-瓜氨酸序列的可切割接头共价连接的寡核苷酸的第一中间体；(i) obtaining a first intermediate comprising an oligonucleotide covalently linked to a cleavable linker comprising a valine-citrulline sequence;
(ii)将步骤(i)中所获得的第一中间体与包含双环壬炔的化合物连接以获得第二中间体；以及(ii) connecting the first intermediate obtained in step (i) with a compound containing a bicyclononyne to obtain a second intermediate; and
(iii)将步骤(ii)中所获得的第二中间体与抗体连接以获得所述复合物；(iii) connecting the second intermediate obtained in step (ii) to an antibody to obtain the complex;
其中所述包含双环壬炔的化合物以小于步骤(ii)中所述化合物起始量的5％的量存在于步骤(iii)的反应中，任选地其中所述寡核苷酸在5’端处与所述包含缬氨酸-瓜氨酸序列的可切割接头共价连接，和/或所述抗体通过赖氨酸连接。wherein the compound comprising bicyclononyne is present in the reaction of step (iii) in an amount less than 5% of the starting amount of the compound in step (ii), optionally wherein the oligonucleotide is covalently linked at the 5' end to the cleavable linker comprising a valine-citrulline sequence, and/or the antibody is linked via a lysine.
本公开内容的另一方面涉及混合物，其包含复合物和ii)未连接寡核苷酸，所述复合物各自包含与一个或更多个寡核苷酸共价连接的抗体，其中所述混合物通过包括以下的方法来产生：Another aspect of the disclosure relates to a mixture comprising complexes each comprising an antibody covalently linked to one or more oligonucleotides and ii) unlinked oligonucleotides, wherein the mixture is produced by a method comprising:
(i)将一个或更多个寡核苷酸与式(A)接头在产生式(B)产物的反应条件下组合：(i) combining one or more oligonucleotides with a linker of formula (A) under reaction conditions that produce a product of formula (B):
(A)，其中n为3：(A), where n is 3:
(B)，其中n为3；(B), where n is 3;
(ii)使式(B)产物与式(C)化合物在产生式(D)产物的反应条件下接触：(ii) contacting the product of formula (B) with a compound of formula (C) under reaction conditions to produce a product of formula (D):
(C)，其中m为4；(C), where m is 4;
(D)，其中n为3并且m为4；以及(D), wherein n is 3 and m is 4; and
(iii)使式(D)产物与抗体在产生式(E)复合物的反应条件下接触：(iii) contacting the product of formula (D) with an antibody under reaction conditions to produce a complex of formula (E):
(E)，其中n为3并且m为4；(E), wherein n is 3 and m is 4;
其中式(C)化合物以小于在步骤(ii)的反应中式(C)化合物起始量的5％的量存在于步骤(iii)的反应中。wherein the compound of formula (C) is present in the reaction of step (iii) in an amount less than 5% of the starting amount of the compound of formula (C) in the reaction of step (ii).
本公开内容的另一方面涉及处理复合物的方法，所述复合物各自包含与一个或更多个寡核苷酸共价连接的抗体，所述方法包括：Another aspect of the disclosure relates to a method of treating complexes, each of which comprises an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i)使上述两种混合物中任一种的所述混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂在使所述复合物吸附至所述混合模式树脂的条件下接触，其中所述混合物包含的含有炔基的未连接抗体为痕量；以及(i) contacting the mixture of either of the above two mixtures with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions such that the complex is adsorbed to the mixed mode resin, wherein the mixture comprises a trace amount of unlinked antibody containing an alkynyl group; and
(ii)在所述复合物从所述混合模式树脂上解离的条件下从所述混合模式树脂洗脱所述复合物。在一些实施方案中，步骤(i)中的混合物未经过预先纯化。在一些实施方案中，步骤(i)中的混合物包含的磷酸根离子和/或氯离子为痕量。在一些实施方案中，所述方法还包括在步骤(i)与步骤(ii)之间用包含最高为20mM磷酸根离子和/或最高为30mM氯离子的洗涤溶液来洗涤所述混合模式树脂，任选地其中所述溶液包含最高为10mM的磷酸根离子和/或最高为25mM的氯离子。在一些实施方案中，所述洗涤溶液的pH为5.0至7.6。在一些实施方案中，在所述洗涤步骤中，大多数或所有未连接寡核苷酸从所述混合模式树脂中去除。(ii) eluting the complex from the mixed mode resin under conditions where the complex dissociates from the mixed mode resin. In some embodiments, the mixture in step (i) is not pre-purified. In some embodiments, the mixture in step (i) contains trace amounts of phosphate ions and/or chloride ions. In some embodiments, the method further comprises washing the mixed mode resin between step (i) and step (ii) with a washing solution containing up to 20 mM phosphate ions and/or up to 30 mM chloride ions, optionally wherein the solution contains up to 10 mM phosphate ions and/or up to 25 mM chloride ions. In some embodiments, the washing solution has a pH of 5.0 to 7.6. In some embodiments, in the washing step, most or all of the unconnected oligonucleotides are removed from the mixed mode resin.
在一些实施方案中，步骤(ii)包括将向所述混合模式树脂施加包含至少30mM磷酸根离子和/或至少50mM氯离子的洗脱溶液以洗脱所述复合物，任选地其中所述洗脱溶液包含至少100mM的磷酸根离子和/或至少100mM的氯离子。在一些实施方案中，所述洗脱溶液的pH为7.5至8.5。In some embodiments, step (ii) comprises applying an elution solution comprising at least 30 mM phosphate ions and/or at least 50 mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the elution solution comprises at least 100 mM phosphate ions and/or at least 100 mM chloride ions. In some embodiments, the pH of the elution solution is 7.5 to 8.5.
在一些实施方案中，所述抗体是抗转铁蛋白受体抗体。在一些实施方案中，所述寡核苷酸是带电荷寡核苷酸。在一些实施方案中，所述寡核苷酸是带负电荷的寡核苷酸。在一些实施方案中，寡核苷酸是单链的。在一些实施方案中，所述寡核苷酸是反义寡核苷酸，任选地是间隔聚体。在一些实施方案中，所述寡核苷酸是双链寡核苷酸的一条链，任选地其中所述双链寡核苷酸是siRNA，并且任选地其中所述一条链是siRNA的有义链。在一些实施方案中，所述寡核苷酸包含至少一个经修饰核苷酸间键联，任选地其中所述至少一个经修饰核苷酸间键联是硫代磷酸酯键联。在一些实施方案中，所述寡核苷酸包含一个或更多个经修饰核苷酸，任选地其中所述经修饰核苷酸包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(locked nucleic acid，LNA)、2’-氟修饰或吗啉代修饰。In some embodiments, the antibody is an anti-transferrin receptor antibody. In some embodiments, the oligonucleotide is a charged oligonucleotide. In some embodiments, the oligonucleotide is a negatively charged oligonucleotide. In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide is an antisense oligonucleotide, optionally a spacer. In some embodiments, the oligonucleotide is a strand of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is an siRNA, and optionally wherein the strand is the sense strand of the siRNA. In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleotides, optionally wherein the modified nucleotide comprises 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (locked nucleic acid, LNA), 2'-fluorine modification or morpholino modification.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1A至1C示出了涉及抗TfR抗体与寡核苷酸载荷的连接的分子的化学结构。图1A示出了寡核苷酸-PAB-VC-PEG3-叠氮化物的结构。图1B示出了endo-BCN-PEG4-PFP酯。图1C示出了寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯。BCN：双环壬炔。PFP：五氟苯基。PAB：4-氨基苯甲酸。VC：val-cit。在所有图1A至1C中，n为3并且m为4。Figures 1A to 1C show the chemical structures of molecules involved in the connection of anti-TfR antibodies to oligonucleotide payloads. Figure 1A shows the structure of oligonucleotide-PAB-VC-PEG3-azide. Figure 1B shows endo-BCN-PEG4-PFP ester. Figure 1C shows oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester. BCN: bicyclononyne. PFP: pentafluorophenyl. PAB: 4-aminobenzoic acid. VC: val-cit. In all Figures 1A to 1C, n is 3 and m is 4.
图2示出了在包含不同比率的抗TfR Fab与寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的反应中产生的粗制Fab-寡核苷酸缀合物的SDS-PAGE凝胶。最左侧泳道示出了分子量梯带，第二泳道示出了未反应的抗TfR Fab。标记为A、B、C、D、E和F的泳道示出了反应产物。标签D0、D1、D2和D3分别指示药物抗体比为0、1、2和3的反应产物。Fig. 2 shows the SDS-PAGE gel of the crude Fab-oligonucleotide conjugate produced in the reaction containing different ratios of anti-TfR Fab and oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester. The leftmost lane shows the molecular weight ladder, and the second lane shows the unreacted anti-TfR Fab. The lanes marked as A, B, C, D, E and F show the reaction products. Labels D0, D1, D2 and D3 indicate the reaction products of drug-antibody ratios of 0, 1, 2 and 3, respectively.
图3示出了在包含不同比率的抗TfR Fab与寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯和不同溶剂条件的反应中产生的粗制Fab-寡核苷酸缀合物的SDS-PAGE凝胶。最左侧泳道示出了分子量梯带。标记为G、H、I、J、K、L和M的泳道示出了反应产物。Fig. 3 shows the SDS-PAGE gel of the crude Fab-oligonucleotide conjugate produced in the reaction containing different ratios of anti-TfR Fab and oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester and different solvent conditions. The leftmost lane shows the molecular weight ladder. The lanes marked as G, H, I, J, K, L and M show the reaction products.
图4示出了RP C18UPLC随时间监测endo-BCN-PEG4-PFP酯与寡核苷酸-PAB-VC-PEG4-叠氮化物之间的反应的结果。endo-BCN-PEG4-PFP酯起始物质的峰面积随时间在220nm下测量，并示出了在时间0时原始的起始物质峰的百分比。Fig. 4 shows the result of RP C18UPLC monitoring the reaction between endo-BCN-PEG4-PFP ester and oligonucleotide-PAB-VC-PEG4-azide over time.The peak area of endo-BCN-PEG4-PFP ester starting material is measured at 220nm over time, and the percentage of the original starting material peak at time 0 is shown.
图5示出了在260nm和280nm下测量的纯化之后抗TfR Fab’-寡核苷酸缀合物的尺寸排阻色谱(size exclusion chromatography，SEC)色谱图。Figure 5 shows the size exclusion chromatography (SEC) chromatogram of the anti-TfR Fab'-oligonucleotide conjugate after purification measured at 260 nm and 280 nm.
图6示出了纯化之后抗TfR Fab-寡核苷酸缀合物的SDS-PAGE凝胶。最右侧泳道示出了分子量梯带。剩余的三个泳道示出了从纯化柱洗脱的级分。标签D0、D1、D2和D3分别指示药物抗体比为0、1、2和3的反应产物。Figure 6 shows an SDS-PAGE gel of the anti-TfR Fab-oligonucleotide conjugate after purification. The rightmost lane shows the molecular weight ladder. The remaining three lanes show the fractions eluted from the purification column. Labels D0, D1, D2 and D3 indicate reaction products with drug-antibody ratios of 0, 1, 2 and 3, respectively.
图7是示出了使用本文中所述方法处理的复合物在体外降低DMPK mRNA水平的活性的图。复合物1通过2步缀合进行制备：参见实施例6。复合物2通过预反应缀合进行制备：参见实施例1。Figure 7 is a graph showing the activity of complexes treated using the methods described herein in reducing DMPK mRNA levels in vitro. Complex 1 was prepared by 2-step conjugation: see Example 6. Complex 2 was prepared by pre-reaction conjugation: see Example 1.
图8示出了粗制和经纯化的抗TfR-BCN反应产物的分析型HPLC-SEC迹线。粗制产物的曲线在约17.4分钟时示出了第二主峰，这在经纯化产物的曲线中是不存在的，表明去除了未缀合的BCN。BCN：双环壬炔。Figure 8 shows analytical HPLC-SEC traces of crude and purified anti-TfR-BCN reaction products. The curve of the crude product shows a second major peak at about 17.4 minutes, which is absent in the curve of the purified product, indicating the removal of unconjugated BCN. BCN: bicyclononyne.
图9示出了粗制接头-寡核苷酸反应产物的分析型RP-HPLC，除期望的寡核苷酸-接头产物的峰之外，还显示了对应于游离寡核苷酸以及游离接头的峰。Figure 9 shows an analytical RP-HPLC of the crude linker-oligonucleotide reaction product, showing peaks corresponding to free oligonucleotide and free linker in addition to the peak of the desired oligonucleotide-linker product.
图10示出了通过醇沉淀纯化的接头-寡核苷酸反应产物的分析型RP-HPLC。相对于图9中示出的曲线，该曲线示出了游离寡核苷酸峰显著降低并且游离接头峰几乎消失，表明通过纯化过程去除了游离寡核苷酸和接头。Figure 10 shows analytical RP-HPLC of the linker-oligonucleotide reaction product purified by alcohol precipitation. Relative to the curve shown in Figure 9, this curve shows that the free oligonucleotide peak is significantly reduced and the free linker peak almost disappears, indicating that the free oligonucleotide and linker are removed by the purification process.
图11示出了经纯化的寡核苷酸-接头产物的LCMS，仅示出了一个主峰。Figure 11 shows the LCMS of the purified oligonucleotide-adapter product, showing only one major peak.
图12示出了抗TfR-寡核苷酸缀合反应产物的SDS-PAGE分析。左侧泳道示出了分子量梯带，并且右侧泳道示出了反应产物。DAR0、DAR1、DAR2、DAR3和DAR4标签分别指示药物抗体比(drug-antibody ratio，DAR)为0、1、2、3或4的产物。Figure 12 shows an SDS-PAGE analysis of the anti-TfR-oligonucleotide conjugation reaction products. The left lane shows the molecular weight ladder, and the right lane shows the reaction products. The DAR0, DAR1, DAR2, DAR3, and DAR4 labels indicate products with a drug-antibody ratio (DAR) of 0, 1, 2, 3, or 4, respectively.
图13示出了抗TfR-寡核苷酸缀合反应产物的分析型SEC曲线。FIG. 13 shows the analytical SEC curve of the anti-TfR-oligonucleotide conjugation reaction product.
图14示出了在220nm下，在5、35和65分钟反应持续时间时，通过endo-BCN-PEG4-PFP酯起始物质随时间的消失对预反应进程的RP-C18UPLC监测。PFP：五氟苯基。Figure 14 shows RP-C18 UPLC monitoring of the pre-reaction progress by the disappearance of the endo-BCN-PEG4-PFP ester starting material over time at 5, 35 and 65 minutes reaction duration at 220 nm. PFP: pentafluorophenyl.
图15示出了在抗TfR Fab与寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯缀合进行20小时之后粗制反应混合物的SDS-PAGE分析。左侧泳道示出了分子量梯带。D0、D1、D2、D3、D4、D5和D6标签分别指示药物抗体比(DAR)为0、1、2、3、4、5或6的产物。Figure 15 shows SDS-PAGE analysis of the crude reaction mixture after anti-TfR Fab was conjugated to oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester for 20 hours. The left lane shows the molecular weight ladder. The D0, D1, D2, D3, D4, D5 and D6 labels indicate products with a drug-antibody ratio (DAR) of 0, 1, 2, 3, 4, 5 or 6, respectively.
图16示出了在色谱纯化期间HA流通物级分的尺寸排阻色谱(SEC)色谱图(260nm)。色谱图示出了在10.5分钟和11.3分钟时的游离(未缀合)的载荷物质。在装载流通物(loadflow-through)中观察到很少至没有Fab-寡核苷酸缀合物(主峰在约9.1分钟)。Figure 16 shows a size exclusion chromatography (SEC) chromatogram (260 nm) of the HA flow-through fraction during chromatographic purification. The chromatogram shows free (unconjugated) load material at 10.5 minutes and 11.3 minutes. Little to no Fab-oligonucleotide conjugates were observed in the load flow-through (major peak at about 9.1 minutes).
图17示出了在色谱纯化期间HA洗脱峰的SEC色谱图(260nm)。色谱图示出了游离(未缀合)的寡核苷酸载荷物质(预期的峰在约10.5分钟和约11.3分钟)的完全去除，并且替代地仅示出了Fab-寡核苷酸缀合物(主峰在约9.1分钟)。Figure 17 shows a SEC chromatogram (260 nm) of the HA elution peak during chromatographic purification. The chromatogram shows complete removal of free (unconjugated) oligonucleotide cargo material (expected peaks at about 10.5 minutes and about 11.3 minutes), and instead only shows Fab-oligonucleotide conjugates (major peak at about 9.1 minutes).
图18示出了粗制缀合反应产物的24μg注入物(4.0μL以6μg/μL；具有两个主峰的“粗制反应混合物”曲线)和HA洗脱物中理论上24μg的Fab-寡核苷酸缀合物(假设100％回收(7.4μL以3.24μg/μL，并且体积为13.9mL；仅有一个主峰的“HA洗出物”曲线))的重叠SEC色谱图(260nm)。Figure 18 shows overlaid SEC chromatograms (260 nm) of a 24 μg infusion of the crude conjugation reaction product (4.0 μL at 6 μg/μL; "Crude Reaction Mixture" curve with two major peaks) and a theoretical 24 μg of Fab-oligonucleotide conjugate in the HA eluate assuming 100% recovery (7.4 μL at 3.24 μg/μL and a volume of 13.9 mL; "HA Eluate" curve with only one major peak).
图19A至19B示出了在260nm(图19A)和280nm(图19B)处最终经纯化的抗TfR-寡核苷酸Fab-寡核苷酸缀合物的SEC色谱图。19A to 19B show SEC chromatograms of the final purified anti-TfR-oligonucleotide Fab-oligonucleotide conjugate at 260 nm ( FIG. 19A ) and 280 nm ( FIG. 19B ).
图20示出了在抗TfR Fab与寡核苷酸缀合以形成Fab-寡核苷酸缀合物之后，在50mM His(pH 6.0)缓冲液中的经纯化反应产物的SDS-PAGE分析。左侧泳道示出了经纯化的Fab-寡核苷酸缀合物反应产物，右侧泳道示出了分子量梯带。Figure 20 shows the SDS-PAGE analysis of the purified reaction products in 50mM His (pH 6.0) buffer after anti-TfR Fab is conjugated to oligonucleotide to form Fab-oligonucleotide conjugates. The left lane shows the purified Fab-oligonucleotide conjugate reaction product, and the right lane shows the molecular weight ladder.
图21示出了在寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯与抗TfR Fab以不同摩尔比缀合之后粗制反应产物的SDS-PAGE分析。左侧泳道示出了分子量梯带。泳道A、B、C和D示出了分别用相对于抗TfR的2、4、6或10摩尔当量的BCN进行的缀合反应的产物。右侧泳道示出了未反应的抗TfR Fab。D0、D1、D2、D3、D4、D5和D6标签分别指示药物抗体比(DAR)为0、1、2、3、4、5或6的产物。Figure 21 shows the SDS-PAGE analysis of the crude reaction product after oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester was conjugated to anti-TfR Fab in different molar ratios. The left lane shows the molecular weight ladder. Lanes A, B, C and D show the products of the conjugation reaction carried out with 2, 4, 6 or 10 molar equivalents of BCN relative to anti-TfR, respectively. The right lane shows unreacted anti-TfR Fab. The D0, D1, D2, D3, D4, D5 and D6 labels indicate products with a drug-antibody ratio (DAR) of 0, 1, 2, 3, 4, 5 or 6, respectively.
图22示出了在寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯与抗TfR Fab以不同摩尔比缀合之后粗制反应产物的SDS-PAGE分析。最左侧和最右侧泳道示出了分子量梯带。泳道I、H、G、F和E示出了分别用相对于抗TfR的8、6、5、4或摩尔当量的BCN进行的缀合反应的产物。D0、D1、D2、D3、D4、D5和D6标签分别指示药物抗体比(DAR)为0、1、2、3、4、5或6的产物。Figure 22 shows the SDS-PAGE analysis of the crude reaction product after oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester was conjugated to anti-TfR Fab in different molar ratios. The leftmost and rightmost lanes show the molecular weight ladder. Lanes I, H, G, F and E show the products of the conjugation reaction carried out with 8, 6, 5, 4 or molar equivalents of BCN relative to anti-TfR, respectively. The D0, D1, D2, D3, D4, D5 and D6 labels indicate products with a drug-antibody ratio (DAR) of 0, 1, 2, 3, 4, 5 or 6, respectively.
图23示出了endo-BCN-PEG4-PFP酯的水解速率。蓝色曲线结果示出了，在1:1DMA下，endo-BCN-PEG4-PFP酯的初始水解速率为约0.9％/小时。绿色曲线示出了在相同的1:1DMA反应条件下，模型val-cit-PAB-PEG3-叠氮化物载荷与endo-BCN-PEG4-PFP酯之间的反应。两条曲线均示出了在220nm处的吸光度。Figure 23 shows the hydrolysis rate of endo-BCN-PEG4-PFP ester. The blue curve results show that under 1: 1DMA, the initial hydrolysis rate of endo-BCN-PEG4-PFP ester is about 0.9%/hour. The green curve shows the reaction between the model val-cit-PAB-PEG3-azide load and endo-BCN-PEG4-PFP ester under the same 1: 1DMA reaction conditions. Both curves show the absorbance at 220nm.
图24A至24C示出了粗制反应混合物(图24A)、样品流通物(图24B)和合并的HA洗脱物(图24C)的SEC-UPLC分析。粗制反应混合物包含游离寡核苷酸和抗TfR-寡核苷酸Fab-寡核苷酸缀合物。样品流通物包含高DAR物质，具有高抗体-寡核苷酸缀合的物质。合并的HA洗脱物包含残留的寡核苷酸和抗体-寡核苷酸缀合物。Figures 24A to 24C show SEC-UPLC analysis of crude reaction mixture (Figure 24A), sample flowthrough (Figure 24B) and merged HA eluate (Figure 24C). The crude reaction mixture contains free oligonucleotides and anti-TfR-oligonucleotide Fab-oligonucleotide conjugates. The sample flowthrough contains high DAR material, with high antibody-oligonucleotide conjugated material. The merged HA eluate contains residual oligonucleotides and antibody-oligonucleotide conjugates.
图25示出了缓冲液交换的最终缀合物的SEC-UPLC分析。总缀合效率产率为62％，其中残留寡核苷酸在右侧峰中显示。SEC-UPLC analysis of the buffer exchanged final conjugate is shown in Figure 25. The overall conjugation efficiency yield is 62%, with residual oligonucleotide shown in the right peak.
图26A至26B示出了合并的HA洗脱物(图26A)和样品流通物(图26B)的SEC-UPLC分析。样品保持在21℃，并且流量为0.25mL/分钟。流动相包含100mM的磷酸钠和10％的MeCN，pH为7.3。合并的HA洗脱物和样品流通物示出了游离寡核苷酸的存在。Figures 26A to 26B show SEC-UPLC analysis of the combined HA eluate (Figure 26A) and sample flow-through (Figure 26B). The sample was maintained at 21°C and the flow rate was 0.25 mL/min. The mobile phase contained 100 mM sodium phosphate and 10% MeCN at a pH of 7.3. The combined HA eluate and sample flow-through showed the presence of free oligonucleotides.
图27示出了最终抗体片段-药物缀合物(FDC)的SEC-UPLC。样品保持在21℃，并且流量为0.25mL/分钟。流动相包含100mM的NaPO和10％的MeCN，pH为7.3。47.2％的缀合物被回收，其中游离寡核苷酸在右侧峰中显示。Figure 27 shows the SEC-UPLC of the final antibody fragment-drug conjugate (FDC). The sample was kept at 21°C and the flow rate was 0.25 mL/min. The mobile phase contained 100 mM NaPO and 10% MeCN at a pH of 7.3. 47.2% of the conjugate was recovered, with free oligonucleotides shown in the right peak.
图28A至28B示出了每种溶液的HA纯化色谱图(图28A)和SDS-PAGE(图28B)。用pH为3.5的500mM MES将反应混合物的pH调节至5.7。28A to 28B show the HA purification chromatogram ( FIG. 28A ) and SDS-PAGE ( FIG. 28B ) of each solution. The pH of the reaction mixture was adjusted to 5.7 with 500 mM MES at pH 3.5.
图29A至29C示出了在不同平衡缓冲液和装载比为6mg/mL下通过SEC的样品流通物色谱图。图29A示出了在pH 5.6下在0％IPA下10mM磷酸钠平衡缓冲液中的样品流通物。图29B示出了在pH 5.6下在25％IPA下10mM NaPO平衡缓冲液中的样品流通物。图29C示出了在pH 5.6下在25％IPA下5mM磷酸钠平衡缓冲液中的样品流通物。Figures 29A to 29C show chromatograms of sample flowthrough through SEC at different equilibration buffers and a loading ratio of 6 mg/mL. Figure 29A shows sample flowthrough in 10 mM sodium phosphate equilibration buffer at 0% IPA at pH 5.6. Figure 29B shows sample flowthrough in 10 mM NaPO equilibration buffer at 25% IPA at pH 5.6. Figure 29C shows sample flowthrough in 5 mM sodium phosphate equilibration buffer at 25% IPA at pH 5.6.
具体实施方式DETAILED DESCRIPTION
如本文中所述的，本公开内容提供了处理(例如，产生、纯化)复合物(例如，包含蛋白质(例如，与分子载荷(例如，寡核苷酸或小分子)共价连接的肌肉靶向剂(例如，抗体))的复合物)的方法。在一些实施方案中，通过本文中所述方法进行的复合物处理中的分子载荷是寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)或疏水性小分子。在一些实施方案中，使用包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂(例如，羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂、氯磷灰石树脂)从包含所述复合物和未连接(例如，过量)的分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)的混合物中纯化含有与分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的蛋白质(例如，抗体)的一种或多种复合物，其中有机溶剂存在于混合模式树脂色谱的流动相中。混合模式色谱流动相中有机溶剂的存在降低了分子载荷(例如寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)和疏水性小分子)与混合模式树脂之间的非特异性相互作用，并提高了复合物的产率。As described herein, the present disclosure provides methods for treating (e.g., producing, purifying) complexes (e.g., complexes comprising a protein (e.g., a muscle targeting agent (e.g., an antibody) covalently linked to a molecular payload (e.g., an oligonucleotide or a small molecule)). In some embodiments, the molecular payload in the complex treatment performed by the methods described herein is an oligonucleotide (e.g., a charge-neutral oligonucleotide (e.g., a PMO) or a charged oligonucleotide) or a hydrophobic small molecule. In some embodiments, one or more complexes containing a protein (e.g., an antibody) covalently linked to a molecular payload (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide) are purified from a mixture comprising the complex and unlinked (e.g., excess) molecular payload (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide) using a mixed mode resin (e.g., a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, a chloroapatite resin) comprising a positively charged metal site and a negatively charged ionic site, wherein an organic solvent is present in the mobile phase of the mixed mode resin chromatography. The presence of an organic solvent in the mixed mode chromatography mobile phase reduces nonspecific interactions between molecular payloads such as oligonucleotides (e.g., charge neutral oligonucleotides (e.g., PMOs) or charged oligonucleotides) and hydrophobic small molecules and the mixed mode resin and increases the yield of the complex.
在一些实施方案中，被纯化的复合物特别可用于递送调节肌细胞中靶基因之表达或活性的分子载荷(例如，寡核苷酸)，例如在患有或怀疑患有肌肉疾病的对象中。例如，在一些实施方案中，复合物可用于治疗患有罕见肌肉疾病的对象，所述罕见肌肉疾病包括强直性肌营养不良(例如，1型强直性肌营养不良)、面肩肱型肌营养不良(Facioscapulohumeral muscular dystrophy，FSHD)、庞贝病(Pompe disease)、中心核肌病、进行性骨化性纤维结构不良、弗里德赖希共济失调(Friedreich’s ataxia)和迪谢内肌营养不良(Duchenne muscular dystrophy)。在一些实施方案中，取决于待治疗的病症，可在这样的复合物中使用不同的寡核苷酸。In some embodiments, the purified complexes are particularly useful for delivering molecular payloads (e.g., oligonucleotides) that modulate the expression or activity of a target gene in a muscle cell, such as in a subject suffering from or suspected of suffering from a muscle disease. For example, in some embodiments, the complexes can be used to treat subjects suffering from rare muscle diseases, including myotonic dystrophy (e.g., myotonic dystrophy type 1), facioscapulohumeral muscular dystrophy (FSHD), Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva, Friedreich's ataxia, and Duchenne muscular dystrophy. In some embodiments, different oligonucleotides can be used in such complexes, depending on the condition to be treated.
下面提供了本公开内容的另一些方面，包括定义的术语的描述。[0013] Provided below are further aspects of the disclosure, including a description of defined terms.
I.定义I. Definitions
施用：如本文中使用的，术语“施用”及其变化形式意指以生理和/或药理学上可用的方式向对象提供复合物(例如，以治疗对象中的病症)。Administering: As used herein, the term "administering" and variations thereof means providing a complex to a subject in a physiologically and/or pharmacologically usable manner (eg, to treat a condition in a subject).
大约：如本文中使用的，术语“大约”或“约”，如应用于一个或更多个目标值时，是指类似于陈述的参考值的值。在某些实施方案中，术语“大约”或“约”是指落入陈述的参考值的任一方向上(大于或小于)15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、或更小以内的值的范围，除非另有说明或在其他情况下从上下文中明显看出(除非这样的数字超过可能值的100％)。Approximately: As used herein, the term "approximately" or "about", as applied to one or more target values, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value, unless otherwise specified or otherwise apparent from the context (unless such a number exceeds 100% of the possible value).
抗体：如本文中使用的，术语“抗体”是指包含至少一个免疫球蛋白可变结构域或至少一个抗原决定簇(例如，与抗原特异性结合的互补位(paratope))的多肽。在一些实施方案中，抗体是全长抗体，例如全长IgG。在一些实施方案中，抗体是嵌合抗体。在一些实施方案中，抗体是人源化抗体。然而，在一些实施方案中，抗体是Fab片段、F(ab’)2片段、Fv片段或scFv片段。在一些实施方案中，抗体是来源于骆驼科抗体的纳米抗体或来源于鲨鱼抗体的纳米抗体。在一些实施方案中，抗体是双抗体。在一些实施方案中，抗体包含具有人种系序列的框架。在另一个实施方案中，抗体包含选自IgG、IgG1、IgG2、IgG2A、IgG2B、IgG2C、IgG3、IgG4、IgA1、IgA2、IgD、IgM和IgE恒定结构域的重链恒定结构域。在一些实施方案中，抗体包含重(heavy，H)链可变区(在本文中简称为VH)和/或轻(light，L)链可变区(在本文中简称为VL)。在一些实施方案中，抗体包含恒定结构域，例如Fc区。免疫球蛋白恒定结构域是指重链或轻链恒定结构域。人IgG重链和轻链恒定结构域氨基酸序列及其功能变异是已知的。关于重链，在一些实施方案中，本文中所述的抗体的重链可以是alpha(α)、delta(Δ)、epsilon(ε)、gamma(γ)或mu(μ)重链。在一些实施方案中，本文中所述的抗体的重链可包含人alpha(α)、delta(Δ)、epsilon(ε)、gamma(γ)或mu(μ)重链。在一个特定的实施方案中，本文中所述的抗体包含人γ1CH1、CH2和/或CH3结构域。在一些实施方案中，VH结构域的氨基酸序列包含人gamma(γ)重链恒定区的氨基酸序列，例如本领域已知的任何。人恒定区序列的非限制性实例已在本领域中描述，例如，参见美国专利No.5,693,780和Kabat E Aet al.,(1991)同上。在一些实施方案中，VH结构域包含与本文中提供的任何可变链恒定区具有至少70％、75％、80％、85％、90％、95％、98％或至少99％同一性的氨基酸序列。在一些实施方案中，对抗体进行修饰，例如，通过糖基化、磷酸化、SUMO化(sumoylation)和/或甲基化进行修饰。在一些实施方案中，抗体是与一个或更多个糖或碳水化合物分子缀合的糖基化抗体。在一些实施方案中，一个或更多个糖或碳水化合物分子通过N-糖基化、O-糖基化、C-糖基化、糖基磷脂酰肌醇化(GPI锚定附着)和/或磷酸糖基化(phosphoglycosylation)与抗体缀合。在一些实施方案中，一个或更多个糖或碳水化合物分子是单糖、二糖、寡糖或聚糖。在一些实施方案中，一个或更多个糖或碳水化合物分子是支链的寡糖或支链的聚糖。在一些实施方案中，一个或更多个糖或碳水化合物分子包含甘露糖单元、葡萄糖单元、N-乙酰葡糖胺单元、N-乙酰半乳糖胺单元、半乳糖单元、岩藻糖单元或磷脂单元。在一些实施方案中，抗体是包含多肽的构建体，所述多肽包含与接头多肽或免疫球蛋白恒定结构域共价连接的一个或更多个本公开内容的抗原结合片段。接头多肽包含通过肽键连接的两个或更多个氨基酸残基，并且用于连接一个或更多个抗原结合部分。接头多肽的实例已有报道(参见例如，Holliger,P.,et al.(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.,et al.(1994)Structure 2:1121-1123)。另外，抗体可以是更大的免疫黏附分子的一部分，免疫黏附分子通过抗体或抗体部分与一个或更多个其他蛋白质或肽的共价或非共价缔合而形成。这样的免疫黏附分子的实例包括使用链霉亲和素核芯区域来制备四聚体scFv分子(Kipriyanov,S.M.,et al.(1995)Human Antibodies and Hybridomas 6:93-101)，以及使用半胱氨酸残基、标记肽和C端多组氨酸标签来制备二价和生物素化的scFv分子(Kipriyanov,S.M.,et al.(1994)Mol.Immunol.31:1047-1058)。Antibody: As used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant (e.g., a paratope that specifically binds to an antigen). In some embodiments, the antibody is a full-length antibody, such as a full-length IgG. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment, or a scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a double antibody. In some embodiments, the antibody comprises a framework with a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (referred to herein as VH) and/or a light (L) chain variable region (referred to herein as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains and their functional variations are known. With regard to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a specific embodiment, the antibody described herein comprises a human γ1CH1, CH2 and/or CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with any variable chain constant region provided herein. In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and/or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and/or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure covalently linked to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to connect one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). In addition, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine residues, a marker peptide, and a C-terminal polyhistidine tag to prepare bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058).
CDR：如本文中使用的，术语“CDR”是指抗体可变序列内的互补决定区。重链和轻链的每个可变区中有三个CDR，对于每个可变区分别称为CDR1、CDR2和CDR3。如本文中使用的术语“CDR组”是指出现在单个可变区内的能够结合抗原的三个CDR的组。这些CDR的确切边界已根据不同的系统进行了不同的定义。通过Kabat描述的系统(Kabat et al.,Sequencesof Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987)and(1991))不仅提供了适用于抗体的任何可变区的明确的残基编号系统，而且还提供了定义三个CDR的精确残基边界。这些CDR可以被称为Kabat CDR。CDR的子部分可以被指定为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指定轻链和重链区域。这些区域可以称为Chothia CDR，其具有与Kabat CDR重叠的边界。Padlan(FASEB J.9:133-139(1995))和MacCallum(J Mol Biol 262(5):732-45(1996))已经描述了定义与Kabat CDR重叠的CDR的其他边界。另一些CDR边界定义可能并不严格遵循上述系统之一，但仍与Kabat CDR重叠，尽管可以根据预测或者根据特定残基或残基的组或甚至整个CDR不会显著影响抗原结合的实验发现来缩短或延长它们。尽管优选的实施方案使用Kabat或Chothia定义的CDR，但是本文中使用的方法可利用根据这些系统中的任何一个定义的CDR。CDR: As used herein, the term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to the group of three CDRs that appear within a single variable region and are capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Subportions of the CDRs may be designated as L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)) have described other boundaries that define CDRs that overlap with the Kabat CDRs. Other CDR boundary definitions may not strictly follow one of the above systems, but may still be consistent with the Kabat CDRs. CDRs overlap, although they can be shortened or lengthened based on predictions or based on experimental findings that specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. Although preferred embodiments use CDRs defined by Kabat or Chothia, the methods used herein can utilize CDRs defined according to any of these systems.
CDR接枝抗体(CDR-grafted antibody)：术语“CDR接枝抗体”是指包含来自一个物种的重链和轻链可变区序列但是其中VH和/或VL的一个或更多个CDR区的序列被来自另一物种的CDR序列替代的抗体，例如具有鼠重链和轻链可变区并且其中一个或更多个鼠CDR(例如CDR3)已被人CDR序列替代的抗体。CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species but in which the sequence of one or more CDR regions of VH and/or VL is replaced by a CDR sequence from another species, such as an antibody having mouse heavy and light chain variable regions in which one or more mouse CDRs (e.g., CDR3) have been replaced by a human CDR sequence.
嵌合抗体：术语“嵌合抗体”是指包含来自一个物种的重链和轻链可变区序列和来自另一物种的恒定区序列的抗体，例如具有与人恒定区连接的鼠重链和轻链可变区的抗体。Chimeric antibody: The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.
互补：如本文中使用的，术语“互补”是指在两个核苷酸或两组核苷酸之间精确配对的能力。特别地，互补是表征氢键配对引起两个核苷酸或两组核苷酸之间结合的程度的术语。例如，如果寡核苷酸的一个位置处的碱基能够与靶核酸(例如，mRNA)的相应位置处的碱基进行氢键合，则认为在该位置处碱基彼此互补。碱基配对可包括规范的沃森-克里克碱基配对和非沃森-克里克碱基配对(例如，Wobble碱基配对和Hoogsteen碱基配对)二者。例如，在一些实施方案中，对于互补碱基配对，腺苷型碱基(A)与胸苷型碱基(T)或尿嘧啶型碱基(U)互补，胞嘧啶型碱基(C)与鸟苷型碱基(G)互补，并且通用碱基如3-硝基吡咯或5-硝基吲哚可与任何A、C、U或T杂交并被认为是互补的。肌苷(I)在本领域中也被认为是通用碱基，并且被认为与任何A、C、U或T互补。Complementary: As used herein, the term "complementary" refers to the ability to accurately pair between two nucleotides or two groups of nucleotides. In particular, complementarity is a term that characterizes the degree of binding between two nucleotides or two groups of nucleotides caused by hydrogen bond pairing. For example, if the base at one position of an oligonucleotide can hydrogen bond with the base at the corresponding position of a target nucleic acid (e.g., mRNA), it is considered that the bases at this position are complementary to each other. Base pairing can include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, adenosine base (A) is complementary to thymidine base (T) or uracil base (U), cytosine base (C) is complementary to guanosine base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary. Inosine (I) is also considered in the art to be a universal base, and is considered complementary to any of A, C, U, or T.
保守氨基酸替换：如本文中使用的，“保守氨基酸替换”是指不改变进行氨基酸替换的蛋白质的相对电荷或尺寸特征的氨基酸替换。可以根据本领域普通技术人员已知的用于改变多肽序列的方法来制备变体，所述方法例如可以在汇编这样的方法的参考文献中找到：例如Molecular Cloning:ALaboratory Manual,J.Sambrook,et al.,eds.,FourthEdition,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York,2012,或Current Protocols in Molecular Biology,F.M.Ausubel,et al.,eds.,John Wiley&Sons,Inc.,New York。氨基酸的保守替换包括在以下组内的氨基酸之间进行的替换：(a)M、I、L、V；(b)F、Y、W；(c)K、R、H；(d)A、G；(e)S、T；(f)Q、N；和(g)E、D。Conservative amino acid substitutions: As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods: for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
共价连接：如本文中使用的，术语“共价连接”是指两个或更多个分子通过至少一个共价键连接在一起的特征。在一些实施方案中，两个分子可以通过充当分子之间的接头的单键例如二硫键或二硫桥共价连接在一起。然而，在一些实施方案中，两个或更多个分子可以通过充当接头的分子共价连接在一起，该接头通过多个共价键将两个或更多个分子连接在一起。在一些实施方案中，接头可以是可切割接头。然而，在一些实施方案中，接头可以是不可切割接头。Covalently linked: As used herein, the term "covalently linked" refers to the feature of two or more molecules being linked together by at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, such as a disulfide bond or a disulfide bridge, that acts as a linker between the molecules. However, in some embodiments, two or more molecules can be covalently linked together by a molecule that acts as a linker that links the two or more molecules together by multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.
交叉反应性：如本文中使用的以及在靶向剂(例如，抗体)的情况下，术语“交叉反应性”是指物质能够以相似亲和力或亲合力与相似类型或类别的超过一种抗原(例如，多个同源物、旁系同源物或直系同源物的抗原)特异性结合的性质。例如，在一些实施方案中，对相似类型或类别的人和非人灵长类抗原(例如人转铁蛋白受体和非人灵长类转移受体)具有交叉反应性的抗体能够以相似亲和力或亲合力与人抗原和非人灵长类抗原结合。在一些实施方案中，抗体对相似类型或类别的人抗原和啮齿动物抗原具有交叉反应性。在一些实施方案中，抗体对相似类型或类别的啮齿动物抗原和非人灵长类抗原具有交叉反应性。在一些实施方案中，抗体对相似类型或类别的人抗原、非人灵长类抗原和啮齿动物抗原具有交叉反应性。Cross-reactivity: As used herein and in the case of targeting agents (e.g., antibodies), the term "cross-reactivity" refers to the property of a substance being able to specifically bind to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity. For example, in some embodiments, antibodies cross-reactive to human and non-human primate antigens of similar types or classes (e.g., human transferrin receptor and non-human primate transfer receptor) are able to bind to human antigens and non-human primate antigens with similar affinity or avidity. In some embodiments, antibodies are cross-reactive to human antigens and rodent antigens of similar types or classes. In some embodiments, antibodies are cross-reactive to rodent antigens and non-human primate antigens of similar types or classes. In some embodiments, antibodies are cross-reactive to human antigens, non-human primate antigens, and rodent antigens of similar types or classes.
疾病等位基因：如本文中使用的，术语“疾病等位基因”是指等位基因与疾病相关和/或者直接或间接促成或造成疾病的基因的任一种替代形式(例如突变形式)。相对于野生型(非疾病)等位基因，疾病等位基因可包含基因改变，包括但不限于插入(例如，下文所述的疾病相关重复)、缺失、错义突变、无义突变和剪接位点突变。在一些实施方案中，疾病等位基因具有功能丧失性突变。在一些实施方案中，疾病等位基因具有功能获得性突变。在一些实施方案中，疾病等位基因编码激活突变(例如，编码组成性活性的蛋白质)。在一些实施方案中，疾病等位基因是具有隐性表型的隐性等位基因。在一些实施方案中，疾病等位基因是具有显性表型的显性等位基因。Disease allele: As used herein, the term "disease allele" refers to any alternative form (e.g., mutant form) of a gene in which the allele is associated with a disease and/or directly or indirectly contributes to or causes a disease. Relative to a wild-type (non-disease) allele, a disease allele may comprise a genetic alteration, including but not limited to insertions (e.g., disease-associated duplications described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, the disease allele has a loss-of-function mutation. In some embodiments, the disease allele has a gain-of-function mutation. In some embodiments, the disease allele encodes an activating mutation (e.g., encoding a constitutively active protein). In some embodiments, the disease allele is a recessive allele with a recessive phenotype. In some embodiments, the disease allele is a dominant allele with a dominant phenotype.
疾病相关重复：如本文中使用的，术语“疾病相关重复”是指在基因组位置处的重复核苷酸序列，其中重复核苷酸序列的单元的数目与遗传疾病相关和/或者直接或间接促成或造成遗传疾病。疾病相关重复的每个重复单元的长度可以是2、3、4、5个或更多个核苷酸。例如，在一些实施方案中，疾病相关重复是二核苷酸重复。在一些实施方案中，疾病相关重复是三核苷酸重复。在一些实施方案中，疾病相关重复是四核苷酸重复。在一些实施方案中，疾病相关重复是五核苷酸重复。在一些实施方案中，疾病相关重复包含CAG重复、CTG重复、CUG重复、CGG重复、CCTG重复或其任何的核苷酸互补序列。在一些实施方案中，疾病相关重复在基因的非编码部分中。然而，在一些实施方案中，疾病相关重复在基因的编码区中。在一些实施方案中，疾病相关重复从正常状态扩增至直接或间接促成或造成遗传疾病的长度。在一些实施方案中，疾病相关重复在RNA(例如，RNA转录物)中。在一些实施方案中，疾病相关重复在DNA(例如，染色体、质粒)中。在一些实施方案中，疾病相关重复在生殖细胞的染色体中扩增。在一些实施方案中，疾病相关重复在体细胞的染色体中扩增。在一些实施方案中，疾病相关重复扩增至与疾病的先天性发作相关的重复单元数目。在一些实施方案中，疾病相关重复扩增至与儿童期疾病发作相关的重复单元数目。在一些实施方案中，疾病相关重复扩增至与成年疾病发作相关的重复单元数目。Disease-related repetition: As used herein, the term "disease-related repetition" refers to a repetitive nucleotide sequence at a genomic position, wherein the number of units of the repetitive nucleotide sequence is associated with a genetic disease and/or directly or indirectly contributes to or causes a genetic disease. The length of each repetitive unit of disease-related repetition can be 2, 3, 4, 5 or more nucleotides. For example, in some embodiments, disease-related repetition is a dinucleotide repetition. In some embodiments, disease-related repetition is a trinucleotide repetition. In some embodiments, disease-related repetition is a tetranucleotide repetition. In some embodiments, disease-related repetition is a pentanucleotide repetition. In some embodiments, disease-related repetitions include CAG repetitions, CTG repetitions, CUG repetitions, CGG repetitions, CCTG repetitions, or any nucleotide complementary sequence thereof. In some embodiments, disease-related repetitions are in the non-coding portion of a gene. However, in some embodiments, disease-related repetitions are in the coding region of a gene. In some embodiments, disease-related repetitions are amplified from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, disease-related repetitions are in RNA (e.g., RNA transcripts). In some embodiments, disease-related repetitions are in DNA (e.g., chromosomes, plasmids). In some embodiments, the disease-associated repeats are expanded in the chromosomes of germ cells. In some embodiments, the disease-associated repeats are expanded in the chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the congenital onset of the disease. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the childhood onset of the disease. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the adult onset of the disease.
框架：如本文中使用的，术语“框架”或“框架序列”是指可变区减去CDR的剩余序列。由于CDR序列的确切定义可通过不同的系统确定，因此框架序列的含义相应地具有不同解释。六个CDR(轻链的CDR-L1、CDR-L2和CDR-L3和重链的CDR-H1、CDR-H2和CDR-H3)也将轻链和重链上的框架区分为每条链上的四个子区域(FR1、FR2、FR3和FR4)，其中CDR1位于FR1和FR2之间，CDR2位于FR2和FR3之间，并且CDR3位于FR3和FR4之间。在未将特定子区域指定为FR1、FR2、FR3或FR4的情况下，其他人提到的框架区代表单个天然免疫球蛋白链的可变区内的组合的FR。如本文所使用的，FR代表四个子区域之一，并且FRs代表构成框架区的四个子区域中的两个或更多个。人重链和轻链受体序列是本领域已知的。在一个实施方案中，本领域已知的受体序列可用于本文中公开的抗体中。Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus CDR. Since the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences has different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also distinguish the framework on the light chain and the heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. In the case where a specific subregion is not designated as FR1, FR2, FR3 or FR4, the framework region mentioned by others represents the FR of the combination in the variable region of a single natural immunoglobulin chain. As used herein, FR represents one of the four subregions, and FRs represents two or more of the four subregions constituting the framework region. Human heavy chain and light chain receptor sequences are known in the art. In one embodiment, receptor sequences known in the art may be used in the antibodies disclosed herein.
人抗体：如本文中使用的，术语“人抗体”旨在包括具有源自人种系免疫球蛋白序列的可变区和恒定区的抗体。本公开内容的人抗体可包含不是由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)，例如在CDR中，特别是在CDR3中。然而，如本文中使用的，术语“人抗体”不意图包括其中源自另一哺乳动物物种(例如小鼠)种系的CDR序列已接枝到人框架序列上的抗体。Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDRs, particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.
人源化抗体：术语“人源化抗体”是指包含来自非人物种(例如，小鼠)的重链和轻链可变区序列但是其中VH和/或VL序列的至少一部分已被改变为更加“人样”(即，更类似于人种系可变序列)的抗体。一种类型的人源化抗体是CDR接枝抗体，其中人CDR序列被引入非人VH和VL序列中以替代相应的非人CDR序列。在一个实施方案中，提供了人源化抗转铁蛋白受体抗体和抗原结合部分。这样的抗体可以通过使用传统的杂交瘤技术获得鼠抗转铁蛋白受体单克隆抗体随后使用体外基因工程化进行人源化来产生，例如在Kasaian等人的PCT公开No.WO 2005/123126 A2中公开的那些。Humanized antibody: The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from non-human species (e.g., mice) but wherein at least a portion of the VH and/or VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining mouse anti-transferrin receptor monoclonal antibodies using traditional hybridoma technology and then humanizing using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO 2005/123126 A2 of Kasaian et al.
内化细胞表面受体：如本文中使用的，术语“内化细胞表面受体”是指在外部刺激(例如配体与受体结合)下被细胞内化的细胞表面受体。在一些实施方案中，内化细胞表面受体通过内吞作用内化。在一些实施方案中，内化细胞表面受体通过网格蛋白介导的内吞作用内化。然而，在一些实施方案中，内化细胞表面受体通过不依赖于网格蛋白的途径内化，例如如吞噬作用、巨胞饮作用、小窝和筏介导的摄取或组成型网格蛋白非依赖性内吞作用。在一些实施方案中，内化细胞表面受体包含胞内结构域、跨膜结构域和/或胞外结构域，其可任选地还包含配体结合结构域。在一些实施方案中，细胞表面受体在配体结合后被细胞内化。在一些实施方案中，配体可以是肌肉靶向蛋白或肌肉靶向抗体。在一些实施方案中，内化细胞表面受体是转铁蛋白受体。Internalized cell surface receptor: As used herein, the term "internalized cell surface receptor" refers to a cell surface receptor that is internalized by a cell under external stimulation (e.g., ligand binding to a receptor). In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, pits and raft-mediated uptake or constitutive clathrin-independent endocytosis. In some embodiments, the internalized cell surface receptor comprises an intracellular domain, a transmembrane domain, and/or an extracellular domain, which may optionally further comprise a ligand binding domain. In some embodiments, the cell surface receptor is internalized by a cell after ligand binding. In some embodiments, the ligand may be a muscle targeting protein or a muscle targeting antibody. In some embodiments, the internalized cell surface receptor is a transferrin receptor.
分离的抗体：本文所用的“分离的抗体”旨在指代基本上不含具有不同抗原特异性的其他抗体的抗体(例如，特异性结合转铁蛋白受体的分离的抗体基本上不含特异性结合转铁蛋白受体以外的抗原的抗体)。然而，特异性结合转铁蛋白受体复合物的分离的抗体可能与其他抗原(例如来自其他物种的转铁蛋白受体分子)具有交叉反应性。此外，分离的抗体可以基本上不含其他细胞材料和/或化学物质。Separated antibody: "Separated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., a separated antibody that specifically binds to transferrin receptor is substantially free of antibodies that specifically bind to antigens other than transferrin receptor). However, a separated antibody that specifically binds to transferrin receptor complex may have cross-reactivity with other antigens (e.g., transferrin receptor molecules from other species). In addition, the separated antibody may be substantially free of other cellular materials and/or chemicals.
Kabat编号：术语“Kabat编号”、“Kabat定义和“Kabat标记”在本文可互换使用。在本领域中公认的这些术语是指对抗体或其抗原结合部分的重链和轻链可变区中的比其他氨基酸残基更加可变(即高变)的氨基酸残基进行编号的系统(Kabat et al.(1971)Ann.NYAcad,Sci.190:382-391以及,Kabat,E.A.,et al.(1991)Sequences of Proteins ofImmunological Interest,Fifth Edition,U.S.Department of Health and HumanServices,NIH Publication No.91-3242)。对于重链可变区，CDR1的高变区为第31至35位氨基酸，CDR2的高变区为第50至65位氨基酸，并且CDR3的高变区为第95至102位氨基酸。对于轻链可变区，CDR1的高变区为第24至34位氨基酸，CDR2的高变区为第50至56位氨基酸，并且CDR3的高变区为第89至97位氨基酸。Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat notation" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or antigen-binding portion thereof that are more variable (i.e., hypervariable) than other amino acid residues (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 2001-0306). No.91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is from amino acids 31 to 35, the hypervariable region of CDR2 is from amino acids 50 to 65, and the hypervariable region of CDR3 is from amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is from amino acids 24 to 34, the hypervariable region of CDR2 is from amino acids 50 to 56, and the hypervariable region of CDR3 is from amino acids 89 to 97.
混合模式树脂：如本文中使用的，术语“混合模式树脂”是指包含带正电荷的金属位点和带负电荷的离子位点的用于生物分子的纯化、分离和/或分离的色谱树脂或材料。在一些实施方案中，金属位点包含钙。在一些实施方案中，带负电荷的离子位点包含磷酸根、硫酸根、氟化物或氯化物。在一些实施方案中，金属位点包含钙，并且带负电荷的离子位点包含磷酸根，以及任选的硫酸根、氟化物或氯化物。在一些实施方案中，混合模式树脂是磷灰石树脂。在一些实施方案中，磷灰石树脂是羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂或氯磷灰石树脂。在一些实施方案中，磷灰石树脂包含下式的矿物质：Ca10(PO4)6(OH)2。在一些实施方案中，磷灰石树脂包含下式的矿物质：Ca10(PO4)6F2。在一些实施方案中，磷灰石树脂包含下式的矿物质：Ca10(PO4)6Cl2。Mixed mode resin: As used herein, the term "mixed mode resin" refers to a chromatographic resin or material for the purification, separation and/or isolation of biomolecules that contains positively charged metal sites and negatively charged ionic sites. In some embodiments, the metal sites contain calcium. In some embodiments, the negatively charged ionic sites contain phosphate, sulfate, fluoride, or chloride. In some embodiments, the metal sites contain calcium, and the negatively charged ionic sites contain phosphate, and optionally sulfate, fluoride, or chloride. In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. In some embodiments, the apatite resin contains a mineral of the formula: Ca10 (PO4 )6 (OH)2 . In some embodiments, the apatite resin contains a mineral of the formula: Ca10 (PO4 )6 F2 . In some embodiments, the apatite resin comprises a mineral of the formula: Ca10 (PO4 )6 Cl2 .
分子载荷：如本文中使用的，术语“分子载荷”是指发挥调节生物学结局作用的分子或物质。在一些实施方案中，分子载荷与肌肉靶向剂共价连接或以其他方式缔合。在一些实施方案中，分子载荷是小分子、蛋白质、肽、核酸或寡核苷酸。在一些实施方案中，分子载荷是疏水性小分子。在一些实施方案中，分子载荷是寡核苷酸(例如，电荷中性寡核苷酸(例如，磷酸二酰胺吗啉代寡聚物(phosphorodiamidate morpholino oligomer，PMO))或带电荷寡核苷酸)。在一些实施方案中，分子载荷发挥调节DNA序列的转录、调节蛋白质的表达或调节蛋白质的活性的作用。在一些实施方案中，分子载荷是寡核苷酸，例如包含具有与靶基因互补的区域的链的寡核苷酸。Molecular payload: As used herein, the term "molecular payload" refers to a molecule or substance that plays a role in regulating biological outcomes. In some embodiments, the molecular payload is covalently linked to a muscle targeting agent or otherwise associated. In some embodiments, the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide. In some embodiments, the molecular payload is a hydrophobic small molecule. In some embodiments, the molecular payload is an oligonucleotide (e.g., a charge-neutral oligonucleotide (e.g., phosphorodiamidate morpholino oligomer (PMO)) or a charged oligonucleotide). In some embodiments, the molecular payload plays a role in regulating the transcription of a DNA sequence, regulating the expression of a protein, or regulating the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide, such as an oligonucleotide comprising a chain having a region complementary to a target gene.
肌肉疾病基因：如本文中使用的，术语“肌肉疾病基因”是指具有至少一个与肌肉疾病相关和/或者直接或间接促成或造成肌肉疾病的疾病等位基因的基因。在一些实施方案中，肌肉疾病是罕见疾病，例如如遗传和罕见疾病信息中心(Genetic and RareDiseases Information Center，GARD)所定义，遗传和罕见疾病信息中心(GARD)是美国国家转化科学促进中心(National Center for Advancing Translational Sciences，NCATS)的计划。在一些实施方案中，肌肉疾病是罕见疾病，其以影响少于200,000人为特征。在一些实施方案中，肌肉疾病是单基因疾病。在一些实施方案中，肌肉疾病基因是表1中列出的基因。Muscle disease gene: As used herein, the term "muscle disease gene" refers to a gene having at least one disease allele associated with a muscle disease and/or directly or indirectly contributing to or causing a muscle disease. In some embodiments, the muscle disease is a rare disease, such as defined by the Genetic and Rare Diseases Information Center (GARD), which is a program of the National Center for Advancing Translational Sciences (NCATS). In some embodiments, the muscle disease is a rare disease characterized by affecting less than 200,000 people. In some embodiments, the muscle disease is a monogenic disease. In some embodiments, the muscle disease gene is a gene listed in Table 1.
肌肉靶向剂：如本文中使用的，术语“肌肉靶向剂”是指与肌细胞上表达的抗原特异性结合的分子。肌细胞内或上的抗原可以是膜蛋白，例如整合膜蛋白或外周膜蛋白。通常来说，肌肉靶向剂与肌细胞上的抗原特异性结合，这有助于将肌肉靶向剂(和任何缔合的分子载荷)内化到肌细胞中。在一些实施方案中，肌肉靶向剂与肌肉上的内化细胞表面受体特异性结合，并且能够通过受体介导的内化而内化到肌细胞中。在一些实施方案中，肌肉靶向剂是小分子、蛋白质、肽、核酸(例如适配体)、或抗体。在一些实施方案中，肌肉靶向剂与分子载荷共价连接。在一些实施方案中，肌肉靶向剂是肌肉靶向蛋白(例如，抗体)。Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell can be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Generally speaking, a muscle targeting agent specifically binds to an antigen on a muscle cell, which helps internalize the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, a muscle targeting agent specifically binds to an internalizing cell surface receptor on the muscle and can be internalized into the muscle cell by receptor-mediated internalization. In some embodiments, a muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, a muscle targeting agent is covalently linked to a molecular payload. In some embodiments, a muscle targeting agent is a muscle targeting protein (e.g., an antibody).
肌肉靶向抗体：如本文中使用的，术语“肌肉靶向抗体”是指为与存在于肌细胞内或上的抗原特异性结合的抗体的肌肉靶向剂。在一些实施方案中，肌肉靶向抗体与肌细胞上的抗原特异性结合，这有助于将肌肉靶向抗体(和任何缔合的分子载荷)内化到肌细胞中。在一些实施方案中，肌肉靶向抗体与存在于肌细胞上的内化细胞表面受体特异性结合。在一些实施方案中，肌肉靶向抗体是与转铁蛋白受体特异性结合的抗体。Muscle targeting antibody: As used herein, the term "muscle targeting antibody" refers to a muscle targeting agent that is an antibody that specifically binds to an antigen present in or on a muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an antigen on a muscle cell, which facilitates internalization of the muscle targeting antibody (and any associated molecular cargo) into the muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to the transferrin receptor.
寡核苷酸：如本文中使用的，术语“寡核苷酸”是指长度最高为200个核苷酸的寡聚核酸化合物。寡核苷酸的实例包括但不限于RNAi寡核苷酸(例如，siRNA、shRNA)、微RNA、间隔聚体、混合聚体、亚磷酸二酰胺吗啉代、肽核酸、适配体、指导核酸(例如，Cas9指导RNA)等。寡核苷酸可以是单链或双链。在一些实施方案中，寡核苷酸可包含一个或更多个经修饰核苷酸(例如2’-O-甲基糖修饰、嘌呤或嘧啶修饰)。在一些实施方案中，寡核苷酸可包含一个或更多个经修饰核苷酸间键联。在一些实施方案中，寡核苷酸可包含一个或更多个硫代磷酸酯键联，其可以处于Rp或Sp立体化学构象。Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, spacer polymers, mixed polymers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, the oligonucleotide may include one or more modified nucleotides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, the oligonucleotide may include one or more modified internucleotide linkages. In some embodiments, the oligonucleotide may include one or more phosphorothioate linkages, which may be in Rp or Sp stereochemical conformations.
重组抗体：如本文中使用的，术语“重组人抗体”旨在包括通过重组方式制备、表达、产生或分离的所有人抗体，例如使用转染到宿主细胞中的重组表达载体表达的抗体(在本公开内容中更详细地描述)，从重组、组合人抗体文库分离的抗体(Hoogenboom H.R.,(1997)TIB Tech.15:62-70；Azzazy H.,and Highsmith W.E.,(2002)Clin.Biochem.35:425-445；Gavilondo J.V.,and Larrick J.W.(2002)BioTechniques 29:128-145；Hoogenboom H.,and Chames P.(2000)Immunology Today 21:371-378)，从人免疫球蛋白基因转基因的动物(例如，小鼠)分离的抗体(参见例如Taylor,L.D.,et al.(1992)Nucl.Acids Res.20:6287-6295；Kellermann S-A.,and Green L.L.(2002)CurrentOpinion in Biotechnology 13:593-597；Little M.et al(2000)Immunology Today 21:364-370)，或通过涉及将人免疫球蛋白基因序列剪接至其他DNA序列的任何其他方式制备、表达、产生或分离的抗体。这样的重组人抗体具有源自人种系免疫球蛋白序列的可变区和恒定区。然而，在某些实施方案中，对这样的重组人抗体进行体外诱变(或当使用人Ig序列转基因的动物时，进行体内体细胞诱变)，并且因此重组抗体的VH和VL区的氨基酸序列是这样的序列，尽管其源自人种系VH和VL序列并与之相关，但可能不是体内人抗体种系库中天然存在的。本公开内容的一个实施方案提供了能够结合人转铁蛋白受体的完全人抗体，其可使用本领域公知的技术产生，例如但不限于使用人Ig噬菌体文库，例如Jermutus等人的PCT公开No.WO 2005/007699 A2中公开的那些。Recombinant antibody: As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail in this disclosure), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W.E., (2002) Clin. Biochem. 35:425-445; Gavilondo J.V., and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor, L.D., et al., 2003). al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13: 593-597; Little M. et al (2000) Immunology Today 21: 364-370), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgenic for human Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not be naturally present in the human antibody germline repertoire in vivo. One embodiment of the present disclosure provides fully human antibodies capable of binding to human transferrin receptor, which can be generated using techniques known in the art, such as, but not limited to, using human Ig phage libraries, such as those disclosed in PCT Publication No. WO 2005/007699 A2 to Jermutus et al.
互补区：如本文中使用的，术语“互补区”是指与例如靶核酸的同源核苷酸序列充分互补的例如寡核苷酸的核苷酸序列，使得两个核苷酸序列能够在生理条件下(例如，在细胞中)彼此退火。在一些实施方案中，互补区与靶核酸的同源核苷酸序列完全互补。然而，在一些实施方案中，互补区与靶核酸的同源核苷酸序列部分互补(例如，至少80％、90％、95％或99％互补)。在一些实施方案中，与靶核酸的同源核苷酸序列相比，互补区包含1、2、3或4个错配。Complementary region: As used herein, the term "complementary region" refers to a nucleotide sequence, such as an oligonucleotide, that is fully complementary to a homologous nucleotide sequence, such as a target nucleic acid, so that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is completely complementary to the homologous nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the homologous nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementary). In some embodiments, the complementary region comprises 1, 2, 3 or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.
特异性结合：如本文中使用的，术语“特异性结合”是指分子以一定程度的亲和力或亲合力与结合伴侣结合的能力，该亲和力或亲合力使得分子能够用于在结合测定或其他结合环境中将结合伴侣与合适的对照区分开。关于抗体，术语“特异性结合”是指与合适的一种或更多种参考抗原相比，抗体以一定程度的亲和力或亲合力与特异性抗原结合的能力，该亲和力或亲合力使得抗体能够用于将特异性抗原与其他抗原区分开，例如至允许通过与如本文中所述的抗原结合而优先靶向某些细胞(例如，肌细胞)的程度。在一些实施方案中，如果抗体与靶标结合的KD为至少约10-4M、10-5M、10-6M、10-7M、10-8M、10-9M、10-10M、10-11M、10-12M、10-13M或更小，则抗体与靶标特异性结合。在一些实施方案中，抗体与转铁蛋白受体(例如转铁蛋白受体的顶端结构域(apical domain)的表位)特异性结合。Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish a binding partner from a suitable control in a binding assay or other binding environment. With respect to antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity compared to a suitable one or more reference antigens that enables the antibody to be used to distinguish a specific antigen from other antigens, such as to the extent that certain cells (e.g., muscle cells) are preferentially targeted by binding to an antigen as described herein. In some embodiments, an antibody specifically binds to a target if the KD for which the antibody binds to the target is at least about 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, 10-12M, 10-13M or less. In some embodiments, the antibody specifically binds to the transferrin receptor (eg, an epitope of the apical domain of the transferrin receptor).
对象：如本文中使用的，术语“对象”是指哺乳动物。在一些实施方案中，对象是非人灵长类或啮齿动物。在一些实施方案中，对象是人。在一些实施方案中，对象是患者，例如患有或怀疑患有疾病的人患者。在一些实施方案中，对象是患有或怀疑患有肌肉疾病(例如，表1中提供的任何疾病)的人患者。Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a human patient suffering from or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of having a muscle disease (e.g., any of the diseases provided in Table 1).
转铁蛋白受体：如本文中使用的，术语“转铁蛋白受体”(也称为TFRC、CD71、p90、TFR或TFR1)是指结合转铁蛋白以促进通过内吞作用摄取铁的内化细胞表面受体。在一些实施方案中，转铁蛋白受体可以是人来源的(NCBI基因ID 7037)、非人灵长类来源的(例如，NCBI基因ID 711568或NCBI基因ID 102136007)或啮齿动物来源的(例如，NCBI基因ID22042)。另外，已经表征了编码受体的不同同工型的多种人转录物变体(例如，如以以下GenBank RefSeq登录号注释的:NP_001121620.1、NP_003225.2、NP_001300894.1和NP_001300895.1)。Transferrin receptor: As used herein, the term "transferrin receptor" (also referred to as TFRC, CD71, p90, TFR or TFR1) refers to a cell surface receptor that binds transferrin to promote the internalization of iron uptake by endocytosis. In some embodiments, the transferrin receptor can be human-derived (NCBI gene ID 7037), non-human primate-derived (e.g., NCBI gene ID 711568 or NCBI gene ID 102136007) or rodent-derived (e.g., NCBI gene ID 22042). In addition, a variety of human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1 and NP_001300895.1).
对应于NCBI序列NP_003225.2(转铁蛋白受体蛋白1同工型1，智人(homosapiens))的示例性人转铁蛋白受体氨基酸序列如下：An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homosapiens) is as follows:
对应于NCBI序列NP_001244232.1(转铁蛋白受体蛋白1，恒河猴(Macacamulatta))的示例性非人灵长类转铁蛋白受体氨基酸序列如下：An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Rhesus monkey (Macaca mulatta)) is as follows:
对应于NCBI序列XP_005545315.1(转铁蛋白受体蛋白1，食蟹猴(Macacafascicularis))的示例性非人灵长类转铁蛋白受体氨基酸序列如下：An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows:
对应于NCBI序列NP_001344227.1(转铁蛋白受体蛋白1，小家鼠(mus musculus))的示例性小鼠转铁蛋白受体氨基酸序列如下：An exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows:
未连接分子载荷：如本文中使用的，术语“未连接分子载荷”是指例如，在缀合反应产生包含与分子载荷(例如，寡核苷酸或小分子)连接的蛋白质的复合物之后存在于溶液中的游离分子载荷(例如，寡核苷酸或小分子)或过量分子载荷(例如，寡核苷酸或小分子)。在一些实施方案中，未连接分子载荷(例如，寡核苷酸或小分子)不与蛋白质(例如，抗体)连接。在一些实施方案中，未连接分子载荷(例如，寡核苷酸或小分子)不与任何其他部分连接。在一些实施方案中，未连接分子载荷(例如，寡核苷酸或小分子)与官能团连接，但不与蛋白质(例如，抗体)连接以形成复合物。在一些实施方案中，未连接分子载荷(例如，寡核苷酸或小分子)与接头或接头的一部分连接，但不与蛋白质(例如，抗体)连接以形成复合物。Unconnected molecular payload: As used herein, the term "unconnected molecular payload" refers to, for example, free molecular payload (e.g., oligonucleotide or small molecule) or excess molecular payload (e.g., oligonucleotide or small molecule) present in solution after a conjugation reaction produces a complex comprising a protein connected to a molecular payload (e.g., oligonucleotide or small molecule). In some embodiments, an unconnected molecular payload (e.g., oligonucleotide or small molecule) is not connected to a protein (e.g., an antibody). In some embodiments, an unconnected molecular payload (e.g., oligonucleotide or small molecule) is not connected to any other part. In some embodiments, an unconnected molecular payload (e.g., oligonucleotide or small molecule) is connected to a functional group but not to a protein (e.g., an antibody) to form a complex. In some embodiments, an unconnected molecular payload (e.g., oligonucleotide or small molecule) is connected to a linker or a portion of a linker but not to a protein (e.g., an antibody) to form a complex.
未连接蛋白质：如本文中使用的，术语“未连接蛋白质”是指例如在缀合反应产生包含与分子载荷连接的蛋白质的复合物之后存在于溶液中的游离蛋白质或过量蛋白质(例如，游离抗体或过量抗体)。在一些实施方案中，未连接蛋白质(例如，抗体)未经化学修饰。在一些实施方案中，未连接(例如，抗体)经化学修饰。在一些实施方案中，未连接(例如，抗体)经化学修饰以包含官能团，但不与分子载荷(例如，寡核苷酸或小分子)连接。在一些实施方案中，官能团用于与分子载荷缀合(例如，通过点击化学)。在一些实施方案中，官能团是炔基。在一些实施方案中，未连接(例如，抗体)包含炔基。Unconnected protein: As used herein, the term "unconnected protein" refers to free protein or excess protein (e.g., free antibody or excess antibody) present in solution, for example, after a conjugation reaction produces a complex comprising a protein connected to a molecular payload. In some embodiments, unconnected proteins (e.g., antibodies) are not chemically modified. In some embodiments, unconnected (e.g., antibodies) are chemically modified. In some embodiments, unconnected (e.g., antibodies) are chemically modified to include functional groups, but are not connected to a molecular payload (e.g., an oligonucleotide or a small molecule). In some embodiments, the functional group is used to conjugate with a molecular payload (e.g., by click chemistry). In some embodiments, the functional group is an alkynyl group. In some embodiments, unconnected (e.g., antibodies) include alkynyl groups.
复合物：如本文中使用的，术语“复合物”是指包含与一个或更多个分子载荷(例如，治疗剂例如小分子或寡核苷酸)共价连接的蛋白质(例如，抗体)的缀合物。在一些实施方案中，复合物中的蛋白质包含靶向剂(例如，抗体)。在一些实施方案中，靶向剂靶向肌肉(例如，抗转铁蛋白受体抗体)。Complex: As used herein, the term "complex" refers to a conjugate comprising a protein (e.g., an antibody) covalently linked to one or more molecular payloads (e.g., a therapeutic agent such as a small molecule or oligonucleotide). In some embodiments, the protein in the complex comprises a targeting agent (e.g., an antibody). In some embodiments, the targeting agent targets muscle (e.g., an anti-transferrin receptor antibody).
处理：如本文中使用的，术语“处理”包括但不限于本文中所述复合物的产生(例如，通过缀合)、分离(例如，从反应混合物中)、和/或(例如，和)修饰。Processing: As used herein, the term "processing" includes, but is not limited to, the production (eg, by conjugation), separation (eg, from a reaction mixture), and/or (eg, and) modification of a complex described herein.
炔烃：如本文中使用的，术语“炔烃”是指包含至少一个碳-碳三键的不饱和烃。Alkyne: As used herein, the term "alkyne" refers to an unsaturated hydrocarbon containing at least one carbon-carbon triple bond.
带电荷寡核苷酸：如本文中使用的，术语“带电荷寡核苷酸”是指包含在生理pH(例如，pH 7.35至pH 7.45)下具有净负电荷或净正电荷的骨架的寡核苷酸类似物。在一些实施方案中，带电荷寡核苷酸在生理pH下具有净负电荷(本文中称为带负电荷的寡核苷酸)。在一些实施方案中，带电荷寡核苷酸在生理pH下具有净正电荷(本文中称为带正电荷的寡核苷酸)。在一些实施方案中，带电荷寡核苷酸包含在生理pH下具有净负电荷的磷酸二酯骨架。在一些实施方案中，带电荷寡核苷酸包含在生理pH下具有净负电荷的硫代磷酸酯骨架。Charged oligonucleotide: As used herein, the term "charged oligonucleotide" refers to an oligonucleotide analog comprising a backbone having a net negative charge or a net positive charge at physiological pH (e.g., pH 7.35 to pH 7.45). In some embodiments, the charged oligonucleotide has a net negative charge (referred to herein as a negatively charged oligonucleotide) at physiological pH. In some embodiments, the charged oligonucleotide has a net positive charge (referred to herein as a positively charged oligonucleotide) at physiological pH. In some embodiments, the charged oligonucleotide comprises a phosphodiester backbone having a net negative charge at physiological pH. In some embodiments, the charged oligonucleotide comprises a phosphorothioate backbone having a net negative charge at physiological pH.
电荷中性寡核苷酸：如本文中使用的，术语“电荷中性寡核苷酸”是指在生理pH(例如，pH 7.35至pH 7.45)下包含电荷中性骨架的寡核苷酸类似物。电荷中性寡核苷酸的一些实例包括但不限于，磷酸二酰胺吗啉代寡聚物(PMO)和肽核酸(peptide nucleic acid，PNA)，例如，如在et al.,(Nucleic Acid Therapeutics,Vol.25,No.2,2015)中所述，其通过引用并入本文。Charge-neutral oligonucleotide: As used herein, the term "charge-neutral oligonucleotide" refers to an oligonucleotide analog that contains a charge-neutral backbone at physiological pH (e.g., pH 7.35 to pH 7.45). Some examples of charge-neutral oligonucleotides include, but are not limited to, phosphorodiamidate morpholino oligomers (PMOs) and peptide nucleic acids (PNAs), such as in et al., (Nucleic Acid Therapeutics, Vol. 25, No. 2, 2015), which is incorporated herein by reference.
有机溶剂：如本文中使用的，术语“有机溶剂”是指能够溶解其他物质的基于碳的物质。由于是基于碳的，因此有机溶剂在其化合物的结构中具有碳原子。根据本公开内容可以使用的有机溶剂的一些非限制性实例包括但不限于二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。Organic solvent: As used herein, the term "organic solvent" refers to a carbon-based substance that can dissolve other substances. Since it is carbon-based, organic solvents have carbon atoms in the structure of their compounds. Some non-limiting examples of organic solvents that can be used according to the present disclosure include, but are not limited to, dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
％(v/v)：如本文中使用的，术语“％(v/v)”是指混合物的一种组分(例如，有机溶剂)的体积在混合物总体积中的％。% (v/v): As used herein, the term "% (v/v)" refers to the % of the volume of one component of a mixture (eg, an organic solvent) in the total volume of the mixture.
药物抗体比(Drug-to-antibody Ratio，DAR)：如本文中使用的，术语“药物抗体比(DAR)”是指与抗体缀合的药物的数目。该DAR数目可以随所使用的抗体和药物的性质以及用于缀合的实验条件(起始反应物质中抗体与分子载荷的比率、反应时间、溶剂与共溶剂(如果有的话)的性质)而变化。所确定的DAR是平均值。可用于确定DAR的方法的一个实例在Dimitrov et al.,2009,Therapeutic Antibodies and Protocols,vol 525,445,Springer Science中描述，其通过引用并入本文。Drug-to-antibody Ratio (DAR): As used herein, the term "drug-to-antibody ratio (DAR)" refers to the number of drugs conjugated to an antibody. The DAR number may vary with the nature of the antibody and drug used and the experimental conditions used for conjugation (ratio of antibody to molecular load in the starting reaction material, reaction time, nature of solvent and co-solvent (if any)). The determined DAR is an average value. An example of a method that can be used to determine DAR is described in Dimitrov et al., 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science, which is incorporated herein by reference.
中间体：如本文中使用的，术语“中间体”是指在获得复合物之前，进行产生复合物的方法时生成的分子。在一些实施方案中，中间体包含与接头(例如，可切割接头，例如，val-cit接头(即，包含缬氨酸-瓜氨酸序列的可切割接头))连接的寡核苷酸。在一些实施方案中，中间体包含与接头(例如，可切割接头，例如，val-cit接头)共价连接的寡核苷酸，所述接头与包含双环壬炔的化合物共价连接。在一些实施方案中，中间体包含共价连接含有双环壬炔的化合物的抗体。Intermediate: As used herein, the term "intermediate" refers to a molecule generated when a method for producing a complex is performed before obtaining the complex. In some embodiments, the intermediate comprises an oligonucleotide connected to a linker (e.g., a cleavable linker, e.g., a val-cit linker (i.e., a cleavable linker comprising a valine-citrulline sequence)). In some embodiments, the intermediate comprises an oligonucleotide covalently linked to a linker (e.g., a cleavable linker, e.g., a val-cit linker), which is covalently linked to a compound comprising a bicyclononyne. In some embodiments, the intermediate comprises an antibody covalently linked to a compound containing a bicyclononyne.
2’-经修饰核苷：如本文中使用的，术语“2’-经修饰核苷”和“2’-经修饰核糖核苷”可互换使用，并且是指在2’位置处具有经修饰糖部分的核苷。在一些实施方案中，2’-经修饰核苷是2’-4’双环核苷，其中糖的2’和4’位置被桥联(例如，通过亚甲基、亚乙基或(S)-约束性乙基桥联)。在一些实施方案中，2’-经修饰核苷是非双环的2’-经修饰核苷，例如，其中糖部分的2’位置被替代。2’-经修饰核苷的一些非限制性实例包括：2’-脱氧、2’-氟(2’-F)、2’-O-甲基(2’-O-Me)、2’-O-甲氧基乙基(2’-MOE)、2’-O-氨基丙基(2’-O-AP)、2’-O-二甲基氨基乙基(2’-O-DMAOE)、2’-O-二甲基氨基丙基(2’-O-DMAP)、2’-O-二甲基氨基乙氧基乙基(2’-O-DMAEOE)、2’-O-N-甲基乙酰胺基(2’-O-NMA)、锁核酸(LNA，亚甲基桥联核酸)、乙烯桥联核酸(ethylene-bridged nucleic acid，ENA)以及(S)-约束性乙基桥联核酸(cEt)。在一些实施方案中，本文中所述的2’-经修饰核苷是高亲和力的经修饰核苷酸，并且包含2’-经修饰核苷酸的寡核苷酸相对于未经修饰寡核苷酸对靶序列具有提高的亲和力。下面提供了2’-经修饰核苷的结构的一些实例：2'-modified nucleosides: As used herein, the terms "2'-modified nucleosides" and "2'-modified ribonucleosides" are used interchangeably and refer to nucleosides having a modified sugar moiety at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, wherein the 2' and 4' positions of the sugar are bridged (e.g., by a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, e.g., wherein the 2' position of the sugar moiety is replaced. Some non-limiting examples of 2'-modified nucleosides include: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-O-NMA), locked nucleic acid (LNA, methylene-bridged nucleic acid), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleotides, and oligonucleotides comprising 2'-modified nucleotides have increased affinity for target sequences relative to unmodified oligonucleotides. Some examples of the structures of 2'-modified nucleosides are provided below:
II.处理复合物的方法II. Methods of treating the composite
在一些方面中，本公开内容提供了处理(例如，产生、分离)包含与一个或更多个分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的蛋白质(例如，抗体)的复合物的方法。In some aspects, the disclosure provides methods of processing (eg, producing, isolating) a complex comprising a protein (eg, an antibody) covalently linked to one or more molecular cargoes (eg, charge-neutral oligonucleotides or charged oligonucleotides).
在一些方面中，本公开内容提供了产生包含与一个或更多个寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的蛋白质(例如，抗体)的复合物的方法。在一些实施方案中，该方法包括：(i)将寡核苷酸与可切割接头(例如，val-cit接头)共价连接以获得第一中间体。在一些实施方案中，该方法还包括(ii)将步骤(i)中获得的第一中间体与双环壬炔化合物共价连接以获得第二中间体。在一些实施方案中，该方法还包括(iii)将步骤(ii)中获得的第二中间体与抗体共价连接以获得复合物。在一些实施方案中，双环壬炔化合物以是步骤(ii)中双环壬炔化合物起始量的10％或更少或者5％或更少(例如，小于5％、小于4％、小于3％、小于2％、小于1％、小于0.5％或小于0.1％)的量存在于步骤(iii)的反应中。在一些实施方案中，可切割接头(例如，val-cit接头)与寡核苷酸的5’端连接。在一些实施方案中，可切割接头(例如，val-cit接头)通过另外的化学部分与寡核苷酸连接。在一些实施方案中，步骤(iii)导致抗体的赖氨酸残基与第二中间体连接。In some aspects, the present disclosure provides a method for producing a complex comprising a protein (e.g., an antibody) covalently linked to one or more oligonucleotides (e.g., a charge neutral oligonucleotide or a charged oligonucleotide). In some embodiments, the method comprises: (i) covalently linking an oligonucleotide to a cleavable linker (e.g., a val-cit linker) to obtain a first intermediate. In some embodiments, the method further comprises (ii) covalently linking the first intermediate obtained in step (i) to a bicyclononyne compound to obtain a second intermediate. In some embodiments, the method further comprises (iii) covalently linking the second intermediate obtained in step (ii) to an antibody to obtain a complex. In some embodiments, the bicyclononyne compound is present in the reaction of step (iii) in an amount that is 10% or less or 5% or less (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%) of the starting amount of the bicyclononyne compound in step (ii). In some embodiments, a cleavable linker (e.g., a val-cit linker) is connected to the 5' end of the oligonucleotide. In some embodiments, a cleavable linker (eg, a val-cit linker) is attached to the oligonucleotide via an additional chemical moiety. In some embodiments, step (iii) results in attachment of a lysine residue of the antibody to the second intermediate.
在一些实施方案中，产生复合物的方法包括(i)将寡核苷酸与可切割接头(例如，val-cit接头)共价连接以获得第一中间体；(ii)将步骤(i)中获得的第一中间体与双环壬炔化合物共价连接以获得第二中间体；以及(iii)将步骤(ii)中获得的第二中间体与抗体共价连接以获得复合物；其中双环壬炔化合物以是步骤(ii)中化合物起始量的10％或更少或者5％或更少(例如，小于5％、小于4％、小于3％、小于2％、小于1％、小于0.5％或小于0.1％)的量存在于步骤(iii)的反应中。在该方法中，可切割接头(例如，val-cit接头)任选地与寡核苷酸的5’端连接。在一些实施方案中，可切割接头(例如，val-cit接头)通过另外的化学部分与寡核苷酸连接。此外，在一些实施方案中，步骤(iii)导致抗体的赖氨酸残基与第二中间体连接。In some embodiments, the method of producing a complex comprises (i) covalently linking an oligonucleotide to a cleavable linker (e.g., a val-cit linker) to obtain a first intermediate; (ii) covalently linking the first intermediate obtained in step (i) to a bicyclononyne compound to obtain a second intermediate; and (iii) covalently linking the second intermediate obtained in step (ii) to an antibody to obtain a complex; wherein the bicyclononyne compound is present in the reaction of step (iii) in an amount that is 10% or less or 5% or less (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1%) of the starting amount of the compound in step (ii). In this method, a cleavable linker (e.g., a val-cit linker) is optionally linked to the 5' end of the oligonucleotide. In some embodiments, a cleavable linker (e.g., a val-cit linker) is linked to the oligonucleotide via an additional chemical moiety. In addition, in some embodiments, step (iii) results in the connection of a lysine residue of the antibody to the second intermediate.
在一些实施方案中，分子载荷是疏水性小分子。在一些实施方案中，分子载荷是寡核苷酸。在一些实施方案中，分子载荷是电荷中性寡核苷酸。在一些实施方案中，分子载荷是带电荷寡核苷酸。在一些实施方案中，寡核苷酸是单链寡核苷酸(例如，电荷中性单链寡核苷酸或带电荷单链寡核苷酸)。在一些实施方案中，电荷中性链的寡核苷酸是反义寡核苷酸。在一些实施方案中，电荷中性寡核苷酸是磷酸二酰胺吗啉代寡聚物(PMO)。在一些实施方案中，电荷中性寡核苷酸是肽核酸(PNA)。在一些实施方案中，带电荷寡核苷酸是间隔聚体。在一些实施方案中，抗体与单链寡核苷酸(例如，间隔聚体或PMO)的5’端共价连接。在一些实施方案中，抗体与单链寡核苷酸(例如，间隔聚体或PMO)的3’端共价连接。在一些实施方案中，抗体与反义寡核苷酸(例如，间隔聚体或PMO)的5’端共价连接。In some embodiments, the molecular load is a hydrophobic small molecule. In some embodiments, the molecular load is an oligonucleotide. In some embodiments, the molecular load is a charge-neutral oligonucleotide. In some embodiments, the molecular load is a charged oligonucleotide. In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (e.g., a charge-neutral single-stranded oligonucleotide or a charged single-stranded oligonucleotide). In some embodiments, the oligonucleotide of the charge-neutral chain is an antisense oligonucleotide. In some embodiments, the charge-neutral oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the charge-neutral oligonucleotide is a peptide nucleic acid (PNA). In some embodiments, the charged oligonucleotide is a spacer polymer. In some embodiments, the antibody is covalently linked to the 5' end of a single-stranded oligonucleotide (e.g., a spacer polymer or PMO). In some embodiments, the antibody is covalently linked to the 3' end of a single-stranded oligonucleotide (e.g., a spacer polymer or PMO). In some embodiments, the antibody is covalently linked to the 5' end of an antisense oligonucleotide (e.g., a spacer polymer or PMO).
在一些实施方案中，单链寡核苷酸是双链寡核苷酸的一条链。在一些实施方案中，双链寡核苷酸的一条链可以与抗体共价缀合，并且可使用本文中所述方法分离复合物，随后使双链寡核苷酸的另一条链退火。在一些实施方案中，双链寡核苷酸是siRNA，并且有义链与抗体共价连接(例如，在3’端或在5’端)。在一些实施方案中，使用本文中所述方法纯化的复合物包含与siRNA的有义链的3’端共价连接的抗体。siRNA的反义链可在纯化之后与有义链退火。In some embodiments, the single-stranded oligonucleotide is a chain of a double-stranded oligonucleotide. In some embodiments, a chain of a double-stranded oligonucleotide can be covalently conjugated to an antibody, and the complex can be separated using the methods described herein, and the other chain of the double-stranded oligonucleotide is subsequently annealed. In some embodiments, the double-stranded oligonucleotide is siRNA, and the sense strand is covalently linked to the antibody (e.g., at the 3' end or at the 5' end). In some embodiments, the complex purified using the methods described herein comprises an antibody covalently linked to the 3' end of the sense strand of the siRNA. The antisense strand of the siRNA can be annealed to the sense strand after purification.
在一些实施方案中，寡核苷酸包含至少一个经修饰核苷酸间键联。在一些实施方案中，至少一个经修饰核苷酸间键联是硫代磷酸酯键联。在一些实施方案中，寡核苷酸包含一个或更多个经修饰核苷酸。在一些实施方案中，经修饰核苷酸包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)、2’-氟修饰或吗啉代修饰。In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the modified nucleotide comprises 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), 2'-fluorine modification or morpholino modification.
在一些实施方案中，寡核苷酸是单链寡核苷酸(例如，反义寡核苷酸)，其含有包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)、2’-氟修饰或吗啉代修饰的经修饰核苷酸。在一些实施方案中，反义寡核苷酸是间隔聚体，其含有包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)或2’-氟修饰的经修饰核苷酸。在一些实施方案中，反义寡核苷酸是磷酸二酰胺吗啉代寡聚物(PMO)。反义寡核苷酸可包含多于一种类型的本文中所述的修饰，例如具有MOE和2’-氟修饰。In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (e.g., an antisense oligonucleotide) containing a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), a locked nucleic acid (LNA), a 2'-fluoro modification, or a morpholino modification. In some embodiments, the antisense oligonucleotide is a spacer containing a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), a locked nucleic acid (LNA), or a 2'-fluoro modification. In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). The antisense oligonucleotide may contain more than one type of modification described herein, for example, with MOE and 2'-fluoro modification.
在一些实施方案中，寡核苷酸是单链寡核苷酸(例如，双链RNA(例如siRNA)的一条链或反义寡核苷酸)，其包含含有2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)或2’-氟修饰的经修饰核苷酸。在一些实施方案中，寡核苷酸是siRNA的有义链，其含有包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)或2’-氟修饰的经修饰核苷酸。在一些实施方案中，寡核苷酸是siRNA的反义链，其含有包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)或2’-氟修饰的经修饰核苷酸。siRNA的有义链和反义链可包含相同类型或不同类型的本文中所述的修饰。siRNA的一条或两条链可包含多于一种类型的本文中所述的修饰，例如具有MOE和2’-氟修饰。In some embodiments, the oligonucleotide is a single-stranded oligonucleotide (e.g., one strand of a double-stranded RNA (e.g., siRNA) or an antisense oligonucleotide) comprising a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), or 2'-fluoro modification. In some embodiments, the oligonucleotide is the sense strand of the siRNA, comprising a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), or 2'-fluoro modification. In some embodiments, the oligonucleotide is the antisense strand of the siRNA, comprising a modified nucleotide comprising 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), or 2'-fluoro modification. The sense strand and antisense strand of the siRNA may comprise the same type or different types of modifications described herein. One or both strands of the siRNA may comprise more than one type of modification described herein, for example, with MOE and 2'-fluoro modification.
在一些实施方案中，产生包含与一个或更多个寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的抗体的复合物的方法包括：In some embodiments, a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide) comprises:
(i)将寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)与式(A)接头共价连接：(i) covalently linking an oligonucleotide (e.g., a neutrally charged oligonucleotide or a charged oligonucleotide) to a linker of formula (A):
其中n为0至15(例如，3)；以提供式(B)经修饰寡核苷酸：wherein n is 0 to 15 (eg, 3); to provide a modified oligonucleotide of formula (B):
其中n为0至15(例如，3)；wherein n is 0 to 15 (e.g., 3);
(ii)使式(B)经修饰寡核苷酸与式(C)化合物接触：(ii) contacting a modified oligonucleotide of formula (B) with a compound of formula (C):
其中m为0至15(例如，4)；以提供式(D)经修饰寡核苷酸：wherein m is 0 to 15 (eg, 4); to provide a modified oligonucleotide of formula (D):
其中n为0至15(例如，3)，并且m为0至15(例如，4)；以及wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4); and
(iii)使式(D)经修饰寡核苷酸与抗体接触以提供式(E)复合物：(iii) contacting the modified oligonucleotide of formula (D) with an antibody to provide a complex of formula (E):
其中n为0至15(例如，3)，并且m为0至15(例如，4)。wherein n is 0 to 15 (eg, 3), and m is 0 to 15 (eg, 4).
在一些实施方案中，产生本文中所述复合物的方法产生了包含复合物、未连接寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)和/或未连接抗体的反应混合物。在一些实施方案中，反应混合物包含式(E)复合物：In some embodiments, the method of producing a complex described herein produces a reaction mixture comprising a complex, an unattached oligonucleotide (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide) and/or an unattached antibody. In some embodiments, the reaction mixture comprises a complex of formula (E):
其中n为0至15(例如，3)，并且m为0至15(例如，4)；式(B)未连接寡核苷酸：wherein n is 0 to 15 (eg, 3), and m is 0 to 15 (eg, 4); Formula (B) is an unligated oligonucleotide:
其中n为0至15(例如，3)；以及未连接抗体。在一些实施方案中，式(C)化合物以是步骤(ii)的反应中式(C)化合物的起始量的10％或更少或者5％或更少(例如，小于5％、小于4％、小于3％、小于2％、小于1％、小于0.5％或小于0.1％)的量存在于步骤(iii)的反应混合物中，任选地，其中使式(C)化合物与步骤(iii)中的抗体接触以在反应混合物中形成式(F)未连接抗体：wherein n is 0 to 15 (e.g., 3); and unlinked antibody. In some embodiments, the compound of formula (C) is present in the reaction mixture of step (iii) in an amount that is 10% or less or 5% or less (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%) of the starting amount of the compound of formula (C) in the reaction of step (ii), optionally wherein the compound of formula (C) is contacted with the antibody in step (iii) to form an unlinked antibody of formula (F) in the reaction mixture:
其中m为0至15(例如，4)。wherein m is 0 to 15 (eg, 4).
在一些实施方案中，产生复合物(所述复合物各自包含与一个或更多个寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的抗体)的方法包括将寡核苷酸与接头(例如，可切割接头(例如，val-cit接头))共价连接以获得第一中间体。在一些实施方案中，该方法还包括将抗体与双环壬炔化合物共价连接以获得第二中间体。在一些实施方案中，该方法还包括共价连接第一中间体与第二中间体以获得复合物。在该方法中，接头(例如，val-cit接头)任选地与寡核苷酸的5’端连接。在一些实施方案中，接头(例如，val-cit接头)通过另外的化学部分与寡核苷酸共价连接。此外，在一些实施方案中，步骤(iii)导致抗体的赖氨酸残基与第二中间体共价连接。In some embodiments, a method of producing a complex (each of which comprises an antibody covalently linked to one or more oligonucleotides (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide)) comprises covalently linking the oligonucleotide to a linker (e.g., a cleavable linker (e.g., a val-cit linker)) to obtain a first intermediate. In some embodiments, the method further comprises covalently linking the antibody to a bicyclononyne compound to obtain a second intermediate. In some embodiments, the method further comprises covalently linking the first intermediate to the second intermediate to obtain a complex. In this method, a linker (e.g., a val-cit linker) is optionally linked to the 5' end of the oligonucleotide. In some embodiments, the linker (e.g., a val-cit linker) is covalently linked to the oligonucleotide via an additional chemical moiety. In addition, in some embodiments, step (iii) results in a lysine residue of the antibody being covalently linked to the second intermediate.
在一些实施方案中，产生复合物(所述复合物各自包含与一个或更多个寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的抗体)的方法包括：(i)将寡核苷酸与接头(例如，可切割接头(例如，val-cit接头))共价连接以获得第一中间体；(ii)将抗体与包含双环壬炔的化合物共价连接以获得第二中间体；以及(iii)共价连接步骤(i)中获得的第一中间体与步骤(ii)中获得的第二中间体以获得复合物。在该方法中，接头(例如，val-cit接头)任选地与寡核苷酸的5’端连接。在一些实施方案中，接头(例如，val-cit接头)通过另外的化学部分与寡核苷酸共价连接。此外，在一些实施方案中，步骤(iii)导致抗体的赖氨酸残基与第二中间体共价连接。In some embodiments, a method of producing a complex (each of which comprises an antibody covalently linked to one or more oligonucleotides (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide)) comprises: (i) covalently linking the oligonucleotide to a linker (e.g., a cleavable linker (e.g., a val-cit linker)) to obtain a first intermediate; (ii) covalently linking the antibody to a compound comprising a bicyclononyne to obtain a second intermediate; and (iii) covalently linking the first intermediate obtained in step (i) to the second intermediate obtained in step (ii) to obtain a complex. In this method, the linker (e.g., a val-cit linker) is optionally linked to the 5' end of the oligonucleotide. In some embodiments, the linker (e.g., a val-cit linker) is covalently linked to the oligonucleotide via an additional chemical moiety. In addition, in some embodiments, step (iii) results in a lysine residue of the antibody being covalently linked to the second intermediate.
在一些实施方案中，产生包含与一个或更多个寡核苷酸(例如，电荷中性寡核苷酸或带电荷寡核苷酸)共价连接的抗体的复合物的方法包括：In some embodiments, a method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide) comprises:
(i)将寡核苷酸与式(A)接头共价连接：(i) covalently linking an oligonucleotide to a linker of formula (A):
其中n为0至15(例如，3)；以提供式(B)经修饰寡核苷酸：wherein n is 0 to 15 (eg, 3); to provide a modified oligonucleotide of formula (B):
其中n为0至15(例如，3)；wherein n is 0 to 15 (e.g., 3);
(ii)将抗体与式(C)化合物共价连接：(ii) covalently linking the antibody to a compound of formula (C):
其中m为0至15(例如，4)；以提供式(F)经修饰抗体：wherein m is 0 to 15 (eg, 4); to provide a modified antibody of formula (F):
其中n为0至15(例如，3)，并且m为0至15(例如，4)；以及wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4); and
(iii)使式(B)经修饰寡核苷酸与式(F)经修饰抗体接触以提供式(E)复合物：(iii) contacting the modified oligonucleotide of formula (B) with the modified antibody of formula (F) to provide a complex of formula (E):
其中n为0至15(例如，3)，并且m为0至15(例如，4)。wherein n is 0 to 15 (eg, 3), and m is 0 to 15 (eg, 4).
在一些实施方案中，产生本文中所述复合物的方法产生了包含复合物、未连接寡核苷酸和/或未连接抗体的反应混合物。在一些实施方案中，反应混合物包含式(E)复合物：In some embodiments, the method of producing a complex described herein produces a reaction mixture comprising a complex, unattached oligonucleotides and/or unattached antibodies. In some embodiments, the reaction mixture comprises a complex of formula (E):
其中n为0至15(例如，3)，并且m为0至15(例如，4)；式(B)未连接寡核苷酸：wherein n is 0 to 15 (eg, 3), and m is 0 to 15 (eg, 4); Formula (B) is an unligated oligonucleotide:
其中n为0至15(例如，3)；以及式(F)未连接抗体wherein n is 0 to 15 (eg, 3); and formula (F) is not linked to an antibody
其中m为0至15(例如，4)。wherein m is 0 to 15 (eg, 4).
在一些实施方案中，寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)的长度为10至50(例如，10至50、10至40、10至30、10至20、20至50、20至40、20至30、30至50、30至40或40至50)个核苷酸。在一些实施方案中，寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)的长度为15至30(例如，15至30、15至25、15至20、20至30、20至25或25至30)个核苷酸。在一些实施方案中，寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)的长度为15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸。在一些实施方案中，寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)的长度为30个核苷酸。在一些实施方案中，寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)通过赖氨酸或半胱氨酸与抗体共价连接。在一些实施方案中，寡核苷酸(例如，电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸)通过接头(例如，包含Val-cit接头的接头)与抗体共价连接。在一些实施方案中，接头具有式(G)：In some embodiments, the length of an oligonucleotide (e.g., a charge-neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) is 10 to 50 (e.g., 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 50, 20 to 40, 20 to 30, 30 to 50, 30 to 40, or 40 to 50) nucleotides. In some embodiments, the length of an oligonucleotide (e.g., a charge-neutral oligonucleotide (e.g., PMO) or a charged oligonucleotide) is 15 to 30 (e.g., 15 to 30, 15 to 25, 15 to 20, 20 to 30, 20 to 25, or 25 to 30) nucleotides. In some embodiments, the length of the oligonucleotide (e.g., a neutral charge oligonucleotide (e.g., PMO) or a charged oligonucleotide) is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the oligonucleotide (e.g., a neutral charge oligonucleotide (e.g., PMO) or a charged oligonucleotide) is 30 nucleotides. In some embodiments, the oligonucleotide (e.g., a neutral charge oligonucleotide (e.g., PMO) or a charged oligonucleotide) is covalently linked to the antibody via a lysine or cysteine. In some embodiments, the oligonucleotide (e.g., a neutral charge oligonucleotide (e.g., PMO) or a charged oligonucleotide) is covalently linked to the antibody via a linker (e.g., a linker comprising a Val-cit linker). In some embodiments, the linker has formula (G):
其中n为0至15(例如，3)，并且m为0至15(例如，4)。在一些实施方案中，n为3和/或(例如，和)m为4。在一些实施方案中，n为3和/或(例如，和)m为4。在一些实施方案中，X是抗体的NH(例如，来自赖氨酸的胺基的NH)、S(例如，来自半胱氨酸的巯基的S)或O(例如，来自丝氨酸、苏氨酸或酪氨酸的羟基的O)。wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4). In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NH from the amine group of lysine), S (e.g., S from the sulfhydryl group of cysteine), or O (e.g., O from the hydroxyl group of serine, threonine, or tyrosine) of the antibody.
在一些实施方案中，复合物具有式(E)：In some embodiments, the complex has formula (E):
其中n为0至15(例如，3)，并且m为0至15(例如，4)。在一些实施方案中，寡核苷酸是电荷中性寡核苷酸(例如，PMO)或带电荷寡核苷酸(例如，间隔聚体)。wherein n is 0 to 15 (eg, 3), and m is 0 to 15 (eg, 4). In some embodiments, the oligonucleotide is a charge-neutral oligonucleotide (eg, PMO) or a charged oligonucleotide (eg, a gapmer).
在一些方面中，分离本文中所述复合物的方法涉及使包含复合物和未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)的混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂接触，去除未连接的分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)，以及从混合模式树脂洗脱吸附的复合物。在一些实施方案中，混合模式树脂是磷灰石树脂。在一些实施方案中，磷灰石树脂是羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂或氯磷灰石树脂。In some aspects, the method of separating the complex described herein involves contacting a mixture comprising the complex and an unattached molecular load (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) with a mixed mode resin comprising a positively charged metal site and a negatively charged ionic site, removing the unattached molecular load (e.g., a charge neutral oligonucleotide or a charged oligonucleotide), and eluting the adsorbed complex from the mixed mode resin. In some embodiments, the mixed mode resin is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.
在一些实施方案中，经受混合模式色谱的包含复合物和未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)的混合物是产生复合物的反应的反应混合物(例如，如果抗体和分子载荷通过缀合)。在一些实施方案中，经受混合模式色谱的复合物和未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)的混合物是产生复合物(例如，如果抗体和分子载荷通过缀合)的反应的反应混合物，所述复合物在混合模式色谱之前没有经受过任何先前的纯化步骤。在一些实施方案中，经受混合模式色谱的混合物中的复合物的平均药物抗体比(drug to antibody ratio，DAR)为至少约1.0(例如，约1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.5、3.0或更高)。In some embodiments, the mixture comprising the complex and the unattached molecular payload (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) subjected to mixed mode chromatography is the reaction mixture of a reaction that produces the complex (e.g., if the antibody and the molecular payload are conjugated). In some embodiments, the mixture of the complex and the unattached molecular payload (e.g., a charge neutral oligonucleotide or a charged oligonucleotide) subjected to mixed mode chromatography is the reaction mixture of a reaction that produces the complex (e.g., if the antibody and the molecular payload are conjugated), and the complex has not been subjected to any previous purification steps prior to mixed mode chromatography. In some embodiments, the average drug to antibody ratio (DAR) of the complex in the mixture subjected to mixed mode chromatography is at least about 1.0 (e.g., about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 or higher).
在一些实施方案中，使用本文中所述的混合模式树脂色谱从未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)中基本上纯化复合物。在一些实施方案中，使用本文中所述方法纯化之后的复合物组合物不包含任何可检测水平的未连接寡核苷酸或未连接蛋白质。In some embodiments, the complex is substantially purified from unattached molecular payloads (e.g., charge-neutral oligonucleotides or charged oligonucleotides) using mixed-mode resin chromatography as described herein. In some embodiments, the complex composition after purification using the methods described herein does not contain any detectable levels of unattached oligonucleotides or unattached proteins.
在一些实施方案中，本文中所述方法适合于分离包含与一个或更多个寡核苷酸共价连接的抗体的复合物。在一些实施方案中，抗体可以是全长IgG、Fab片段、Fab’片段、F(ab’)2片段、scFv或Fv片段。特定的抗体序列不影响纯化结局。在一些实施方案中，抗体是抗转铁蛋白受体抗体(例如，表2中列出的任意抗转铁蛋白受体抗体)或其任意抗原结合片段(例如，Fab片段、Fab’片段、F(ab’)2片段、scFv或Fv片段))。In some embodiments, the method described herein is suitable for separating a complex comprising an antibody covalently linked to one or more oligonucleotides. In some embodiments, the antibody can be a full-length IgG, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv or Fv fragment. Specific antibody sequence does not affect the purification outcome. In some embodiments, the antibody is an anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody listed in Table 2) or any antigen-binding fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, scFv or Fv fragment)).
A.使用混合模式树脂从复合物中去除未连接电荷中性寡核苷酸A. Removal of unligated neutral oligonucleotides from complexes using mixed-mode resins
在一些实施方案中，本文中表明，包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂(例如，磷灰石树脂，例如羟基磷灰石树脂)的使用有效地从复合物中去除未连接分子载荷，特别是电荷中性或疏水性分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)，并且在整个纯化过程中有机溶剂的使用显著提高了不含未连接分子载荷的复合物的产率。与其他已知去除未连接分子载荷和/或过量盐(脱盐)的方法相比，本文中所述的混合模式树脂纯化方法是有利的。一种这样的已知方法是尺寸排阻色谱(SEC)。混合模式树脂纯化方法至少由于其可扩展性和较高的回收率而优于SEC。使用本文中所述的混合模式树脂方法实现了至少50％的复合物的回收，而SEC只能实现20％至30％的复合物的回收。In some embodiments, it is shown herein that the use of a mixed mode resin (e.g., an apatite resin, such as a hydroxyapatite resin) comprising a positively charged metal site and a negatively charged ionic site effectively removes unattached molecular loads, particularly charge neutral or hydrophobic molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules) from the complex, and the use of an organic solvent throughout the purification process significantly improves the yield of complexes that do not contain unattached molecular loads. Compared to other known methods for removing unattached molecular loads and/or excess salts (desalting), the mixed mode resin purification method described herein is advantageous. One such known method is size exclusion chromatography (SEC). The mixed mode resin purification method is superior to SEC at least due to its scalability and higher recovery rate. At least 50% of the complex is recovered using the mixed mode resin method described herein, while SEC can only achieve 20% to 30% recovery of the complex.
在一些方面中，本公开内容提供了处理(例如，分离)复合物的方法，所述复合物各自包含与一个或更多个分子载荷共价连接的抗体。在一些实施方案中，分子载荷是小分子(例如，疏水性小分子)。在一些实施方案中，分子载荷是寡核苷酸(例如，电荷中性寡核苷酸)。In some aspects, the disclosure provides methods of treating (e.g., separating) complexes, each of which comprises an antibody covalently linked to one or more molecular payloads. In some embodiments, the molecular payload is a small molecule (e.g., a hydrophobic small molecule). In some embodiments, the molecular payload is an oligonucleotide (e.g., a charge neutral oligonucleotide).
在一些实施方案中，本文中所述处理复合物的方法包括：(i)使包含有机溶剂、复合物和未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)的混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂在其中复合物吸附至混合模式树脂的条件下接触，以及(ii)在其中复合物从混合模式树脂解离的条件下，使复合物从混合模式树脂中洗脱。在一些实施方案中，步骤(i)的混合物还包含不超过30％(例如，不超过30％、不超过25％、不超过20％、不超过15％、不超过10％、不超过9％、不超过8％、不超过7％、不超过6％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1％或小于0.5％)的未连接抗体。在一些实施方案中，步骤(i)的混合物还包含的未连接抗体的水平不可检测。在一些实施方案中，步骤(i)的混合物是产生复合物的反应混合物。在一些实施方案中，步骤(i)的混合物是产生复合物的反应混合物，其没有经受过任何先前的纯化步骤。在一些实施方案中，步骤(i)的混合物包含的含有炔基的未连接抗体为痕量。In some embodiments, the method for treating the complex described herein comprises: (i) contacting a mixture comprising an organic solvent, a complex and an unconnected molecular load (e.g., a charge-neutral oligonucleotide or a hydrophobic small molecule) with a mixed mode resin comprising a positively charged metal site and a negatively charged ionic site under conditions where the complex is adsorbed to the mixed mode resin, and (ii) eluting the complex from the mixed mode resin under conditions where the complex dissociates from the mixed mode resin. In some embodiments, the mixture of step (i) also comprises no more than 30% (e.g., no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% or less than 0.5%) of unconnected antibodies. In some embodiments, the level of unconnected antibodies also included in the mixture of step (i) is undetectable. In some embodiments, the mixture of step (i) is a reaction mixture that produces a complex. In some embodiments, the mixture of step (i) is a reaction mixture that produces a complex, which has not been subjected to any prior purification steps. In some embodiments, the mixture of step (i) contains trace amounts of unlinked antibodies containing alkyne groups.
在一些实施方案中，分子载荷(连接或未连接的)与混合模式树脂非特异性地相互作用，影响复合物的产率。本文中表明，在混合模式色谱的流动相中包含有机溶剂有效地降低/消除了电荷中性寡核苷酸与混合模式树脂之间的非特异性相互作用。在一些实施方案中，与没有降低这样的非特异性相互作用相比，降低分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)之间的非特异性相互作用导致复合物的产率提高(例如，提高了至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少100％、至少2倍、至少5倍或更多)。In some embodiments, molecular load (connected or unconnected) interacts with mixed mode resin non-specifically, affecting the yield of complex.Shown herein, the non-specific interactions between charge neutral oligonucleotide and mixed mode resin are effectively reduced/eliminated in the mobile phase of mixed mode chromatography.In some embodiments, compared with not reducing such non-specific interactions, reducing the non-specific interactions between molecular load (for example, charge neutral oligonucleotide or hydrophobic small molecule) causes the yield of complex to increase (for example, increase at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 5 times or more).
色谱方法中常用的有机溶剂可用于本文中所述的所有方法中。在一些实施方案中，在本文中所述的处理复合物的方法的步骤(i)中使用的有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。在一些实施方案中，本文中所述方法的步骤(i)中使用的有机溶剂是二甲基乙酰胺(DMA)。在一些实施方案中，本文中所述方法的步骤(i)中使用的有机溶剂是异丙醇(IPA)。在一些实施方案中，本文中所述方法的步骤(i)中使用的有机溶剂是二甲基亚砜(DMSO)。在一些实施方案中，本文中所述方法的步骤(i)中使用的有机溶剂是乙腈(ACN)。在一些实施方案中，本文中所述方法的步骤(i)中使用的有机溶剂是丙二醇(PG)。Organic solvents commonly used in chromatographic methods can be used in all methods described herein. In some embodiments, the organic solvent used in step (i) of the method for treating the complex described herein is dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG). In some embodiments, the organic solvent used in step (i) of the method described herein is dimethylacetamide (DMA). In some embodiments, the organic solvent used in step (i) of the method described herein is isopropyl alcohol (IPA). In some embodiments, the organic solvent used in step (i) of the method described herein is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent used in step (i) of the method described herein is acetonitrile (ACN). In some embodiments, the organic solvent used in step (i) of the method described herein is propylene glycol (PG).
在一些实施方案中，有机溶剂在步骤(i)中的混合物中为2％至50％(v/v)。例如，有机溶剂在本文中所述方法的步骤(i)中的混合物中可以为2％至50％(v/v)、5％至50％(v/v)、10％至50％(v/v)、20％至50％(v/v)、30％至50％(v/v)、40％至50％(v/v)、2％至40％(v/v)、5％至40％(v/v)、10％至40％(v/v)、20％至40％(v/v)、30％至40％(v/v)、2％至30％(v/v)、5％至30％(v/v)、10％至30％(v/v)、20％至30％(v/v)、2％至20％(v/v)、5％至20％(v/v)、10％至20％(v/v)、2％至10％(v/v)、5％至10％(v/v)、5％至20％(v/v)、5％至15％(v/v)、5％至10％(v/v)、10％至15％(v/v)、或15％至20％(v/v)。在一些实施方案中，有机溶剂在步骤(i)中的混合物中为5％至20％(v/v)。在一些实施方案中，有机溶剂在本文中所述方法的步骤(i)中的混合物中为5％(v/v)、6％(v/v)、7％(v/v)、8％(v/v)、9％(v/v)、10％(v/v)、11％(v/v)、12％(v/v)、13％(v/v)、14％(v/v)、15％(v/v)、16％(v/v)、17％(v/v)、18％(v/v)、19％(v/v)或20％(v/v)。在一些实施方案中，超过20％(v/v)的有机溶剂可用于本文中所述方法的步骤(i)中的混合物中。在一些实施方案中，有机溶剂在本文中所述方法的步骤(i)中的混合物中为15％(v/v)。在一些实施方案中，有机溶剂在本文中所述方法的步骤(i)中的混合物中为30％(v/v)。In some embodiments, the organic solvent is present in the mixture in step (i) at 2% to 50% (v/v). For example, the organic solvent can be present in the mixture in step (i) of the method described herein at 2% to 50% (v/v), 5% to 50% (v/v), 10% to 50% (v/v), 20% to 50% (v/v), 30% to 50% (v/v), 40% to 50% (v/v), 2% to 40% (v/v), 5% to 40% (v/v), 10% to 40% (v/v), 20% to 40% (v/v), 30% to 40% (v/v), 2% to 5 ...0% to 50% (v/v), 30% to 40% (v/v), 30% to 4 In some embodiments, the organic solvent is 5% to 20% (v/v) in the mixture in step (i). In some embodiments, the organic solvent is 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v), 16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v) or 20% (v/v) in the mixture in step (i) of the method described herein. In some embodiments, more than 20% (v/v) of the organic solvent can be used in the mixture in step (i) of the method described herein. In some embodiments, the organic solvent is 15% (v/v) in the mixture in step (i) of the method described herein. In some embodiments, the organic solvent is 30% (v/v) in the mixture in step (i) of the method described herein.
在一些实施方案中，通过在步骤(i)中的混合物中以允许复合物吸附至混合模式树脂的浓度包含磷酸根离子和/或氯离子，来实现步骤(i)中复合物吸附的条件。在一些实施方案中，步骤(i)中的混合物的pH为约5.0至8.0(例如，约5.0、5.5、6.0、6.5、7.0、7.5、8.0)。在一些实施方案中，步骤(i)中的混合物的pH为约5.0至6.0(例如，约5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9或6.0)。在一些实施方案中，本文中所述方法的步骤(i)中的混合物不包含磷酸根离子或氯离子。在一些实施方案中，本文中所述方法的步骤(i)中的混合物还包含最高为10mM的磷酸根离子。在一些实施方案中，本文中所述方法的步骤(i)中的混合物还包含最高为10mM(例如，最高为10mM或最高为5mM)的磷酸根离子，并且任选地在一些实施方案中，本文中所述方法的步骤(i)中的混合物还包含最高为20mM(例如，最高为20mM、最高为15mM、最高为10mM或最高为5mM)的氯离子。在一些实施方案中，本文中所述方法的步骤(i)中的混合物还包含5至10mM(例如，5、6、7、8、9或10mM)的磷酸根离子，并且任选地在一些实施方案中，本文中所述方法的步骤(i)中的混合物还包含5至20mM(例如，5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20mM)的氯离子。在这些条件下，未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)保留在流通物中，并且不会吸附至混合模式树脂。In some embodiments, the conditions for complex adsorption in step (i) are achieved by including phosphate ions and/or chloride ions in the mixture in step (i) at a concentration that allows the complex to be adsorbed to the mixed mode resin. In some embodiments, the pH of the mixture in step (i) is about 5.0 to 8.0 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0). In some embodiments, the pH of the mixture in step (i) is about 5.0 to 6.0 (e.g., about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0). In some embodiments, the mixture in step (i) of the method described herein does not include phosphate ions or chloride ions. In some embodiments, the mixture in step (i) of the method described herein also includes phosphate ions up to 10 mM. In some embodiments, the mixture in step (i) of the methods described herein further comprises up to 10 mM (e.g., up to 10 mM or up to 5 mM) of phosphate ions, and optionally in some embodiments, the mixture in step (i) of the methods described herein further comprises up to 20 mM (e.g., up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM) of chloride ions. In some embodiments, the mixture in step (i) of the methods described herein further comprises 5 to 10 mM (e.g., 5, 6, 7, 8, 9, or 10 mM) of phosphate ions, and optionally in some embodiments, the mixture in step (i) of the methods described herein further comprises 5 to 20 mM (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM) of chloride ions. Under these conditions, unattached molecular cargo (eg, charge-neutral oligonucleotides or hydrophobic small molecules) remain in the flow-through and do not adsorb to the mixed mode resin.
在一些实施方案中，在步骤(i)中，未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)不会吸附至混合模式树脂。在一些实施方案中，在步骤(i)中，少于5％(例如，少于5％、少于4％、少于3％、少于2％、少于1％或少于0.5％)的未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)吸附至混合模式树脂。在一些实施方案中，少于5％(例如，少于5％、少于4％、少于3％、少于2％、少于1％或少于0.5％)的未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)与混合模式树脂非特异性地相互作用。In some embodiments, in step (i), unattached molecular load (e.g., charge neutral oligonucleotide or hydrophobic small molecule) is not adsorbed to the mixed mode resin. In some embodiments, in step (i), less than 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5%) of unattached molecular load (e.g., charge neutral oligonucleotide or hydrophobic small molecule) is adsorbed to the mixed mode resin. In some embodiments, less than 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less than 0.5%) of unattached molecular load (e.g., charge neutral oligonucleotide or hydrophobic small molecule) interacts non-specifically with the mixed mode resin.
在一些实施方案中，混合模式树脂可在步骤(i)与步骤(ii)之间在去除松散结合但未吸附至混合模式树脂的未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)的条件下进一步洗涤。在一些实施方案中，使用洗涤溶液进行洗涤步骤。在一些实施方案中，洗涤溶液包含有机溶剂。在一些实施方案中，洗涤溶液中使用的有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。在一些实施方案中，洗涤溶液中使用的有机溶剂是二甲基乙酰胺(DMA)。在一些实施方案中，清洗溶液中使用的有机溶剂是异丙醇(IPA)。在一些实施方案中，洗涤溶液中使用的有机溶剂是二甲基亚砜(DMSO)。在一些实施方案中，洗涤溶液中使用的有机溶剂是乙腈(ACN)。在一些实施方案中，洗涤溶液中使用的有机溶剂是丙二醇(PG)。In some embodiments, the mixed mode resin can be further washed between step (i) and step (ii) under conditions of removing loosely bound but unattached molecular loads (e.g., charge neutral oligonucleotides or hydrophobic small molecules) that are not adsorbed to the mixed mode resin. In some embodiments, a washing solution is used to carry out the washing step. In some embodiments, the washing solution comprises an organic solvent. In some embodiments, the organic solvent used in the washing solution is dimethylacetamide (DMA), isopropanol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG). In some embodiments, the organic solvent used in the washing solution is dimethylacetamide (DMA). In some embodiments, the organic solvent used in the cleaning solution is isopropanol (IPA). In some embodiments, the organic solvent used in the washing solution is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent used in the washing solution is acetonitrile (ACN). In some embodiments, the organic solvent used in the washing solution is propylene glycol (PG).
在一些实施方案中，有机溶剂在洗涤溶液中为5％至20％(v/v)。例如，有机溶剂在洗涤溶液中可以为5％至20％(v/v)、5％至15％(v/v)、5％至10％(v/v)、10％至20％(v/v)、10％至15％(v/v)或15％至20％(v/v)。在一些实施方案中，有机溶剂在洗涤溶液中为5％(v/v)、6％(v/v)、7％(v/v)、8％(v/v)、9％(v/v)、10％(v/v)、11％(v/v)、12％(v/v)、13％(v/v)、14％(v/v)、15％(v/v)、16％(v/v)、17％(v/v)、18％(v/v)、19％(v/v)或20％(v/v)。在一些实施方案中，超过20％(v/v)的有机溶剂可用于洗涤溶液中。在一些实施方案中，有机溶剂在洗涤溶液中为15％(v/v)。在一些实施方案中，有机溶剂在洗涤溶液中为30％(v/v)。In some embodiments, the organic solvent is 5% to 20% (v/v) in the washing solution. For example, the organic solvent can be 5% to 20% (v/v), 5% to 15% (v/v), 5% to 10% (v/v), 10% to 20% (v/v), 10% to 15% (v/v) or 15% to 20% (v/v) in the washing solution. In some embodiments, the organic solvent is 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v), 16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v) or 20% (v/v) in the washing solution. In some embodiments, more than 20% (v/v) of organic solvent may be used in the wash solution. In some embodiments, the organic solvent is 15% (v/v) in the wash solution. In some embodiments, the organic solvent is 30% (v/v) in the wash solution.
在一些实施方案中，洗涤溶液包含磷酸根离子和/或氯化物，其浓度可去除松散结合的分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)，但不会使复合物从混合模式树脂解离。在一些实施方案中，洗涤溶液的pH为5.0至8.0(例如，约5.0、5.5、6.0、6.5、7.0、7.5或8.0)。在一些实施方案中，洗涤溶液的pH为5.0至6.0(例如，5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9或6.0)。在一些实施方案中，洗涤溶液不包含磷酸根离子或氯离子。在一些实施方案中，洗涤溶液还包含最高为10mM的磷酸根离子。在一些实施方案中，洗涤溶液还包含最高为10mM(例如，最高为10mM，或最高为5mM)的磷酸根离子，并且任选地在一些实施方案中，洗涤溶液还包含最高为20mM(例如，最高为20mM，最高为15mM，最高为10mM，或最高为5mM)的氯离子。在一些实施方案中，洗涤溶液还包含5至10mM(例如，5、6、7、8、9或10mM)的磷酸根离子，并且任选地在一些实施方案中，洗涤溶液还包含5至20mM(例如，5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20mM)的氯离子。In some embodiments, the wash solution comprises phosphate ions and/or chloride at concentrations that remove loosely bound molecular payloads (e.g., charge neutral oligonucleotides or hydrophobic small molecules) but do not dissociate the complex from the mixed mode resin. In some embodiments, the pH of the wash solution is 5.0 to 8.0 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). In some embodiments, the pH of the wash solution is 5.0 to 6.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the wash solution does not comprise phosphate ions or chloride ions. In some embodiments, the wash solution further comprises phosphate ions up to 10 mM. In some embodiments, the wash solution further comprises up to 10 mM (e.g., up to 10 mM, or up to 5 mM) of phosphate ions, and optionally in some embodiments, the wash solution further comprises up to 20 mM (e.g., up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM) of chloride ions. In some embodiments, the wash solution further comprises 5 to 10 mM (e.g., 5, 6, 7, 8, 9, or 10 mM) of phosphate ions, and optionally in some embodiments, the wash solution further comprises 5 to 20 mM (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM) of chloride ions.
在一些实施方案中，为了从混合模式树脂洗脱复合物，在步骤(ii)中，使混合模式和结合的复合物经受允许复合物从混合模式树脂解离的条件。在一些实施方案中，与步骤(i)的混合物或洗涤溶液中磷酸根离子和/或氯离子的浓度相比，通过向混合模式树脂施加包含更高浓度的磷酸根离子和/或氯离子的洗脱溶液来实现步骤(ii)中允许复合物解离的条件。在一些实施方案中，与步骤(i)的混合物或洗涤溶液中的磷酸根浓度相比，洗脱溶液包含更高浓度的磷酸根离子，并且不包含氯离子。可以使用包含单一磷酸根离子浓度的洗脱溶液，或者使用具有递增磷酸根离子浓度梯度的洗脱溶液来完成洗脱步骤。In some embodiments, in order to elute the complex from the mixed mode resin, in step (ii), the mixed mode and the combined complex are subjected to conditions that allow the complex to dissociate from the mixed mode resin. In some embodiments, the conditions that allow the complex to dissociate in step (ii) are achieved by applying an elution solution containing a higher concentration of phosphate ions and/or chloride ions to the mixed mode resin compared to the concentration of phosphate ions and/or chloride ions in the mixture or washing solution of step (i). In some embodiments, the elution solution contains a higher concentration of phosphate ions and does not contain chloride ions compared to the phosphate concentration in the mixture or washing solution of step (i). The elution step can be completed using an elution solution containing a single phosphate ion concentration, or using an elution solution with an increasing phosphate ion concentration gradient.
在一些实施方案中，洗脱溶液的pH为约6.5至8.5(例如，约6.5、7.0、7.5、8.0或8.5)。在一些实施方案中，洗脱溶液的pH为约7.6至8.5(例如，约7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4或8.5)。In some embodiments, the pH of the elution solution is about 6.5 to 8.5 (e.g., about 6.5, 7.0, 7.5, 8.0, or 8.5). In some embodiments, the pH of the elution solution is about 7.6 to 8.5 (e.g., about 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5).
在一些实施方案中，洗脱溶液包含有机溶剂。在一些实施方案中，洗脱溶液中使用的有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。在一些实施方案中，洗脱溶液中使用的有机溶剂是二甲基乙酰胺(DMA)。在一些实施方案中，洗脱溶液中使用的有机溶剂是异丙醇(IPA)。在一些实施方案中，洗脱溶液中使用的有机溶剂是二甲基亚砜(DMSO)。在一些实施方案中，洗脱溶液中使用的有机溶剂是乙腈(ACN)。在一些实施方案中，洗脱溶液中使用的有机溶剂是丙二醇(PG)。In some embodiments, the elution solution comprises an organic solvent. In some embodiments, the organic solvent used in the elution solution is dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG). In some embodiments, the organic solvent used in the elution solution is dimethylacetamide (DMA). In some embodiments, the organic solvent used in the elution solution is isopropyl alcohol (IPA). In some embodiments, the organic solvent used in the elution solution is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent used in the elution solution is acetonitrile (ACN). In some embodiments, the organic solvent used in the elution solution is propylene glycol (PG).
在一些实施方案中，有机溶剂在洗脱溶液中为2％至50％(v/v)。例如，有机溶剂在洗脱溶液中可以为2％至50％(v/v)、5％至50％(v/v)、10％至50％(v/v)、20％至50％(v/v)、30％至50％(v/v)、40％至50％(v/v)、2％至40％(v/v)、5％至40％(v/v)、10％至40％(v/v)、20％至40％(v/v)、30％至40％(v/v)、2％至30％(v/v)、5％至30％(v/v)、10％至30％(v/v)、20％至30％(v/v)、2％至20％(v/v)、5％至20％(v/v)、10％至20％(v/v)、2％至10％(v/v)、5％至10％(v/v)、5％至20％(v/v)、5％至15％(v/v)、5％至10％(v/v)、10％至15％(v/v)、或15％至20％(v/v)。在一些实施方案中，有机溶剂在洗脱溶液中为5％至20％(v/v)。在一些实施方案中，有机溶剂在洗脱溶液中为5％(v/v)、6％(v/v)、7％(v/v)、8％(v/v)、9％(v/v)、10％(v/v)、11％(v/v)、12％(v/v)、13％(v/v)、14％(v/v)、15％(v/v)、16％(v/v)、17％(v/v)、18％(v/v)、19％(v/v)或20％(v/v)。在一些实施方案中，超过20％(v/v)的有机溶剂可用于洗脱溶液中。在一些实施方案中，有机溶剂在洗脱溶液中为15％(v/v)。在一些实施方案中，有机溶剂在洗脱溶液中为30％(v/v)。In some embodiments, the organic solvent is 2% to 50% (v/v) in the elution solution. For example, the organic solvent can be 2% to 50% (v/v), 5% to 50% (v/v), 10% to 50% (v/v), 20% to 50% (v/v), 30% to 50% (v/v), 40% to 50% (v/v), 2% to 40% (v/v), 5% to 40% (v/v), 10% to 40% (v/v), 20% to 40% (v/v), 30% to 40% (v/v), 2% to 30% (v/v) in the elution solution. In some embodiments, the organic solvent is 5% to 20% (v/v) in the elution solution. In some embodiments, the organic solvent is 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v), 16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v) or 20% (v/v) in the elution solution. In some embodiments, more than 20% (v/v) of the organic solvent can be used in the elution solution. In some embodiments, the organic solvent is 15% (v/v) in the elution solution. In some embodiments, the organic solvent is 30% (v/v) in the elution solution.
在一些实施方案中，步骤(ii)的洗脱溶液包含至少30mM的磷酸根离子。在一些实施方案中，步骤(ii)的洗脱溶液包含至少30mM(例如，至少30mM、至少40mM、至少50mM、至少60mM、至少70mM、至少80mM、至少90mM、至少100mM、至少110mM、至少120mM、至少130mM、至少140mM或至少150mM)的磷酸根离子。在一些实施方案中，步骤(ii)的洗脱溶液包含至少100mM的磷酸根离子。在一些实施方案中，步骤(ii)的洗脱溶液包含100mM的磷酸根离子。In some embodiments, the elution solution of step (ii) comprises at least 30mM of phosphate ions. In some embodiments, the elution solution of step (ii) comprises at least 30mM (e.g., at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 110mM, at least 120mM, at least 130mM, at least 140mM or at least 150mM) of phosphate ions. In some embodiments, the elution solution of step (ii) comprises at least 100mM of phosphate ions. In some embodiments, the elution solution of step (ii) comprises 100mM of phosphate ions.
在一些实施方案中，洗脱溶液包含逐渐提高浓度的磷酸根离子。在一些实施方案中，在步骤(ii)期间，磷酸根离子的浓度从至少10mM(例如，10mM、15mM或20mM)提高至至少100mM(例如，100mM、150mM、200mM、250mM、300mM或更高)。在一些实施方案中，在步骤(ii)期间，磷酸根离子的浓度从10mM提高至100mM。In some embodiments, the elution solution comprises phosphate ions of gradually increasing concentrations. In some embodiments, during step (ii), the concentration of phosphate ions is increased from at least 10 mM (e.g., 10 mM, 15 mM, or 20 mM) to at least 100 mM (e.g., 100 mM, 150 mM, 200 mM, 250 mM, 300 mM or higher). In some embodiments, during step (ii), the concentration of phosphate ions is increased from 10 mM to 100 mM.
在一些实施方案中，将洗脱溶液施加至混合模式树脂使至少50％(例如，至少50％、至少60％、至少70％、至少80％、至少90％、至少95％或至少99％)的复合物从混合模式树脂解离。在一些实施方案中，将洗脱溶液施加至混合模式树脂使50％、60％、70％、80％、90％、95％、99％或更多的复合物从混合模式树脂解离。In some embodiments, applying the elution solution to the mixed mode resin causes at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the complexes to dissociate from the mixed mode resin. In some embodiments, applying the elution solution to the mixed mode resin causes 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the complexes to dissociate from the mixed mode resin.
在一些实施方案中，洗脱溶液不包含氯离子。在一些实施方案中，洗脱溶液可还包含至少50mM(例如，至少50mM、至少60mM、至少70mM、至少80mM、至少90mM、至少100mM、至少110mM、至少120mM、至少130mM、至少140mM、至少150mM、至少160mM、至少170mM、至少180mM、至少190mM或至少200mM)的氯离子。In some embodiments, the elution solution does not include chloride ions. In some embodiments, the elution solution may also include at least 50 mM (e.g., at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, or at least 200 mM) of chloride ions.
在一些实施方案中，本文中所述方法还包括收集解离的复合物。在一些实施方案中，本文中所述方法还包括在配制缓冲液(formulation buffer)(例如，通过缓冲液交换)中制备复合物。在一些实施方案中，缓冲液交换可以通过超滤/渗滤(ultrafiltration/diafiltration，UF/DF)或切向流过滤(tangential flow filtration，TFF)进行。In some embodiments, the methods described herein further include collecting the dissociated complex. In some embodiments, the methods described herein further include preparing the complex in a formulation buffer (e.g., by buffer exchange). In some embodiments, buffer exchange can be performed by ultrafiltration/diafiltration (UF/DF) or tangential flow filtration (TFF).
在一些方面中，本公开内容提供了处理复合物的方法，所述复合物各自包含与一个或更多个电荷中性寡核苷酸共价连接的抗体，该方法包括：In some aspects, the disclosure provides methods of treating complexes, each of which comprises an antibody covalently linked to one or more charge-neutral oligonucleotides, the method comprising:
(i)使包含15％(v/v)的DMA或IPA、复合物和未连接电荷中性寡核苷酸的混合物与包含带正电荷的金属位点和带负电荷的离子位点的羟基磷灰石(HA)树脂接触，其中混合物的pH为约5.7，并且任选地还包含10mM的磷酸根离子和/或(例如，和)20mM的氯离子；(i) contacting a mixture comprising 15% (v/v) DMA or IPA, the complex, and an unattached charge-neutral oligonucleotide with a hydroxyapatite (HA) resin comprising positively charged metal sites and negatively charged ionic sites, wherein the mixture has a pH of about 5.7 and optionally further comprises 10 mM phosphate ions and/or (e.g., and) 20 mM chloride ions;
(ii)用pH为约5.7并包含15％(v/v)的DMA或IPA的洗涤溶液洗涤HA树脂，并且任选地还包含10mM的磷酸根离子和/或(例如，和)20mM的氯离子；(ii) washing the HA resin with a wash solution having a pH of about 5.7 and comprising 15% (v/v) DMA or IPA, and optionally further comprising 10 mM phosphate ions and/or (e.g., and) 20 mM chloride ions;
(iii)通过将洗脱溶液施加至HA树脂，将复合物从混合模式树脂洗脱，其中洗脱溶液的pH为约7.6，并且包含：(iii) eluting the complex from the mixed mode resin by applying an elution solution to the HA resin, wherein the elution solution has a pH of about 7.6 and comprises:
(a)15％(v/v)的DMA或IPA，和(a) 15% (v/v) DMA or IPA, and
(a)100mM的磷酸根离子或浓度范围为30mM至100mM的磷酸根离子梯度；以及(a) 100 mM phosphate ion or a phosphate ion gradient ranging from 30 mM to 100 mM; and
(iv)收集所选择的复合物。(iv) collecting the selected complexes.
在一些实施方案中，抗TfR Fab-寡核苷酸缀合物的纯化需要两部分纯化过程。在一些实施方案中，将包含复合物和未连接Fab和/或寡核苷酸的反应混合物在不含核酸酶的水中稀释(例如，1:3)，并通过添加适当浓度(例如，约50mM)的适当缓冲液(例如，MES缓冲液)来调节pH(例如，至5.7)。在一些实施方案中，使用15:85v/v％的有机溶剂DMA与10mM磷酸钠在pH 5.8下平衡陶瓷羟基磷灰石(ceramic hydroxyapatite，HA)柱。在一些实施方案中，将粗制反应混合物装载到HA柱上以去除未缀合寡核苷酸。在一些实施方案中，HA柱用10mM磷酸钠缓冲液(pH 5.8)中的15:85v/v％DMA进行洗涤。在一些实施方案中，通过用100mM的磷酸钠pH 7.6缓冲液(包含15:85v/v％的DMA)的阶梯梯度以5mL/分钟的流量开始洗脱。在一些实施方案中，HA洗脱物经缓冲液交换到最终制剂中。在一些实施方案中，对最终纯化的抗TfR Fab-寡核苷酸缀合物进行分析(例如，通过SEC、SDS-PAGE密度测定法和/或BCA)。In some embodiments, the purification of anti-TfR Fab-oligonucleotide conjugates requires a two-part purification process. In some embodiments, the reaction mixture containing the complex and the unconnected Fab and/or oligonucleotide is diluted in water without nuclease (e.g., 1: 3), and the pH is adjusted (e.g., to 5.7) by adding an appropriate buffer (e.g., MES buffer) of an appropriate concentration (e.g., about 50mM). In some embodiments, 15: 85v/v% of organic solvent DMA is used to balance ceramic hydroxyapatite (HA) columns with 10mM sodium phosphate at pH 5.8. In some embodiments, the crude reaction mixture is loaded onto the HA column to remove unconjugated oligonucleotides. In some embodiments, the HA column is washed with 15: 85v/v% DMA in 10mM sodium phosphate buffer (pH 5.8). In some embodiments, elution is initiated at a flow rate of 5 mL/min by a step gradient with 100 mM sodium phosphate pH 7.6 buffer (containing 15:85 v/v% DMA). In some embodiments, the HA eluate is buffer exchanged into the final formulation. In some embodiments, the final purified anti-TfR Fab-oligonucleotide conjugate is analyzed (e.g., by SEC, SDS-PAGE densitometry and/or BCA).
在一些实施方案中，本文中所述方法中使用的混合物和溶液还包含磷酸根离子和/或氯离子的抗衡离子。在一些实施方案中，磷酸根的抗衡离子是钙、钠、镁、钾或锰。在一些实施方案中，本文中所述方法中使用的抗衡离子是钠。在一些实施方案中，磷酸根离子源是NaH2PO4、Na2HPO4或Na3PO4。在一些实施方案中，氯的抗衡离子是钙、钠、镁、钾或锰。在一些实施方案中，氯离子源是NaCl。本领域技术人员将容易理解，许多其他等效的盐和离子可用于本文中所述方法。In some embodiments, the mixture and solution used in the methods described herein also include counterions of phosphate ions and/or chloride ions. In some embodiments, the counterion of phosphate is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the counterion used in the methods described herein is sodium. In some embodiments, the phosphate ion source is NaH2 PO4 , Na2 HPO4 or Na3 PO4 . In some embodiments, the counterion of chloride is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the chloride ion source is NaCl. It will be readily appreciated by those skilled in the art that many other equivalent salts and ions can be used in the methods described herein.
在一些实施方案中，洗涤溶液和/或洗脱物溶液可还包含缓冲剂以保持一致的pH。用于本文中的缓冲剂的实例包括乙二胺四乙酸(EDTA)、琥珀酸盐、柠檬酸盐、天冬氨酸、谷氨酸、马来酸盐、二甲胂酸盐(cacodylate)、2-(N-吗啉代)-乙磺酸(MES)、N-(2-乙酰氨基)-2-氨基乙磺酸(ACES)、哌嗪-N,N’-2-乙磺酸(PIPES)、2-(N-吗啉代)-2-羟基-丙磺酸(MOPSO)、N,N-双-(羟乙基)-2-氨基乙磺酸(BES)、3-(N-吗啉代)-丙磺酸(MOPS)、N-2-羟乙基-哌嗪-N-2-乙磺酸(HEPES)、3-(N-三-(羟甲基)甲基氨基)-2-羟基丙磺酸(TAPSO)、3-(N,N-双[2-羟乙基]氨基)-2-羟基丙磺酸(DIPSO)、N-(2-羟乙基)哌嗪-N′-(2-羟基丙磺酸)(HEPPSO)、4-(2-羟乙基)-1-哌嗪丙磺酸(EPPS)、N-[三(羟甲基)-甲基]甘氨酸(Tricine)、N,N-双(2-羟乙基)甘氨酸(Bicine)、[(2-羟基-1,1-双(羟甲基)乙基)氨基]-1-丙磺酸(TAPS)、N-(1,1-二甲基-2-羟乙基)-3-氨基-2-羟基丙磺酸(AMPSO)、三(羟甲基)氨基甲烷(Tris)和双[2-羟乙基]亚氨基三-[羟甲基]甲烷(Bis-Tris)。用于本文中的其他缓冲剂组成、缓冲剂浓度和溶液的其他组分对于本领域技术人员而言将是明显的。In some embodiments, the washing solution and/or the eluate solution may further comprise a buffer to maintain a consistent pH. Examples of buffers used herein include ethylenediaminetetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, cacodylate, 2-(N-morpholino)-ethanesulfonic acid (MES), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N, N'-2-ethanesulfonic acid (PIPES), 2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO), N, N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)-propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (TAPSO) , 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-[tris(hydroxymethyl)-methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), tris(hydroxymethyl)aminomethane (Tris), and bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of the solutions for use herein will be apparent to those skilled in the art.
根据本公开内容，可使用包含带正电荷的金属位点和带负电荷的离子位点的任意混合模式树脂。在一些实施方案中，本文中所述方法中使用的混合模式树脂是磷灰石树脂。在一些实施方案中，磷灰石树脂是羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂或氯磷灰石树脂。磷灰石树脂可包括任何形式的磷灰石，并且通常在使用亲和力、离子交换、疏水相互作用或其组合的生物分子(例如，本文中所述的复合物)的分离和纯化中用作色谱固相。According to the present disclosure, any mixed mode resin comprising a positively charged metal site and a negatively charged ionic site can be used. In some embodiments, the mixed mode resin used in the methods described herein is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin or a chloroapatite resin. Apatite resins can include any form of apatite and are typically used as chromatographic solid phases in the separation and purification of biomolecules (e.g., complexes described herein) using affinity, ion exchange, hydrophobic interactions or combinations thereof.
在一些实施方案中，羟基磷灰石树脂为Bio-Gel HT树脂，例如，来自Bio-RadLaboratories,Inc.(Hercules,ca,USA)。在一些实施方案中，陶瓷羟基磷灰石树脂是Bio-Scale Mini CHT树脂，例如，来自Bio-Rad Laboratories,Inc。在一些实施方案中，磷灰石树脂(例如，陶瓷羟基磷灰石)包括磷灰石的球形颗粒。在一些实施方案中，磷灰石的球形颗粒的直径为约10微米至约100微米、约25微米至约50微米、约20微米、约30微米、约40微米、约50微米、约60微米或约80微米。在一些实施方案中，磷灰石树脂(例如，陶瓷羟基磷灰石)是I型(中等孔隙率和高结合能力)或II型(较大孔隙率和较低结合能力)。在一些实施方案中，磷灰石颗粒可以与另一种分离介质或支持物混合来使用。In some embodiments, the hydroxyapatite resin is Bio-Gel HT resin, for example, from Bio-Rad Laboratories, Inc. (Hercules, ca, USA). In some embodiments, the ceramic hydroxyapatite resin is Bio-Scale Mini CHT resin, for example, from Bio-Rad Laboratories, Inc. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) includes spherical particles of apatite. In some embodiments, the diameter of the spherical particles of apatite is about 10 microns to about 100 microns, about 25 microns to about 50 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns or about 80 microns. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) is type I (medium porosity and high binding capacity) or type II (larger porosity and lower binding capacity). In some embodiments, apatite particles can be used in combination with another separation medium or support.
在一些实施方案中，混合模式树脂可在与复合物和未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)的混合物接触之前被平衡。在一些实施方案中，如上所述，使用洗涤溶液平衡混合模式树脂。在一些实施方案中，混合模式树脂被平衡以使pH达到约5.0至8.0。In some embodiments, the mixed mode resin can be balanced before contacting the mixture with the complex and the unattached molecular load (e.g., a charge neutral oligonucleotide or a hydrophobic small molecule). In some embodiments, the mixed mode resin is balanced with a wash solution as described above. In some embodiments, the mixed mode resin is balanced to a pH of about 5.0 to 8.0.
在一些实施方案中，将混合模式树脂填充到柱(例如，垂直柱)中。在一些实施方案中，可在压力下，任选地在从上至下或从下至上的压力下使用柱。在一些实施方案中，可在没有外部压力的情况下使用柱，例如仅使用重力流。在一些实施方案中，将混合模式树脂用作游离树脂，例如使用分批法。在一些实施方案中，分批法可还包括在使树脂与复合物和未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)的混合物接触之后的离心和/或过滤步骤。In some embodiments, the mixed mode resin is filled into a column (e.g., a vertical column). In some embodiments, the column can be used under pressure, optionally under pressure from top to bottom or from bottom to top. In some embodiments, the column can be used without external pressure, for example, using only gravity flow. In some embodiments, the mixed mode resin is used as a free resin, for example, using a batch process. In some embodiments, the batch process may also include a centrifugation and/or filtration step after contacting the resin with a mixture of complexes and unconnected molecular payloads (e.g., charge neutral oligonucleotides or hydrophobic small molecules).
在一些实施方案中，在本文中所述方法的步骤(i)中的混合物中的复合物和/或(例如，和)本文中所述方法的步骤(ii)中洗脱的复合物包含与1至10个(例如，1、2、3、4、5、6、7、8、9或10个)分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)共价连接的抗体。在一些实施方案中，在本文中所述方法的步骤(i)的混合物中的复合物和/或(例如，和)在步骤(ii)中洗脱的复合物的至少50％(例如，至少50％、至少60％、至少70％、至少80％、至少90％或更多)包含与1至3个(例如，1、2或3个)分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)共价连接的抗体。在一些实施方案中，在本文中所述方法的步骤(i)的混合物中的复合物和/或(例如，和)步骤(ii)中洗脱的复合物具有至少约1.5(例如，至少约1.5、至少约1.6、至少约1.7、至少约1.8、至少约1.9或至少约2)的平均药物抗体比(DAR)。在一些实施方案中，从步骤(ii)获得的洗脱物包含的未连接分子载荷(例如，电荷中性寡核苷酸或疏水性小分子)的水平不可检测。在一些实施方案中，从步骤(ii)获得的洗脱物包含的未连接抗体的水平不可检测。In some embodiments, the complexes in the mixture in step (i) of the methods described herein and/or (e.g., and) the complexes eluted in step (ii) of the methods described herein comprise antibodies covalently linked to 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) molecular payloads (e.g., charge-neutral oligonucleotides or hydrophobic small molecules). In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more) of the complexes in the mixture in step (i) of the methods described herein and/or (e.g., and) the complexes eluted in step (ii) comprise antibodies covalently linked to 1 to 3 (e.g., 1, 2, or 3) molecular payloads (e.g., charge-neutral oligonucleotides or hydrophobic small molecules). In some embodiments, the complex in the mixture of step (i) of the method described herein and/or (e.g., and) the complex eluted in step (ii) has an average drug-antibody ratio (DAR) of at least about 1.5 (e.g., at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, or at least about 2). In some embodiments, the eluate obtained from step (ii) contains an undetectable level of unattached molecular load (e.g., a charge-neutral oligonucleotide or a hydrophobic small molecule). In some embodiments, the eluate obtained from step (ii) contains an undetectable level of unattached antibody.
B.使用混合模式树脂相对于复合物而去除未连接带电荷的寡核苷酸B. Removal of unligated charged oligonucleotides relative to complexes using mixed mode resins
在一些实施方案中，本文表明，包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂(例如磷灰石树脂，例如羟基磷灰石树脂)的使用有效地纯化了复合物使其远离未连接寡核苷酸。这在很大程度上是出乎意料的发现，因为没有其他纯化策略替代方案能够从包含蛋白质-寡核苷酸复合物的组合物中去除基本上所有未连接寡核苷酸。此外，与其他已知的去除未连接寡核苷酸和/或过量盐(脱盐)的方法相比，本文中所述的混合模式树脂纯化法是有利的。一种这样的已知方法是尺寸排阻色谱法(SEC)。混合模式树脂纯化法至少由于其可扩展性和较高的回收率而优于SEC。使用本文中所述的混合模式树脂方法实现了至少90％的复合物的回收，而SEC只能实现20％至30％的复合物的回收。In some embodiments, it is shown herein that the use of a mixed mode resin (e.g., apatite resin, e.g., hydroxyapatite resin) comprising a positively charged metal site and a negatively charged ionic site effectively purifies the complex away from unconnected oligonucleotides. This is a largely unexpected discovery, as no other purification strategy alternatives are able to remove substantially all unconnected oligonucleotides from a composition comprising a protein-oligonucleotide complex. In addition, the mixed mode resin purification method described herein is advantageous compared to other known methods for removing unconnected oligonucleotides and/or excess salt (desalting). One such known method is size exclusion chromatography (SEC). The mixed mode resin purification method is superior to SEC at least due to its scalability and higher recovery. Using the mixed mode resin method described herein, at least 90% of the complex recovery is achieved, while SEC can only achieve 20% to 30% of the complex recovery.
在一些实施方案中，本文中所述的分离一种或多种复合物(所述复合物各自包含与一个或更多个寡核苷酸(例如，带电荷寡核苷酸)共价连接的抗体)的方法包括：(i)使包含复合物和未连接寡核苷酸的混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂在使复合物吸附至混合模式树脂的条件下接触，以及(ii)在复合物从混合模式树脂上解离的条件下从混合模式树脂洗脱复合物。在一些实施方案中，步骤(i)中的混合物包含的含有炔基的未连接抗体为痕量。在一些实施方案中，步骤(i)中的混合物在与混合模式树脂接触之前没有经受过纯化。如本文中所述的，在一些实施方案中，可调节步骤(i)中使复合物吸附至混合模式树脂的条件，以允许或排除未连接寡核苷酸吸附至混合模式树脂。In some embodiments, the method of separating one or more complexes described herein, each of which comprises an antibody covalently linked to one or more oligonucleotides (e.g., a charged oligonucleotide), comprises: (i) contacting a mixture comprising the complex and the unlinked oligonucleotide with a mixed mode resin comprising a positively charged metal site and a negatively charged ionic site under conditions such that the complex is adsorbed to the mixed mode resin, and (ii) eluting the complex from the mixed mode resin under conditions such that the complex dissociates from the mixed mode resin. In some embodiments, the mixture in step (i) comprises trace amounts of unlinked antibodies containing alkynyl groups. In some embodiments, the mixture in step (i) has not been subjected to purification prior to contact with the mixed mode resin. As described herein, in some embodiments, the conditions for adsorbing the complex to the mixed mode resin in step (i) can be adjusted to allow or exclude adsorption of unlinked oligonucleotides to the mixed mode resin.
在一些实施方案中，步骤(i)中使复合物吸附至混合模式树脂的条件不允许未连接寡核苷酸吸附至混合模式树脂，因此使复合物与未连接寡核苷酸分离。在一些实施方案中，通过在步骤(i)中的混合物中包含浓度允许复合物吸附至混合模式树脂但是未连接寡核苷酸未吸附至混合模式树脂的磷酸根离子和/或氯离子来实现所述条件。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为20mM的磷酸根离子和/或最高为30mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为20mM(例如，最高为20mM、最高为15mM、最高为10mM或最高为5mM)的磷酸根离子。另外，在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为30mM(例如，最高为30mM、最高为25mM、最高为20mM、最高为15mM、最高为10mM或最高为5mM)的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含5至20mM(例如，5至20mM、5至15mM、5至10mM、10至20mM、10至15mM或15至20mM)磷酸根离子和/或5至30mM的氯离子(例如，5至30mM、5至25mM、5至20mM、5至15mM、5至10mM、10至30mM、10至25mM、10至20mM、10至15mM、15至30mM、15至25mM、15至20mM、20至30mM、20至25mM、或25至30mM)。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含20mM、15mM、10mM、5mM或1mM的磷酸根离子和/或30mM、25mM、20mM、15mM、10mM或5mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含20mM的磷酸根离子和/或30mM的氯离子，例如20mM的磷酸根离子和30mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为10mM的磷酸根离子和/或最高为25mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含5至10mM的磷酸根离子和/或5至25mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含10mM的磷酸根离子和/或25mM的氯离子，例如10mM的磷酸根离子和25mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物包含的磷酸根离子为痕量。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物包含的氯离子为痕量。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物包含的磷酸根离子和氯离子二者为痕量。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物不包含磷酸根离子，不包含氯离子，或者不包含磷酸根离子或氯离子。在这些条件下，未连接寡核苷酸保留在流通物中，并且未吸附至混合模式树脂。在一些实施方案中，可在步骤(i)与步骤(ii)之间在这些相同条件下进一步洗涤混合模式树脂，以去除松散结合但未吸附至混合模式树脂的未连接寡核苷酸。In some embodiments, the conditions that make complex adsorbed to mixed mode resin in step (i) do not allow unconnected oligonucleotide to be adsorbed to mixed mode resin, so complex is separated from unconnected oligonucleotide.In some embodiments, the conditions are realized by including phosphate ions and/or chloride ions whose concentration allows complex adsorption to mixed mode resin but unconnected oligonucleotide is not adsorbed to mixed mode resin in the mixture in step (i).In some embodiments, the mixture comprising complex and unconnected oligonucleotide also comprises phosphate ions up to 20mM and/or chloride ions up to 30mM.In some embodiments, the mixture comprising complex and unconnected oligonucleotide also comprises phosphate ions up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM or up to 5mM). Additionally, in some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises up to 30 mM (e.g., up to 30 mM, up to 25 mM, up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM) chloride ions. In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 5 to 20 mM (e.g., 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 20 mM, 10 to 15 mM, or 15 to 20 mM) of phosphate ions and/or 5 to 30 mM of chloride ions (e.g., 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 30 mM, 20 to 25 mM, or 25 to 30 mM). In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises 20mM, 15mM, 10mM, 5mM or 1mM phosphate ions and/or 30mM, 25mM, 20mM, 15mM, 10mM or 5mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises 20mM phosphate ions and/or 30mM chloride ions, such as 20mM phosphate ions and 30mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises up to 10mM phosphate ions and/or up to 25mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotides also comprises 5 to 10mM phosphate ions and/or 5 to 25mM chloride ions. In some embodiments, the mixture comprising complex and unconnected oligonucleotide also comprises 10mM phosphate ion and/or 25mM chloride ion, such as 10mM phosphate ion and 25mM chloride ion.In some embodiments, the phosphate ion included in the mixture comprising complex and unconnected oligonucleotide is a trace amount.In some embodiments, the chloride ion included in the mixture comprising complex and unconnected oligonucleotide is a trace amount.In some embodiments, the phosphate ion and chloride ion included in the mixture comprising complex and unconnected oligonucleotide are both trace amounts.In some embodiments, the mixture comprising complex and unconnected oligonucleotide does not comprise phosphate ion, does not comprise chloride ion, or does not comprise phosphate ion or chloride ion.Under these conditions, unconnected oligonucleotide is retained in the flowthrough and is not adsorbed to mixed mode resin.In some embodiments, mixed mode resin can be further washed under these same conditions between step (i) and step (ii), to remove loosely bound but unconnected oligonucleotide that is not adsorbed to mixed mode resin.
在一些实施方案中，步骤(i)中使复合物吸附至混合模式树脂的条件还允许一些或全部(例如，至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或100％)未连接寡核苷酸吸附至混合模式树脂。在一些实施方案中，通过在步骤(i)的混合物中包含浓度允许复合物和未连接寡核苷酸暖二者吸附至混合模式树脂的磷酸根离子和/或氯离子来实现所述条件。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为5mM的磷酸根离子和/或最高为10mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为5mM(例如，最高为5mM、最高为4mM、最高为3mM、最高为2mM或最高为1mM)的磷酸根离子。另外，在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含至最高为10mM(例如，最高为10mM、最高为9mM、最高为8mM、最高为7mM、最高为6mM、最高为5mM、最高为4mM、最高为3mM、最高为2mM、或最高为1mM)的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含1至5mM(例如，1至5mM、1至4mM、1至3mM、1至2mM、2至5mM、2至4mM、2至3mM、3至5mM、3至4mM、或4至5mM)的磷酸根离子和/或1至10mM(例如，1至10mM、1至8mM、1至6mM、1至4mM、1至2mM、2至10mM、2至8mM、2至6mM、2至4mM、4至10mM、4至8mM、4至6mM、6至10mM、6至8mM、或8至10mM)的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含5mM、4mM、3mM、2mM或1mM的磷酸根离子和/或10mM、9mM、8mM、7mM、6mM、5mM、4mM、3mM、2mM或1mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含最高为3mM的磷酸根离子和/或最高为8mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含1至3mM(例如，1、2或3mM)的磷酸根离子和/或1至8mM(例如，1、2、3、4、5、6、7或8mM)的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物还包含3mM的磷酸根离子和/或8mM的氯离子，例如3mM的磷酸根离子和8mM的氯离子。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物包含的磷酸根离子为痕量。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物包含的氯离子为痕量。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物包含的磷酸根离子和氯离子二者为痕量。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物不包含磷酸根离子，不包含氯离子，或者不包含磷酸根离子或氯离子。在这些条件下，所有未连接寡核苷酸中的一些也吸附至混合模式树脂。In some embodiments, the conditions under which the complex is adsorbed to the mixed mode resin in step (i) also allow some or all (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%) of the unattached oligonucleotides to be adsorbed to the mixed mode resin. In some embodiments, the conditions are achieved by including phosphate ions and/or chloride ions in the mixture of step (i) at a concentration that allows both the complex and the unattached oligonucleotides to be adsorbed to the mixed mode resin. In some embodiments, the mixture comprising the complex and the unattached oligonucleotides also comprises phosphate ions up to 5 mM and/or chloride ions up to 10 mM. In some embodiments, the mixture comprising the complex and the unattached oligonucleotides also comprises phosphate ions up to 5 mM (e.g., up to 5 mM, up to 4 mM, up to 3 mM, up to 2 mM or up to 1 mM). Additionally, in some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises chloride ions up to 10 mM (e.g., up to 10 mM, up to 9 mM, up to 8 mM, up to 7 mM, up to 6 mM, up to 5 mM, up to 4 mM, up to 3 mM, up to 2 mM, or up to 1 mM). In some embodiments, the mixture comprising the complex and the unligated oligonucleotide further comprises 1 to 5 mM (e.g., 1 to 5 mM, 1 to 4 mM, 1 to 3 mM, 1 to 2 mM, 2 to 5 mM, 2 to 4 mM, 2 to 3 mM, 3 to 5 mM, 3 to 4 mM, or 4 to 5 mM) of phosphate ions and/or 1 to 10 mM (e.g., 1 to 10 mM, 1 to 8 mM, 1 to 6 mM, 1 to 4 mM, 1 to 2 mM, 2 to 10 mM, 2 to 8 mM, 2 to 6 mM, 2 to 4 mM, 4 to 10 mM, 4 to 8 mM, 4 to 6 mM, 6 to 10 mM, 6 to 8 mM, or 8 to 10 mM) of chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises 5mM, 4mM, 3mM, 2mM or 1mM phosphate ions and/or 10mM, 9mM, 8mM, 7mM, 6mM, 5mM, 4mM, 3mM, 2mM or 1mM chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises up to 3mM phosphate ions and/or up to 8mM chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises 1 to 3mM (e.g., 1, 2 or 3mM) phosphate ions and/or 1 to 8mM (e.g., 1, 2, 3, 4, 5, 6, 7 or 8mM) chloride ions. In some embodiments, the mixture comprising the complex and the unconnected oligonucleotide also comprises 3mM phosphate ions and/or 8mM chloride ions, such as 3mM phosphate ions and 8mM chloride ions. In some embodiments, the phosphate ion included in the mixture comprising complex and unconnected oligonucleotide is a trace amount. In some embodiments, the chloride ion included in the mixture comprising complex and unconnected oligonucleotide is a trace amount. In some embodiments, the phosphate ion and chloride ion included in the mixture comprising complex and unconnected oligonucleotide are both trace amounts. In some embodiments, the mixture comprising complex and unconnected oligonucleotide does not include phosphate ion, does not include chloride ion, or does not include phosphate ion or chloride ion. Under these conditions, some of all unconnected oligonucleotides are also adsorbed to mixed mode resin.
在一些实施方案中，包含复合物和未连接寡核苷酸的混合物的pH为5.0至8.0(例如，约5.0、5.5、6.0、6.5、7.0、7.5或8.0)。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物的pH为5.0至6.0或者约5.0至6.0(例如，约5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9或6.0)。在一些实施方案中，包含复合物和未连接寡核苷酸的混合物的pH为5.7或为约5.7。In some embodiments, the pH of the mixture comprising the complex and the unattached oligonucleotide is 5.0 to 8.0 (e.g., about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). In some embodiments, the pH of the mixture comprising the complex and the unattached oligonucleotide is 5.0 to 6.0 or about 5.0 to 6.0 (e.g., about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the pH of the mixture comprising the complex and the unattached oligonucleotide is 5.7 or about 5.7.
在一些实施方案中，当一些或全部的未连接寡核苷酸也吸附至混合模式树脂时，本文中所述方法还包括在步骤(i)与步骤(ii)之间用使未连接寡核苷酸从混合模式树脂解离但是使复合物不从混合模式树脂解离的溶液洗涤混合模式树脂。在一些实施方案中，用于洗涤的溶液包含最高为20mM的磷酸根离子和/或最高为30mM的氯离子，例如20mM的磷酸根离子和30mM的氯离子。在一些实施方案中，用于洗涤的溶液包含最高为20mM(例如，最高为20mM、最高为15mM、最高为10mM或最高为5mM)的磷酸根离子。另外，在一些实施方案中，用于洗涤的溶液包含最高为30mM(例如，最高为30mM、最高为25mM、最高为20mM、最高为15mM、最高为10mM或最高为5mM)氯离子。在一些实施方案中，用于洗涤的溶液包含5至20mM(例如，5至20mM、5至15mM、5至10mM、10至20mM、10至15mM或15至20mM)磷酸根离子和/或5至30mM的氯离子(例如，5至30mM、5至25mM、5至20mM、5至15mM、5至10mM、10至30mM、10至25mM、10至20mM、10至15mM、15至30mM、15至25mM、15至20mM、20至30mM、20至25mM、或25至30mM)。在一些实施方案中，用于洗涤的溶液包含20mM、15mM、10mM、5mM或1mM的磷酸根离子和/或30mM、25mM、20mM、15mM、10mM或5mM的氯离子。在一些实施方案中，用于洗涤的溶液包含20mM的磷酸根离子和/或30mM的氯离子，例如20mM的磷酸根离子和30mM的氯离子。在一些实施方案中，用于洗涤的溶液包含最高为10mM的磷酸根离子和/或最高为25mM的氯离子，例如10mM的磷酸根离子和最高为的25mM的氯离子。In some embodiments, when some or all of the unconnected oligonucleotides are also adsorbed to the mixed mode resin, the method described herein also includes washing the mixed mode resin with a solution that dissociates the unconnected oligonucleotides from the mixed mode resin but does not dissociate the complex from the mixed mode resin between step (i) and step (ii). In some embodiments, the solution used for washing comprises a phosphate ion of up to 20mM and/or a chloride ion of up to 30mM, such as a phosphate ion of 20mM and a chloride ion of 30mM. In some embodiments, the solution used for washing comprises a phosphate ion of up to 20mM (e.g., up to 20mM, up to 15mM, up to 10mM or up to 5mM). In addition, in some embodiments, the solution used for washing comprises a chloride ion of up to 30mM (e.g., up to 30mM, up to 25mM, up to 20mM, up to 15mM, up to 10mM or up to 5mM). In some embodiments, the solution used for washing comprises 5 to 20 mM (e.g., 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 20 mM, 10 to 15 mM, or 15 to 20 mM) of phosphate ions and/or 5 to 30 mM of chloride ions (e.g., 5 to 30 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, 5 to 10 mM, 10 to 30 mM, 10 to 25 mM, 10 to 20 mM, 10 to 15 mM, 15 to 30 mM, 15 to 25 mM, 15 to 20 mM, 20 to 30 mM, 20 to 25 mM, or 25 to 30 mM). In some embodiments, the solution for washing comprises 20mM, 15mM, 10mM, 5mM or 1mM phosphate ions and/or 30mM, 25mM, 20mM, 15mM, 10mM or 5mM chloride ions. In some embodiments, the solution for washing comprises 20mM phosphate ions and/or 30mM chloride ions, such as 20mM phosphate ions and 30mM chloride ions. In some embodiments, the solution for washing comprises up to 10mM phosphate ions and/or up to 25mM chloride ions, such as 10mM phosphate ions and up to 25mM chloride ions.
在一些实施方案中，当一些或全部的未连接寡核苷酸也吸附至混合模式树脂时，本文中所述方法还包括在步骤(i)与步骤(ii)之间用会使未连接寡核苷酸(而不是复合物)从混合模式树脂解离的一系列溶液来洗涤混合模式树脂。在一些实施方案中，用于洗涤的第一溶液包含10mM或约10mM的磷酸根离子和10mM或约10mM的氯离子。在一些实施方案中，用于洗涤的第二溶液包含15mM或约15mM的磷酸根离子和15mM或约15mM的氯离子。在一些实施方案中，用于洗涤的第三溶液包含19mM或约19mM的磷酸根离子和19mM或约19mM的氯离子。在一些实施方案中，在步骤(i)与步骤(ii)之间洗涤混合模式树脂包括用含有10mM磷酸根离子和10mM氯离子的第一溶液、含有14.5mM磷酸根离子和14.5mM氯离子的第二溶液以及含有19mM磷酸根离子和19mM氯离子的第三溶液来洗涤树脂。In some embodiments, when some or all of the unconnected oligonucleotides are also adsorbed to the mixed mode resin, the method described herein also includes washing the mixed mode resin with a series of solutions that will dissociate the unconnected oligonucleotides (rather than complexes) from the mixed mode resin between step (i) and step (ii). In some embodiments, the first solution for washing comprises 10mM or about 10mM of phosphate ions and 10mM or about 10mM of chloride ions. In some embodiments, the second solution for washing comprises 15mM or about 15mM of phosphate ions and 15mM or about 15mM of chloride ions. In some embodiments, the third solution for washing comprises 19mM or about 19mM of phosphate ions and 19mM or about 19mM of chloride ions. In some embodiments, washing the mixed mode resin between step (i) and step (ii) comprises washing the resin with a first solution containing 10 mM phosphate ions and 10 mM chloride ions, a second solution containing 14.5 mM phosphate ions and 14.5 mM chloride ions, and a third solution containing 19 mM phosphate ions and 19 mM chloride ions.
在一些实施方案中，用于洗涤的溶液的pH为6.0至8.5。在一些实施方案中，用于洗涤的溶液的pH为或为约6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4或8.5。在一些实施方案中，用于洗涤的溶液的pH为约6.5。In some embodiments, the pH of the solution used for washing is 6.0 to 8.5. In some embodiments, the pH of the solution used for washing is or is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of the solution used for washing is about 6.5.
在一些实施方案中，为了从混合模式树脂洗脱复合物，在步骤(ii)中，使混合模式和结合的复合物经受允许复合物从混合模式树脂解离的条件。在一些实施方案中，步骤(ii)中允许复合物解离的条件是通过向混合模式树脂施加包含较高浓度的磷酸根离子和/或氯离子的洗脱溶液实现的。洗脱步骤可使用包含单一磷酸根离子浓度的洗脱溶液来完成，或者使用具有提高的磷酸根离子浓度梯度的洗脱溶液来完成。在洗脱(步骤(ii))期间使用提高的磷酸根离子浓度梯度允许分离具有不同药物:抗体比(DAR)数的复合物。例如，随着洗脱溶液中不断提高的磷酸根离子浓度的提高，首先洗脱具有较低DAR的复合物，然后洗脱具有较高DAR的复合物。In some embodiments, in order to elute the complex from the mixed mode resin, in step (ii), the mixed mode and the combined complex are subjected to conditions that allow the complex to dissociate from the mixed mode resin. In some embodiments, the conditions that allow the complex to dissociate in step (ii) are achieved by applying an elution solution comprising a higher concentration of phosphate ions and/or chloride ions to the mixed mode resin. The elution step can be completed using an elution solution comprising a single phosphate ion concentration, or an elution solution with an increased phosphate ion concentration gradient. Using an increased phosphate ion concentration gradient during elution (step (ii)) allows separation of complexes with different drug: antibody ratios (DAR) numbers. For example, as the phosphate ion concentration that is constantly increased in the elution solution increases, the complex with a lower DAR is first eluted, and then the complex with a higher DAR is eluted.
在一些实施方案中，步骤(ii)包括向混合模式树脂施加包含至少30mM的磷酸根离子和/或至少50mM的氯离子的洗脱溶液以洗脱复合物。在一些实施方案中，洗脱溶液包含至少30mM(例如，至少30mM、至少40mM、至少50mM、至少60mM、至少70mM、至少80mM、至少90mM、至少100mM、至少110mM、至少120mM、至少130mM、至少140mM或至少150mM)的磷酸根离子。另外，在一些实施方案中，洗脱溶液包含至少50mM(例如，至少50mM、至少60mM、至少70mM、至少80mM、至少90mM、至少100mM、至少110mM、至少120mM、至少130mM、至少140mM、至少150mM、至少160mM、至少170mM、至少180mM、至少190mM或至少200mM)的氯离子。在一些实施方案中，洗脱溶液包含至少100mM的磷酸根离子和/或至少100mM的氯离子。在一些实施方案中，洗脱溶液包含100mM的磷酸根离子和100mM的氯离子。In some embodiments, step (ii) comprises applying to the mixed mode resin an elution solution comprising at least 30 mM of phosphate ions and/or at least 50 mM of chloride ions to elute the complex. In some embodiments, the elution solution comprises at least 30 mM (e.g., at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, or at least 150 mM) of phosphate ions. In addition, in some embodiments, the elution solution comprises at least 50 mM (e.g., at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, or at least 200 mM) chloride ions. In some embodiments, the elution solution comprises at least 100 mM of phosphate ions and/or at least 100 mM of chloride ions. In some embodiments, the elution solution comprises 100 mM of phosphate ions and 100 mM of chloride ions.
在一些实施方案中，洗脱溶液的pH为6.0至8.5。在一些实施方案中，洗脱溶液的pH为或为约6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4或8.5。在一些实施方案中，洗脱溶液的pH为约7.5。In some embodiments, the pH of the elution solution is between 6.0 and 8.5. In some embodiments, the pH of the elution solution is or is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the pH of the elution solution is about 7.5.
在一些实施方案中，为了分离包含与带电荷寡核苷酸共价连接的抗TfR Fab的复合物，将包含复合物和未连接Fab和/或寡核苷酸的反应混合物在不含核酸酶的水中稀释(例如，1:3)，并通过添加适当浓度(例如，约50mM)的适当缓冲液(例如，MES缓冲液)来调节pH(例如，至5.7)。在一些实施方案中，将稀释的反应混合物装载到陶瓷羟基磷灰石(HA)柱(例如，以生物分子浓度为8mg/mL的树脂)上。在一些实施方案中，用洗涤溶液(例如，5mMNa2HPO4，25mM NaCl pH 7.0)洗涤柱以去除未连接寡核苷酸。在一些实施方案中，包含与寡核苷酸连接的抗TfR Fab的复合物在配制缓冲液(例如，100mM Na2HPO4，100mM NaCl，pH7.6)中从HA柱洗脱。在一些实施方案中，对分离和纯化的抗TfR Fab-寡核苷酸缀合物(例如，通过SDS-PAGE和/或分析型SEC)进行分析以表明未连接寡核苷酸的完全去除。在一些实施方案中，通过ELISA分析分离和纯化的抗TfR Fab-寡核苷酸缀合物的人TfR1/cyno TfR1结合和内毒素水平。在一些实施方案中，复合物可以通过阳离子交换和阴离子交换来交替纯化。In some embodiments, in order to separate the complex comprising the anti-TfR Fab covalently linked to the charged oligonucleotide, the reaction mixture comprising the complex and the unconnected Fab and/or oligonucleotide is diluted in water without nuclease (e.g., 1: 3), and the pH is adjusted (e.g., to 5.7) by adding an appropriate buffer (e.g., MES buffer) of an appropriate concentration (e.g., about 50mM). In some embodiments, the diluted reaction mixture is loaded onto a ceramic hydroxyapatite (HA) column (e.g., a resin with a biomolecule concentration of 8mg/mL). In some embodiments, the column is washed with a washing solution (e.g., 5mM Na2HPO4, 25mM NaCl pH 7.0) to remove unconnected oligonucleotides. In some embodiments, the complex comprising the anti-TfR Fab connected to the oligonucleotide is eluted from the HA column in a formulation buffer (e.g., 100mM Na2HPO4, 100mM NaCl, pH7.6). In some embodiments, the isolated and purified anti-TfR Fab-oligonucleotide conjugates (e.g., by SDS-PAGE and/or analytical SEC) are analyzed to show complete removal of unattached oligonucleotides. In some embodiments, the human TfR1/cyno TfR1 binding and endotoxin levels of the isolated and purified anti-TfR Fab-oligonucleotide conjugates are analyzed by ELISA. In some embodiments, the complex can be purified alternately by cation exchange and anion exchange.
在一些实施方案中，本文中所述方法中使用的混合物和溶液还包含磷酸根离子和/或氯离子的抗衡离子。在一些实施方案中，磷酸根的抗衡离子是钙、钠、镁、钾或锰。在一些实施方案中，在本文中所述方法中使用的抗衡离子是钠。在一些实施方案中，磷酸根离子源是NaH2PO4、Na2HPO4或Na3PO4。在一些实施方案中，氯的抗衡离子是钙、钠、镁、钾或锰。在一些实施方案中，氯离子源是NaCl。本领域技术人员将容易理解，许多其他等效的盐和离子可用于本文中所述方法。In some embodiments, the mixture and solution used in the methods described herein also include counterions of phosphate ions and/or chloride ions. In some embodiments, the counterion of phosphate is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the counterion used in the methods described herein is sodium. In some embodiments, the phosphate ion source is NaH2 PO4 , Na2 HPO4 or Na3 PO4 . In some embodiments, the counterion of chloride is calcium, sodium, magnesium, potassium, or manganese. In some embodiments, the chloride ion source is NaCl. It will be readily appreciated by those skilled in the art that many other equivalent salts and ions can be used in the methods described herein.
在一些实施方案中，洗涤溶液和/或洗脱物溶液可还包含缓冲剂以保持一致的pH。在一些实施方案中，洗涤缓冲液和/或洗脱缓冲液包含中性pH。在一些实施方案中，洗涤缓冲液和/或洗脱缓冲液的pH为约6、约6.5、约7、约7.5、约8或约6至8。用于本文中的缓冲剂的实例包括乙二胺四乙酸(EDTA)、琥珀酸盐、柠檬酸盐、天冬氨酸、谷氨酸、马来酸盐、二甲胂酸盐(cacodylate)、2-(N-吗啉代)-乙磺酸(MES)、N-(2-乙酰氨基)-2-氨基乙磺酸(ACES)、哌嗪-N,N′-2-乙磺酸(PIPES)、2-(N-吗啉代)-2-羟基-丙磺酸(MOPSO)、N,N-双-(羟乙基)-2-氨基乙磺酸(BES)、3-(N-吗啉代)-丙磺酸(MOPS)、N-2-羟乙基-哌嗪-N-2-乙磺酸(HEPES)、3-(N-三-(羟甲基)甲基氨基)-2-羟基丙磺酸(TAPSO)、3-(N,N-双[2-羟乙基]氨基)-2-羟基丙磺酸(DIPSO)、N-(2-羟乙基)哌嗪-N′-(2-羟基丙磺酸)(HEPPSO)、4-(2-羟乙基)-1-哌嗪丙磺酸(EPPS)、N-[三(羟甲基)-甲基]甘氨酸(Tricine)、N,N-双(2-羟乙基)甘氨酸(Bicine)、[(2-羟基-1,1-双(羟甲基)乙基)氨基]-1-丙磺酸(TAPS)、N-(1,1-二甲基-2-羟乙基)-3-氨基-2-羟基丙磺酸(AMPSO)、三(羟甲基)氨基甲烷(Tris)和双[2-羟乙基]亚氨基三-[羟甲基]甲烷(Bis-Tris)。用于本文中的其他缓冲剂组成、缓冲剂浓度和溶液的其他组分对于本领域技术人员而言将是明显的。In some embodiments, the washing solution and/or the eluate solution may further comprise a buffer to maintain a consistent pH. In some embodiments, the washing buffer and/or the elution buffer comprise a neutral pH. In some embodiments, the pH of the washing buffer and/or the elution buffer is about 6, about 6.5, about 7, about 7.5, about 8, or about 6 to 8. Examples of buffers used herein include ethylenediaminetetraacetic acid (EDTA), succinate, citrate, aspartic acid, glutamic acid, maleate, cacodylate, 2-(N-morpholino)-ethanesulfonic acid (MES), N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES), piperazine-N,N′-2-ethanesulfonic acid (PIPES), 2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO), N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)-propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (TAPSO) , 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-[tris(hydroxymethyl)-methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), tris(hydroxymethyl)aminomethane (Tris), and bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (Bis-Tris). Other buffer compositions, buffer concentrations, and other components of the solutions for use herein will be apparent to those skilled in the art.
根据本公开内容，可使用包含带正电荷的金属位点和带负电荷的离子位点的任意混合模式树脂。在一些实施方案中，本文中所述方法中使用的混合模式树脂是磷灰石树脂。在一些实施方案中，磷灰石树脂是羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂或氯磷灰石树脂。磷灰石树脂可包括任何形式的磷灰石，并且通常在使用亲和力、离子交换、疏水相互作用或其组合进行的对生物分子(例如，本文中所述的复合物)的分离和纯化中用作色谱固相。According to the present disclosure, any mixed mode resin comprising a positively charged metal site and a negatively charged ionic site can be used. In some embodiments, the mixed mode resin used in the methods described herein is an apatite resin. In some embodiments, the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin. Apatite resins can include any form of apatite and are typically used as chromatographic solid phases in the separation and purification of biomolecules (e.g., complexes described herein) using affinity, ion exchange, hydrophobic interactions, or a combination thereof.
在一些实施方案中，羟基磷灰石树脂是Bio-Gel HT树脂，例如，来自Bio-RadLaboratories，Inc.(Hercules,Calif.,USA)。在一些实施方案中，陶瓷羟基磷灰石树脂是Bio-Scale Mini CHT树脂，例如来自Bio-Rad Laboratories,Inc。在一些实施方案中，磷灰石树脂(例如，陶瓷羟基磷灰石)包括磷灰石的球形颗粒。在一些实施方案中，磷灰石的球形颗粒的直径为约10微米至约100微米、约25微米至约50微米、约20微米、约30微米、约40微米、约50微米、约60微米或约80微米。在一些实施方案中，磷灰石树脂(例如，陶瓷羟基磷灰石)是I型(中等孔隙率和高结合能力)或II型(较大孔隙率和较低结合能力)。在一些实施方案中，磷灰石颗粒可以与另一种分离介质或支持物混合来使用。In some embodiments, the hydroxyapatite resin is Bio-Gel HT resin, for example, from Bio-Rad Laboratories, Inc. (Hercules, Calif., USA). In some embodiments, the ceramic hydroxyapatite resin is Bio-Scale Mini CHT resin, for example, from Bio-Rad Laboratories, Inc. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) includes spherical particles of apatite. In some embodiments, the diameter of the spherical particles of apatite is about 10 microns to about 100 microns, about 25 microns to about 50 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns or about 80 microns. In some embodiments, the apatite resin (e.g., ceramic hydroxyapatite) is type I (medium porosity and high binding capacity) or type II (larger porosity and lower binding capacity). In some embodiments, apatite particles can be used in combination with another separation medium or support.
在一些实施方案中，混合模式树脂可在与复合物和未连接寡核苷酸的混合物接触之前被平衡。在一些实施方案中，如上所述，使用洗涤溶液来平衡混合模式树脂。在一些实施方案中，混合模式树脂被平衡以使树脂的pH达到中性pH、pH为6至8、pH为约6.5、pH为约7.0、pH为约7.5或pH为约8.0。In some embodiments, the mixed mode resin can be balanced before contacting with the mixture of complex and unattached oligonucleotide. In some embodiments, as described above, a washing solution is used to balance the mixed mode resin. In some embodiments, the mixed mode resin is balanced so that the pH of the resin reaches a neutral pH, a pH of 6 to 8, a pH of about 6.5, a pH of about 7.0, a pH of about 7.5, or a pH of about 8.0.
在一些实施方案中，将混合模式树脂填充到柱(例如，垂直柱)中。在一些实施方案中，可在压力，任选地在从上至下或从下至上的压力下使用柱。在一些实施方案中，可以在没有外部压力的情况下使用柱，例如，仅使用重力流。在一些实施方案中，将混合模式树脂用作游离树脂，例如，使用分批法。在一些实施方案中，分批法可还包括在使树脂与复合物和未连接寡核苷酸的混合物接触之后的离心和/或过滤步骤。In some embodiments, the mixed mode resin is filled into a column (e.g., a vertical column). In some embodiments, the column can be used under pressure, optionally from top to bottom or from bottom to top. In some embodiments, the column can be used without external pressure, for example, using only gravity flow. In some embodiments, the mixed mode resin is used as a free resin, for example, using a batch process. In some embodiments, the batch process may also include centrifugation and/or filtering steps after contacting the resin with a mixture of complexes and unconnected oligonucleotides.
在一些实施方案中，在本文中所述的混合模式树脂色谱的步骤(ii)中洗脱的复合物包含与1、2或3个寡核苷酸共价连接的抗体。在一些实施方案中，具有不同数目的连接寡核苷酸(例如，1、2或3)的复合物在不同的洗脱级分中分离。在一些实施方案中，从步骤(ii)获得的洗脱物包含的未连接寡核苷酸的水平不可检测。In some embodiments, the complex eluted in step (ii) of the mixed mode resin chromatography described herein comprises an antibody covalently linked to 1, 2 or 3 oligonucleotides. In some embodiments, complexes with different numbers of linked oligonucleotides (e.g., 1, 2 or 3) are separated in different elution fractions. In some embodiments, the level of unlinked oligonucleotides included in the eluate obtained from step (ii) is undetectable.
C.经纯化复合物的组合物C. Composition of the Purified Complex
本文中所述方法可产生基本上纯的复合物，其中经纯化复合物的组合物不包含可检出量的未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)或未连接抗体。在一些实施方案中，经纯化复合物的组合物包含至少9:1、至少95:5、96:4、97:3、98:2、99:1、99.5:0.5或更高摩尔比或重量比的复合物:未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)。在一些实施方案中，经纯化的复合物的组合物包含至少9:1、至少95:5、96:4、97:3、98:2、99:1、99.5:0.5或更高摩尔比或重量比的复合物:未连接蛋白质。The methods described herein can produce substantially pure complexes, wherein the composition of the purified complex does not contain a detectable amount of unattached molecular cargo (e.g., charge neutral oligonucleotide or charged oligonucleotide) or unattached antibody. In some embodiments, the composition of the purified complex comprises at least 9: 1, at least 95: 5, 96: 4, 97: 3, 98: 2, 99: 1, 99.5: 0.5 or more molar or weight ratio of complex: unattached molecular cargo (e.g., charge neutral oligonucleotide or charged oligonucleotide). In some embodiments, the composition of the purified complex comprises at least 9: 1, at least 95: 5, 96: 4, 97: 3, 98: 2, 99: 1, 99.5: 0.5 or more molar or weight ratio of complex: unattached protein.
在一些实施方案中，经纯化的复合物的组合物不包含可检测水平(例如，可检出量)的未连接蛋白质。在一些实施方案中，经纯化的复合物的组合物不包含可检测水平(例如，可检出量)的未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)。In some embodiments, the composition of the purified complex does not include a detectable level (e.g., a detectable amount) of unattached protein. In some embodiments, the composition of the purified complex does not include a detectable level (e.g., a detectable amount) of unattached molecular cargo (e.g., a charge-neutral oligonucleotide or a charged oligonucleotide).
在一些实施方案中，经纯化的复合物的组合物包含按摩尔比计少于10％、少于9％、少于8％、少于7％、少于6％、少于5％、少于4％、少于3％、少于2％、少于5％、或少于0.5％的未连接蛋白质(例如，抗体)。在一些实施方案中，经纯化的复合物的组合物包含按摩尔比计少于10％、少于9％、少于8％、少于7％、少于6％、少于5％、少于4％、少于3％、少于2％、少于5％、或少于0.5％的未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)。在一些实施方案中，经纯化的复合物的组合物包含按摩尔比计少于10％、少于9％、少于8％、少于7％、少于6％、少于5％、少于4％、少于3％、少于2％、少于5％、或少于0.5％的未连接蛋白质(例如，抗体)，并且包含按摩尔比计少于10％、少于9％、少于8％、少于7％、少于6％、少于5％、少于4％、少于3％、少于2％、少于5％、或少于0.5％的未连接分子载荷(例如，电荷中性寡核苷酸或带电荷寡核苷酸)。In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unattached protein (e.g., antibody) by molar ratio. In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% of unattached molecular cargo (e.g., charge-neutral oligonucleotide or charged oligonucleotide) by molar ratio. In some embodiments, the composition of the purified complex comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% unattached protein (e.g., antibody) on a molar basis and comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 5%, or less than 0.5% unattached molecular cargo (e.g., charge-neutral oligonucleotide or charged oligonucleotide) on a molar basis.
在一些实施方案中，经纯化的复合物的组合物包含按摩尔比计至少90％(例如，至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、至少99.5％或至少99.9％)的复合物(即，与一个或更多个寡核苷酸共价连接的蛋白质(例如，抗体))。In some embodiments, the composition of the purified complex comprises at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%) of the complex (i.e., protein (e.g., antibody) covalently linked to one or more oligonucleotides) on a molar basis.
在一些实施方案中，经纯化的复合物的组合物包含与一个寡核苷酸、两个寡核苷酸、三个寡核苷酸和/或更多个寡核苷酸共价连接的蛋白质(例如，抗体)。在一些实施方案中，在经纯化的复合物的组合物中，至少50％(例如，至少60％、至少70％、至少80％、至少90％、至少95％)或更多的复合物包含与一个寡核苷酸共价连接的蛋白质(例如，抗体)(DAR1)。在一些实施方案中，在经纯化的复合物的组合物中，约50％、60％、70％、80％、90％或95％的复合物包含与一个寡核苷酸共价连接的蛋白质(例如，抗体)(DAR1)。In some embodiments, the composition of the purified complexes comprises a protein (e.g., an antibody) covalently linked to one oligonucleotide, two oligonucleotides, three oligonucleotides, and/or more oligonucleotides. In some embodiments, in the composition of the purified complexes, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) or more of the complexes comprise a protein (e.g., an antibody) covalently linked to one oligonucleotide (DAR1). In some embodiments, in the composition of the purified complexes, about 50%, 60%, 70%, 80%, 90%, or 95% of the complexes comprise a protein (e.g., an antibody) covalently linked to one oligonucleotide (DAR1).
在一些实施方案中，在经纯化的复合物的组合物中，约5％、10％、15％、20％、25％、30％或更多的复合物包含与两个寡核苷酸共价连接的蛋白质(例如，抗体)(DAR2)。在一些实施方案中，在经纯化的复合物的组合物中，约1％、2％、3％、5％、7％、10％、20％或更多的复合物包含与三个或更多个寡核苷酸共价连接的蛋白质(例如，抗体)(DAR3+)。In some embodiments, about 5%, 10%, 15%, 20%, 25%, 30% or more of the complexes in the purified composition of complexes comprise a protein (e.g., an antibody) covalently linked to two oligonucleotides (DAR2). In some embodiments, about 1%, 2%, 3%, 5%, 7%, 10%, 20% or more of the complexes in the purified composition of complexes comprise a protein (e.g., an antibody) covalently linked to three or more oligonucleotides (DAR3+).
III.复合物III. Complex
在一些方面中，本文中提供了包含与分子载荷(例如，寡核苷酸)共价连接的靶向剂(例如，抗体)的复合物。在一些实施方案中，复合物包含与寡核苷酸共价连接的肌肉靶向抗体。复合物可包含特异性结合单个抗原位点或结合可存在于相同或不同抗原上的至少两个抗原位点的抗体。复合物可用于调节至少一种基因、蛋白质和/或核酸的活性或功能。在一些实施方案中，与复合物一起存在的分子载荷负责基因、蛋白质和/或核酸的调节。分子载荷可以是能够调节细胞中基因、蛋白质和/或核酸的活性或功能的小分子、蛋白质、核酸、寡核苷酸或任何分子实体。在一些实施方案中，分子载荷是靶向肌细胞中肌肉疾病等位基因的寡核苷酸。In some aspects, provided herein are complexes comprising a targeting agent (e.g., an antibody) covalently linked to a molecular payload (e.g., an oligonucleotide). In some embodiments, the complex comprises a muscle targeting antibody covalently linked to an oligonucleotide. The complex may comprise an antibody that specifically binds to a single antigenic site or binds to at least two antigenic sites that may be present on the same or different antigens. The complex can be used to regulate the activity or function of at least one gene, protein, and/or nucleic acid. In some embodiments, the molecular payload present with the complex is responsible for the regulation of genes, proteins, and/or nucleic acids. The molecular payload can be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity that can regulate the activity or function of a gene, protein, and/or nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets a muscle disease allele in a muscle cell.
在一些实施方案中，复合物包含与分子载荷(例如靶向肌肉疾病等位基因的反义寡核苷酸)共价连接的肌肉靶向剂，例如抗转铁蛋白受体抗体。In some embodiments, the complex comprises a muscle targeting agent, such as an anti-transferrin receptor antibody, covalently linked to a molecular payload, such as an antisense oligonucleotide that targets a muscle disease allele.
在一些实施方案中，复合物可用于治疗肌肉疾病，其中分子载荷影响表1中提供的相应基因的活性。例如，取决于病症，分子载荷可调节(例如，降低、提高)基因的转录或表达，调节由基因编码的蛋白质的表达，或调节编码的蛋白质的活性。在一些实施方案中，分子载荷是寡核苷酸，其包含具有与表1中提供的靶基因互补的区域的链。In some embodiments, the complex can be used to treat muscle diseases, wherein the molecular payload affects the activity of the corresponding gene provided in Table 1. For example, depending on the condition, the molecular payload can modulate (e.g., reduce, increase) the transcription or expression of the gene, modulate the expression of the protein encoded by the gene, or modulate the activity of the encoded protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to the target gene provided in Table 1.
表1-肌肉疾病和相应基因的列表。Table 1 - List of muscle diseases and corresponding genes.
A.细胞靶向剂A. Cell Targeting Agents
本公开内容的一些方面提供了细胞靶向剂(例如，肌肉靶向蛋白)以例如用于将寡核苷酸递送至肌细胞。在一些实施方案中，这样的细胞靶向蛋白能够与特定细胞结合(例如，通过与所述细胞上的抗原特异性结合)并且将缔合的寡核苷酸递送至所述细胞。在一些实施方案中，寡核苷酸与细胞靶向剂结合(例如，共价结合)，并且在细胞靶向剂与细胞上的抗原结合后将寡核苷酸内化到所述细胞中(例如，通过内吞作用)。Some aspects of the present disclosure provide cell targeting agents (e.g., muscle targeting proteins) for, e.g., delivering oligonucleotides to muscle cells. In some embodiments, such cell targeting proteins are capable of binding to specific cells (e.g., by specifically binding to an antigen on the cell) and delivering the associated oligonucleotide to the cell. In some embodiments, the oligonucleotide is bound to the cell targeting agent (e.g., covalently bound), and the oligonucleotide is internalized into the cell (e.g., by endocytosis) after the cell targeting agent binds to an antigen on the cell.
本公开内容的一些方面提供了肌肉靶向剂，例如用于将分子载荷递送至肌细胞。在一些实施方案中，这样的肌肉靶向剂能够例如通过与肌细胞上的抗原特异性结合而与肌细胞结合，并且将缔合的分子载荷递送至肌细胞。在一些实施方案中，分子载荷与肌肉靶向剂结合(例如，共价结合)，并且在肌肉靶向剂与肌细胞上的抗原结合后内化到肌细胞中，例如通过内吞作用。示例性的肌肉靶向剂在本文中进一步详细描述，然而，应理解，本文中提供的示例性肌肉靶向剂并不意味着是限制性的。应理解，根据本公开内容，可使用多种类型的肌肉靶向剂，并且通过本文中所述的任何类型的肌肉靶向剂均可靶向任何肌肉靶标(例如，肌肉表面蛋白)。例如，肌肉靶向剂可包括以下或由以下组成：小分子、核酸(例如，DNA或RNA)、肽(例如，抗体)、脂质(例如，微泡)或糖部分(例如，多糖)。Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, by specifically binding to antigens on muscle cells, and deliver the associated molecular payload to muscle cells. In some embodiments, the molecular payload is bound to the muscle targeting agent (e.g., covalently bound), and is internalized into the muscle cell after the muscle targeting agent binds to the antigen on the muscle cell, for example, by endocytosis. Exemplary muscle targeting agents are further described in detail herein, however, it should be understood that the exemplary muscle targeting agents provided herein are not meant to be limiting. It should be understood that, according to the present disclosure, various types of muscle targeting agents can be used, and any muscle target (e.g., muscle surface protein) can be targeted by any type of muscle targeting agent described herein. For example, a muscle targeting agent may include or consist of a small molecule, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide).
本公开内容的一些方面提供了与肌肉(例如，骨骼肌、平滑肌或心肌)上的抗原特异性结合的肌肉靶向剂。在一些实施方案中，本文中提供的任何肌肉靶向剂均与骨骼肌细胞、平滑肌细胞和/或心肌细胞上的抗原结合(例如，特异性结合)。Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscles (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, any muscle targeting agent provided herein binds to (e.g., specifically binds to) antigens on skeletal muscle cells, smooth muscle cells, and/or cardiac muscle cells.
通过与肌肉特异性细胞表面识别元件(例如，细胞膜蛋白)相互作用，可实现组织定位和选择性摄取到肌细胞中二者。在一些实施方案中，作为肌肉摄取转运体之底物的分子可用于将分子载荷(例如，寡核苷酸)递送到肌肉组织中。与肌肉表面识别元件结合之后是胞吞作用，其可允许甚至大分子(例如，抗体)进入肌细胞。作为另一个实例，与转铁蛋白或抗转铁蛋白受体抗体缀合的寡核苷酸可通过与转铁蛋白受体结合而被肌细胞摄取，然后可例如通过网格蛋白介导的内吞作用内吞。By interacting with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters can be used to deliver molecular payloads (e.g., oligonucleotides) into muscle tissue. Binding to muscle surface recognition elements is followed by endocytosis, which allows even macromolecules (e.g., antibodies) to enter muscle cells. As another example, oligonucleotides conjugated to transferrin or anti-transferrin receptor antibodies can be taken up by muscle cells by binding to transferrin receptors, and can then be internalized, for example, by clathrin-mediated endocytosis.
肌肉靶向剂的使用可用于将分子载荷(例如，寡核苷酸)集中在肌肉中，同时降低与其他组织中的作用相关的毒性。在一些实施方案中，与对象内的另一种细胞类型相比，肌肉靶向剂将结合的分子载荷集中在肌细胞中。在一些实施方案中，肌肉靶向剂将结合的分子载荷以比非肌细胞(例如，肝、神经元、血液或脂肪细胞)中的量最高为少1、2、3、4、5、6、7、8、9、10、15、20、30、40、50、60、70、80、90或100倍的量集中在肌细胞(例如，骨骼肌、平滑肌或心肌细胞)中。在一些实施方案中，当分子载荷在与肌肉靶向剂结合时递送至对象时，其在对象中的毒性降低至少1％、2％、3％、4％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、90％或95％。The use of muscle targeting agents can be used to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells compared to another cell type within the subject. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells (e.g., skeletal muscle, smooth muscle, or cardiomyocytes) in an amount up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times less than in non-muscle cells (e.g., liver, neurons, blood, or adipocytes). In some embodiments, when the molecular payload is delivered to a subject in conjunction with a muscle targeting agent, its toxicity in the subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%.
在一些实施方案中，为了实现肌肉选择性，可能需要肌肉识别元件(例如，肌细胞抗原)。作为一个实例，肌肉靶向剂可以是为肌肉特异性摄取转运体之底物的小分子。作为另一个实例，肌肉靶向剂可以是通过转运体介导的内吞作用进入肌细胞的抗体。作为另一个实例，肌肉靶向剂可以是与肌细胞上的细胞表面受体结合的配体。应理解，尽管基于转运体的方法为细胞进入提供了直接途径，但是基于受体的靶向可能涉及刺激的胞吞作用以达到期望的作用部位。In some embodiments, a muscle recognition element (e.g., a myocyte antigen) may be required to achieve muscle selectivity. As an example, a muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, a muscle targeting agent may be an antibody that enters myocytes via transporter-mediated endocytosis. As another example, a muscle targeting agent may be a ligand that binds to a cell surface receptor on myocytes. It should be understood that while transporter-based approaches provide a direct route for cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.
本公开内容涵盖的肌细胞包括但不限于骨骼肌细胞、平滑肌细胞、心肌细胞、成肌细胞和肌细胞。Myocytes encompassed by the present disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiomyocytes, myoblasts, and myocytes.
i.肌肉靶向抗体i. Muscle-targeted antibodies
在一些实施方案中，肌肉靶向剂是抗体。通常来说，抗体对其靶抗原的高特异性提供了用于选择性靶向肌细胞(例如骨骼肌、平滑肌和/或心肌细胞)的潜力。这种特异性也可限制脱靶毒性。能够靶向肌细胞表面抗原的抗体的实例已经报道并且在本公开内容的范围内。例如，靶向肌细胞表面的抗体在以下中有描述：Arahata K.,et al.“Immunostainingof skeletal and cardiac muscle surface membrane with antibody againstDuchenne muscular dystrophy peptide”Nature 1988；333:861-3；Song K.S.,et al.“Expression of caveolin-3in skeletal,cardiac,and smooth musclecells.Caveolin-3is a component of the sarcolemma and co-fractionates withdystrophin and dystrophin-associated glycoproteins”J Biol Chem 1996；271:15160-5；以及Weisbart R.H.et al.,“Cell type specific targeted intracellulardelivery into muscle of a monoclonal antibody that binds myosin IIb”MolImmunol.2003Mar,39(13):78309；其各自的全部内容均通过引用并入本文。In some embodiments, the muscle targeting agent is an antibody. Generally speaking, the high specificity of antibodies to their target antigens provides the potential for selective targeting of muscle cells (e.g., skeletal muscle, smooth muscle, and/or cardiomyocytes). This specificity can also limit off-target toxicity. Examples of antibodies that can target muscle cell surface antigens have been reported and are within the scope of the present disclosure. For example, antibodies targeting the surface of muscle cells are described in: Arahata K., et al. "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988; 333: 861-3; Song K.S., et al. "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996; 271: 15160-5; and Weisbart R.H. et al., "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar, 39(13):78309; the entire contents of each of which are incorporated herein by reference.
a.抗转铁蛋白受体抗体a. Anti-transferrin receptor antibodies
本公开内容的一些方面是基于这样的认识：与转铁蛋白受体结合的物质(例如，抗转铁蛋白受体抗体)能够靶向肌细胞。转铁蛋白受体是内化细胞表面受体，其通过细胞膜转运转铁蛋白并参与胞内铁水平的调节和稳态。本公开内容的一些方面提供了能够与转铁蛋白受体结合的转铁蛋白受体结合蛋白。因此，本公开内容的方面提供了与转铁蛋白受体结合的结合蛋白(例如，抗体)。在一些实施方案中，与转铁蛋白受体结合的结合蛋白与任何结合的分子载荷(例如，寡核苷酸)一起被内化到肌细胞中。如本文中使用的，与转铁蛋白受体结合的抗体可称为抗转铁蛋白受体抗体。与转铁蛋白受体结合(例如，特异性结合)的抗体可在与转铁蛋白受体结合后例如通过受体介导的内吞作用而被内化到细胞中。Some aspects of the present disclosure are based on such recognition: substances (e.g., anti-transferrin receptor antibodies) that bind to transferrin receptors can target myocytes. Transferrin receptors are internalized cell surface receptors that transfer transferrin through the cell membrane and participate in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-binding proteins that can bind to transferrin receptors. Therefore, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, the binding proteins that bind to transferrin receptors are internalized into myocytes together with any molecular payload (e.g., oligonucleotides) that bind. As used herein, antibodies that bind to transferrin receptors can be referred to as anti-transferrin receptor antibodies. Antibodies that bind to transferrin receptors (e.g., specifically bind) can be internalized into cells after binding to transferrin receptors, for example, by receptor-mediated endocytosis.
应理解，可使用数种已知的方法(例如使用噬菌体展示的文库设计)来产生、合成和/或衍生抗转铁蛋白受体抗体。示例性方法已经在本领域中表征并且通过引用并入(Díez,P.et al.“High-throughput phage-display screening in array format”,Enzymeand microbial technology,2015,79,34-41.；Christoph M.H.and Stanley,J.R.“Antibody Phage Display:Technique and Applications”J Invest Dermatol.2014,134:2.；Engleman,Edgar(Ed.)“Human Hybridomas and Monoclonal Antibodies.”1985,Springer)。在另一些实施方案中，抗转铁蛋白抗体先前已被表征或公开。与转铁蛋白受体特异性结合的抗体是本领域中已知的(参见，例如1979年12月4日提交的美国专利No.4,364,934，“Monoclonal antibody to a human early thymocyte antigen and methodsfor preparing same”；2006年6月14日提交的美国专利No.8,409,573，“Anti-CD71monoclonal antibodies and uses thereof for treating malignant tumor cells”；2014年5月20日提交的美国专利No.9,708,406，“Anti-transferrin receptor antibodiesand methods of use”；2014年12月19日提交的US 9,611,323，“Low affinity bloodbrain barrier receptor antibodies and uses therefor”；2014年12月24日提交的WO2015/098989，“Novel anti-Transferrin receptor antibody that passes throughblood-brain barrier”；Schneider C.et al.“Structural features of the cellsurface receptor for transferrin that is recognized by the monoclonalantibody OKT9.”J Biol Chem.1982,257:14,8516-8522.；Lee et al.“Targeting RatAnti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-BrainBarrier in Mouse”2000,J Pharmacol.Exp.Ther.,292:1048-1052)。It should be understood that several known methods (e.g., library design using phage display) can be used to produce, synthesize and/or derive anti-transferrin receptor antibodies. Exemplary methods have been characterized in the art and incorporated by reference (Díez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph M. H. and Stanley, J. R. "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134: 2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies." 1985, Springer). In other embodiments, anti-transferrin antibodies have been previously characterized or disclosed. Antibodies that specifically bind to transferrin receptor are known in the art (see, e.g., U.S. Patent No. 4,364,934, filed December 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Patent No. 8,409,573, filed June 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Patent No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; US 9,611,323, filed December 19, 2014, "Low affinity bloodbrain barrier receptor antibodies and uses therefor"; WO 2015/098989, filed December 24, 2014, "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al. al. "Structural features of the cellsurface receptor for transferrin that is recognized by the monoclonalantibody OKT9." J Biol Chem.1982,257:14,8516-8522.; Lee et al. 1052).
任何合适的抗转铁蛋白受体抗体都可用于本文公开的复合物中。表2中列出了抗转铁蛋白受体抗体的实例，包括相关参考文献和结合表位。在一些实施方案中，抗转铁蛋白受体抗体包含本文中提供的任何抗转铁蛋白受体抗体(例如表2列出的抗转铁蛋白受体抗体)的互补决定区(CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和CDR-L3)。Any suitable anti-transferrin receptor antibody can be used in the complex disclosed herein. Examples of anti-transferrin receptor antibodies are listed in Table 2, including relevant references and binding epitopes. In some embodiments, the anti-transferrin receptor antibody comprises the complementary determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of any anti-transferrin receptor antibody provided herein (e.g., the anti-transferrin receptor antibody listed in Table 2).
表2-抗转铁蛋白受体抗体克隆的列表，包括相关参考文献和结合表位信息。Table 2 - List of anti-transferrin receptor antibody clones, including relevant references and binding epitope information.
在一些实施方案中，肌肉靶向剂是抗转铁蛋白受体抗体。在一些实施方案中，抗转铁蛋白受体抗体与具有如本文公开的氨基酸序列的转铁蛋白蛋白质特异性结合。在一些实施方案中，抗转铁蛋白受体抗体可与转铁蛋白受体的任何胞外表位或开始暴露于抗体的表位(包括顶端结构域、转铁蛋白结合结构域和蛋白酶样结构域)特异性结合。在一些实施方案中，抗转铁蛋白受体抗体与人或非人灵长类转铁蛋白受体的氨基酸片段(如SEQ ID NO.1至3中提供的)在氨基酸C89至F760的范围内结合。在一些实施方案中，转铁蛋白受体抗体以至少约10-4M、10-5M、10-6M、10-7M、10-8M、10-9M、10-10M、10-11M、10-12M、10-13M或更小的结合亲和力特异性结合。本文使用的抗转铁蛋白受体抗体可能够与其他抗转铁蛋白受体抗体(例如OKT9、8D3)竞争结合，后者抗体以10-3M、10-4M、10-5M、10-6M、10-7M或更小与转铁蛋白受体结合。In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody. In some embodiments, the anti-transferrin receptor antibody specifically binds to a transferrin protein having an amino acid sequence as disclosed herein. In some embodiments, the anti-transferrin receptor antibody can specifically bind to any extracellular epitope of the transferrin receptor or an epitope (including the apical domain, transferrin binding domain, and protease-like domain) that begins to be exposed to the antibody. In some embodiments, the anti-transferrin receptor antibody binds to an amino acid fragment of a transferrin receptor of a human or non-human primate (such as provided in SEQ ID NO.1 to 3) in the range of amino acid C89 to F760. In some embodiments, the transferrin receptor antibody specifically binds with a binding affinity of at least about 10-4 M, 10-5 M, 10-6 M, 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, 10-13 M or less. The anti-transferrin receptor antibodies used herein may be capable of competing for binding with other anti-transferrin receptor antibodies (eg, OKT9, 8D3) that bind to transferrin receptor at10-3 M,10-4 M,10-5 M,10-6 M,10-7 M or less.
对应于NCBI序列NP_003225.2(转铁蛋白受体蛋白1同工型1，智人)的示例性人转铁蛋白受体氨基酸序列如下：An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, Homo sapiens) is as follows:
对应于NCBI序列NP_001244232.1(转铁蛋白受体蛋白1，恒河猴的示例性非人灵长类转铁蛋白受体氨基酸序列如下：An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1 (transferrin receptor protein 1, rhesus monkey) is as follows:
对应于NCBI序列XP_005545315.1(转铁蛋白受体蛋白1，食蟹猴)的示例性非人灵长类转铁蛋白受体氨基酸序列如下：An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, cynomolgus monkey) is as follows:
对应于NCBI序列NP_001344227.1(转铁蛋白受体蛋白1，小家鼠)的示例性鼠转铁蛋白受体氨基酸序列如下：An exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Mus musculus) is as follows:
在一些实施方案中，抗转铁蛋白受体抗体与如下的受体氨基酸区段结合：In some embodiments, the anti-transferrin receptor antibody binds to the following receptor amino acid segment:
并且不抑制转铁蛋白受体与转铁蛋白和/或人血色素沉着蛋白(也称为HFE)之间的结合相互作用。And does not inhibit the binding interaction between transferrin receptor and transferrin and/or human hemochromatin (also known as HFE).
可使用适当的方法来获得和/或产生抗体、抗体片段或抗原结合剂，例如，通过使用重组DNA方案。在一些实施方案中，也可通过杂交瘤的产生来产生抗体(参见，例如，Kohler,G and Milstein,C.“Continuous cultures of fused cells secretingantibody of predefined specificity”Nature,1975,256:495-497)。目的抗原可以任何形式或实体(例如重组或天然形式或实体)用作免疫原。使用标准方法(例如ELISA筛选)筛选杂交瘤，以发现至少一种产生靶向特定抗原之抗体的杂交瘤。也可通过筛选表达抗体的蛋白质表达文库(例如噬菌体展示文库)来产生抗体。在一些实施方案中，也可使用噬菌体展示文库设计(参见，例如，1991年3月1日提交的美国专利No 5,223,409，“Directedevolution of novel binding proteins”；1992年4月10日提交的WO 1992/18619，“Heterodimeric receptor libraries using phagemids”；1991年5月1日提交的WO 1991/17271，“Recombinant library screening methods”；1992年5月15日提交的WO 1992/20791，“Methods for producing members of specific binding pairs”；1992年2月28日提交的WO 1992/15679，“Improved epitope displaying phage”)。在一些实施方案中，目的抗原可用于对非人动物，例如啮齿动物或山羊进行免疫接种。在一些实施方案中，然后从非人动物获得抗体，并且可任选地使用多种方法(例如使用重组DNA技术)对其进行修饰。抗体产生和方法的其他实例是本领域已知的(参见，例如Harlow et al.“Antibodies:ALaboratory Manual”,Cold Spring Harbor Laboratory,1988.)。Appropriate methods can be used to obtain and/or produce antibodies, antibody fragments or antigen binding agents, for example, by using recombinant DNA protocols. In some embodiments, antibodies can also be produced by the production of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secretingantibody of predefined specificity" Nature, 1975, 256: 495-497). The target antigen can be used as an immunogen in any form or entity (e.g., recombinant or natural form or entity). Hybridomas are screened using standard methods (e.g., ELISA screening) to find at least one hybridoma that produces antibodies targeting specific antigens. Antibodies can also be produced by screening protein expression libraries (e.g., phage display libraries) that express antibodies. In some embodiments, phage display library design can also be used (see, e.g., U.S. Patent No 5,223,409, filed March 1, 1991, "Directed evolution of novel binding proteins"; WO 1992/18619, filed April 10, 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991/17271, filed May 1, 1991, "Recombinant library screening methods"; WO 1992/20791, filed May 15, 1992, "Methods for producing members of specific binding pairs"; WO 1992/15679, filed February 28, 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest can be used to immunize non-human animals, such as rodents or goats. In some embodiments, antibodies are then obtained from non-human animals and optionally modified using a variety of methods (e.g., using recombinant DNA technology). Other examples of antibody production and methods are known in the art (see, e.g., Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988.).
在一些实施方案中，对抗体进行修饰，例如，通过糖基化、磷酸化、SUMO化和/或甲基化进行修饰。在一些实施方案中，抗体是与一个或更多个糖或碳水化合物分子缀合的糖基化抗体。在一些实施方案中，一个或更多个糖或碳水化合物分子通过N-糖基化、O-糖基化、C-糖基化、糖基磷脂酰肌醇化(GPI锚定附着)和/或磷酸糖基化与抗体缀合。在一些实施方案中，一个或更多个糖或碳水化合物分子是单糖、二糖、寡糖或聚糖。在一些实施方案中，一个或更多个糖或碳水化合物分子是支链的寡糖或支链的聚糖。在一些实施方案中，一个或更多个糖或碳水化合物分子包含甘露糖单元、葡萄糖单元、N-乙酰葡糖胺单元、N-乙酰半乳糖胺单元、半乳糖单元、岩藻糖单元或磷脂单元。在一些实施方案中，存在约1至10、约1至5、约5至10、约1至4、约1至3或约2个糖分子。在一些实施方案中，糖基化抗体是完全或部分糖基化的。在一些实施方案中，通过化学反应或通过酶促手段使抗体糖基化。在一些实施方案中，抗体在体外或细胞内被糖基化，其可任选地缺少N-或O-糖基化途径中的酶，例如糖基转移酶。在一些实施方案中，用糖或碳水化合物分子对抗体进行官能化，如2014年5月1日公开的标题为“Modified antibody,antibody-conjugate and process for thepreparation thereof”的国际专利申请公开WO2014065661中所述。In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and/or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and/or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3 or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack enzymes in the N- or O-glycosylation pathway, such as glycosyltransferases. In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules, as described in International Patent Application Publication WO2014065661 entitled "Modified antibody, antibody-conjugate and process for the preparation thereof" published on May 1, 2014.
本公开内容的一些方面提供了与转铁蛋白受体(例如，转铁蛋白受体的胞外部分)结合的蛋白质。在一些实施方案中，本文中提供的转铁蛋白受体抗体与转铁蛋白受体(例如，人转铁蛋白受体)特异性结合。转铁蛋白受体是内化细胞表面受体，其通过细胞膜来转运转铁蛋白并参与细胞内铁水平的调节和稳态。在一些实施方案中，本文中提供的转铁蛋白受体抗体与来自人、非人灵长类、小鼠、大鼠等的转铁蛋白受体特异性结合。在一些实施方案中，本文中提供的转铁蛋白受体抗体与人转铁蛋白受体结合。在一些实施方案中，本文中提供的转铁蛋白受体抗体与人转铁蛋白受体特异性结合。在一些实施方案中，本文中提供的转铁蛋白受体抗体与人转铁蛋白受体的顶端结构域结合。在一些实施方案中，本文中提供的转铁蛋白受体抗体与人转铁蛋白受体的顶端结构域特异性结合。Some aspects of the present disclosure provide proteins that are combined with transferrin receptor (e.g., the extracellular part of transferrin receptor). In some embodiments, the transferrin receptor antibody provided herein specifically binds to transferrin receptor (e.g., human transferrin receptor). Transferrin receptor is an internalized cell surface receptor that transfers transferrin through the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. In some embodiments, the transferrin receptor antibody provided herein specifically binds to transferrin receptor from people, non-human primates, mice, rats, etc. In some embodiments, the transferrin receptor antibody provided herein binds to human transferrin receptor. In some embodiments, the transferrin receptor antibody provided herein specifically binds to human transferrin receptor. In some embodiments, the transferrin receptor antibody provided herein binds to the apical domain of human transferrin receptor. In some embodiments, the transferrin receptor antibody provided herein specifically binds to the apical domain of human transferrin receptor.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含选自表2的任一种抗转铁蛋白受体抗体的一个或更多个CDR-H(例如，CDR-H1、CDR-H2和CDR-H3)氨基酸序列。在一些实施方案中，转铁蛋白受体抗体包含为选自表2的任一种抗转铁蛋白受体抗体提供的CDR-H1、CDR-H2和CDR-H3。在一些实施方案中，转铁蛋白受体抗体包含为选自表2的任一种抗转铁蛋白受体抗体提供的CDR-L1、CDR-L2和CDR-L3。在一些实施方案中，转铁蛋白受体抗体包含为选自表2的任一种抗转铁蛋白受体抗体提供的CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和CDR-L3。本公开内容还包含编码包含为选自表2的任一种抗转铁蛋白受体抗体提供的CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2或CDR-L3的分子的任何核酸序列。在一些实施方案中，抗体重链和轻链CDR3结构域可能在抗体对抗原的结合特异性/亲和力中发挥特别重要的作用。因此，本公开内容的抗转铁蛋白受体抗体可至少包含选自表2的任一种抗转铁蛋白受体抗体的重链和/或轻链CDR3。In some embodiments, the transferrin receptor antibodies of the present disclosure comprise one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences selected from any one of the anti-transferrin receptor antibodies of Table 2. In some embodiments, the transferrin receptor antibodies comprise CDR-H1, CDR-H2, and CDR-H3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the transferrin receptor antibodies comprise CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the transferrin receptor antibodies comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 2. The present disclosure also includes any nucleic acid sequence encoding a molecule comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 provided for any anti-transferrin receptor antibody selected from Table 2. In some embodiments, antibody heavy chain and light chain CDR3 domains may play a particularly important role in the binding specificity/affinity of the antibody to the antigen. Therefore, the anti-transferrin receptor antibody of the present disclosure may at least comprise a heavy chain and/or light chain CDR3 of any anti-transferrin receptor antibody selected from Table 2.
在一些实例中，本公开内容的任何抗转铁蛋白受体抗体均具有与选自表2的一种抗转铁蛋白受体抗体的任何CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和/或CDR-L3序列基本上相似的一个或更多个CDR(例如，CDR-H或CDR-L)。在一些实施方案中，本文中所述的抗体的一个或更多个CDR沿着VH(例如，CDR-H1、CDR-H2或CDR-H3)和/或VL(例如，CDR-L1、CDR-L2或CDR-L3)区的位置可改变一个、两个、三个、四个、五个或六个氨基酸位置，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，基本上维持了其所来源原始抗体之结合的例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。例如，在一些实施方案中，限定本文中所述的任何抗体的CDR的位置可通过将CDR的N端和/或C端边界相对于本文中所述的任一种抗体的CDR位置移动一个、两个、三个、四个、五个或六个氨基酸来改变，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，基本上维持了其所来源原始抗体之结合的例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在另一个实施方案中，本文中所述的抗体的一个或更多个CDR沿着VH(例如，CDR-H1、CDR-H2或CDR-H3)和/或VL(例如，CDR-L1、CDR-L2或CDR-L3)的长度可改变(例如变得更短或更长)一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，基本上维持了其所来源原始抗体之结合的例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。In some examples, any anti-transferrin receptor antibody of the present disclosure has one or more CDRs (e.g., CDR-H or CDR-L) that are substantially similar to any CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequence of an anti-transferrin receptor antibody selected from Table 2. In some embodiments, the position of one or more CDRs of the antibodies described herein along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and/or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions may be changed by one, two, three, four, five, or six amino acid positions, as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). For example, in some embodiments, the positions of the CDRs defining any of the antibodies described herein can be changed by moving the N-terminal and/or C-terminal boundaries of the CDRs relative to the positions of the CDRs of any of the antibodies described herein by one, two, three, four, five, or six amino acids, so long as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived). In another embodiment, the length of one or more CDRs of the antibodies described herein along the VH (e.g., CDR-H1, CDR-H2 or CDR-H3) and/or VL (e.g., CDR-L1, CDR-L2 or CDR-L3) can be changed (e.g., made shorter or longer) by one, two, three, four, five or more amino acids, as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).
因此，在一些实施方案中，本文中所述的CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和/或CDR-H3可比本文中所述的一个或更多个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)短一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在一些实施方案中，本文中所述的CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和/或CDR-H3可比本文中所述的一个或更多个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)长一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在一些实施方案中，与本文中所述的一个或更多个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)相比，本文中所述的CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和/或CDR-H3的氨基部分可延长一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在一些实施方案中，与本文中所述的一个或更多个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)相比，本文中所述的CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和/或CDR-H3的羧基部分可延长一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在一些实施方案中，与本文中所述的一个或更多个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)相比，本文中所述的CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和/或CDR-H3的氨基部分可缩短一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在一些实施方案中，与本文中所述的一个或更多个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)相比，本文中所述的CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和/或CDR-H3的羧基部分可缩短一个、两个、三个、四个、五个或更多个氨基酸，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。可使用任何方法来确定是否维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合，例如使用本领域描述的结合测定和条件。Thus, in some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3 described herein may be shorter than one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2) by one, two, three, four, five or more amino acids, as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3 described herein may be one, two, three, four, five or more amino acids longer than one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., a CDR of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., a CDR of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., a CDR of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and/or CDR-H3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2), as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). Any method can be used to determine whether immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained, such as using binding assays and conditions described in the art.
在一些实例中，本公开内容的任何抗转铁蛋白受体抗体均具有与选自选自表2的任一种抗转铁蛋白受体抗体基本上相似的一个或更多个CDR(例如CDR-H或CDR-L)序列。例如，抗体可包含选自表2的任何抗转铁蛋白受体抗体的一个或更多个CDR序列，其与本文中提供的任一个CDR(例如选自表2的任何抗转铁蛋白受体抗体的CDR)的相应CDR区相比包含多至5、4、3、2或1个氨基酸残基变异，只要维持了与转铁蛋白受体(例如，人转铁蛋白受体)的免疫特异性结合(例如，相对于其所来源原始抗体的结合，基本上维持了例如至少50％、至少60％、至少70％、至少80％、至少90％、至少95％)即可。在一些实施方案中，本文中提供的任何CDR中的任何氨基酸变异均可以是保守变异。保守变异可在残基不太可能参与与转铁蛋白受体蛋白(例如人转铁蛋白受体蛋白)相互作用的位置(例如如基于晶体结构确定的)处引入CDR中。本公开内容的一些方面提供了转铁蛋白受体抗体，其包含本文中提供的一个或更多个重链可变(VH)和/或轻链可变(VL)结构域。在一些实施方案中，本文中提供的任何VH结构域均包含本文中提供的一个或更多个CDR-H序列(例如，CDR-H1、CDR-H2和CDR-H3)，例如选自表2的任一种抗转铁蛋白受体抗体中提供的任何CDR-H序列。在一些实施方案中，本文中提供的任何VL结构域均包含本文中提供的一个或更多个CDR-L序列(例如，CDR-L1、CDR-L2和CDR-L3)，例如选自表2的任一种抗转铁蛋白受体抗体中提供的任何CDR-L序列。In some examples, any anti-transferrin receptor antibody of the present disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences that are substantially similar to any anti-transferrin receptor antibody selected from Table 2. For example, the antibody may include one or more CDR sequences of any anti-transferrin receptor antibody selected from Table 2, which contain up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR region of any CDR provided herein (e.g., CDRs of any anti-transferrin receptor antibody selected from Table 2), as long as the immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, any amino acid variation in any CDR provided herein may be a conservative variation. Conservative variation can be introduced into CDR at a position (e.g., as determined based on a crystal structure) where the residue is unlikely to participate in interacting with a transferrin receptor protein (e.g., a human transferrin receptor protein). Some aspects of the present disclosure provide transferrin receptor antibodies comprising one or more heavy chain variable (VH) and/or light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein comprises one or more CDR-H sequences (e.g., CDR-H1, CDR-H2, and CDR-H3) provided herein, such as any CDR-H sequence provided in any anti-transferrin receptor antibody selected from Table 2. In some embodiments, any VL domain provided herein comprises one or more CDR-L sequences (e.g., CDR-L1, CDR-L2, and CDR-L3) provided herein, such as any CDR-L sequence provided in any anti-transferrin receptor antibody selected from Table 2.
在一些实施方案中，本公开内容的抗转铁蛋白受体抗体包括包含任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的重链可变结构域和/或轻链可变结构域的任何抗体。在一些实施方案中，本公开内容的抗转铁蛋白受体抗体包括包含任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的重链可变和轻链可变对的任何抗体。In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibodies comprising a heavy chain variable domain and/or a light chain variable domain of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibodies comprising a heavy chain variable and a light chain variable pair of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2).
本公开内容的一些方面提供了抗转铁蛋白受体抗体，其具有与本文中所述的那些中的任何同源的重链可变(VH)和/或轻链可变(VL)结构域氨基酸序列。在一些实施方案中，抗转铁蛋白受体抗体包含与任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的重链可变序列和/或轻链可变序列具有至少75％(例如，80％、85％、90％、95％、98％或99％)同一性的重链可变序列或轻链可变序列。在一些实施方案中，同源重链可变和/或轻链可变氨基酸序列在本文中提供的任何CDR序列内均不变化。例如，在一些实施方案中，序列变异的程度(例如，75％、80％、85％、90％、95％、98％或99％)可发生在本文中提供的不包括任何CDR序列的重链可变和/或轻链可变序列中。在一些实施方案中，本文中提供的任何抗转铁蛋白受体抗体包含重链可变序列和轻链可变序列，其包含与任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的框架区序列具有至少75％、80％、85％、90％、95％、98％或99％同一性的框架区序列。Some aspects of the present disclosure provide anti-transferrin receptor antibodies, which have heavy chain variable (VH) and/or light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, anti-transferrin receptor antibodies include heavy chain variable sequences and/or light chain variable sequences with at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identity with any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, homologous heavy chain variable and/or light chain variable amino acid sequences do not change in any CDR sequence provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) may occur in the heavy chain variable and/or light chain variable sequences not including any CDR sequence provided herein. In some embodiments, any anti-transferrin receptor antibody provided herein comprises a heavy chain variable sequence and a light chain variable sequence comprising a framework region sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the framework region sequence of any anti-transferrin receptor antibody (e.g., any one of the anti-transferrin receptor antibodies selected from Table 2).
在一些实施方案中，与转铁蛋白受体(例如，人转铁蛋白受体)特异性结合的抗转铁蛋白受体抗体包含轻链可变VL结构域，所述轻链可变VL结构域包含选自表2的任何抗转铁蛋白受体抗体的任何CDR-L结构域(CDR-L1、CDR-L2和CDR-L3)，或本文中提供的CDR-L结构域变体。在一些实施方案中，与转铁蛋白受体(例如，人转铁蛋白受体)特异性结合的抗转铁蛋白受体抗体包含轻链可变VL结构域，所述轻链可变VL结构域包含任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的CDR-L1、CDR-L2和CDR-L3。在一些实施方案中，抗转铁蛋白受体抗体包含轻链可变(VL)区序列，所述轻链可变(VL)区序列包含任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的轻链可变区序列的一个、两个、三个或四个框架区。在一些实施方案中，抗转铁蛋白受体抗体包含轻链可变区序列的一个、两个、三个或四个框架区，其与任何转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)轻链可变区序列的一个、两个、三个或四个框架区具有至少75％、80％、85％、90％、95％或100％同一性。在一些实施方案中，源自所述氨基酸序列的轻链可变框架区由所述氨基酸序列组成，但是存在多至10个氨基酸替换、缺失和/或插入，优选多至10个氨基酸替换。在一些实施方案中，源自所述氨基酸序列的轻链可变框架区由所述氨基酸序列组成，其中1、2、3、4、5、6、7、8、9或10个氨基酸残基替换存在于相应的非人灵长类或人轻链可变框架区的类似位置的氨基酸。In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising any CDR-L domain (CDR-L1, CDR-L2, and CDR-L3) of any anti-transferrin receptor antibody selected from Table 2, or a CDR-L domain variant provided herein. In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence, which comprises one, two, three or four framework regions of a light chain variable region sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibody comprises one, two, three or four framework regions of a light chain variable region sequence, which has at least 75%, 80%, 85%, 90%, 95% or 100% identity with one, two, three or four framework regions of a light chain variable region sequence of any transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, but there are up to 10 amino acid substitutions, deletions and/or insertions, preferably up to 10 amino acid substitutions. In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues replace amino acids present in analogous positions in the corresponding non-human primate or human light chain variable framework region.
在一些实施方案中，与转铁蛋白受体特异性结合的抗转铁蛋白受体抗体包含任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的CDR-L1、CDR-L2和CDR-L3。在一些实施方案中，抗体还包含源自人或灵长类的VL的一个、两个、三个或全部四个VL框架区。被选择用于本文中所述的轻链CDR序列的抗体的灵长类或人轻链框架区可与非人亲本抗体的轻链框架区具有例如至少70％(例如至少75％、80％、85％、90％、95％、98％或至少99％)同一性。所选择的灵长类或人抗体在其轻链互补决定区中的氨基酸编号可与本文中提供的任何抗体(例如选自表2的任何抗转铁蛋白受体抗体)的轻链互补决定区中的氨基酸编号相同或基本上相同。在一些实施方案中，灵长类或人轻链框架区氨基酸残基来自天然灵长类或人抗体轻链框架区，其与任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的轻链框架区具有至少75％同一性，至少80％同一性，至少85％同一性，至少90％同一性，至少95％同一性，至少98％同一性，至少99％(或更多)同一性。在一些实施方案中，抗转铁蛋白受体抗体还包含源自人轻链可变κ亚家族的一个、两个、三个或全部四个VL框架区。在一些实施方案中，抗转铁蛋白受体抗体还包含源自人轻链可变λ亚家族的一个、两个、三个或全部四个VL框架区。In some embodiments, the anti-transferrin receptor antibody that specifically binds to the transferrin receptor comprises CDR-L1, CDR-L2 and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the antibody further comprises one, two, three or all four VL framework regions of the VL derived from humans or primates. The primate or human light chain framework region of the antibody selected for the light chain CDR sequence described herein may have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98% or at least 99%) identity with the light chain framework region of the non-human parent antibody. The amino acid numbering of the selected primate or human antibody in its light chain complementary determining region may be identical or substantially identical to the amino acid numbering in the light chain complementary determining region of any antibody provided herein (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the primate or human light chain framework region amino acid residues are from a natural primate or human antibody light chain framework region, which has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity with the light chain framework region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2). In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three or all four VL framework regions derived from a human light chain variable κ subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three or all four VL framework regions derived from a human light chain variable λ subfamily.
在一些实施方案中，本文中提供的任何抗转铁蛋白受体抗体包含轻链可变结构域，其还包含轻链恒定区。在一些实施方案中，轻链恒定区是κ或λ轻链恒定区。在一些实施方案中，κ或λ轻链恒定区来自哺乳动物，例如来自人、猴、大鼠或小鼠。在一些实施方案中，轻链恒定区是人κ轻链恒定区。在一些实施方案中，轻链恒定区是人λ轻链恒定区。应理解，本文中提供的任何轻链恒定区均可以是本文中提供的任何轻链恒定区的变体。在一些实施方案中，轻链恒定区包含与任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的任何轻链恒定区具有至少75％、80％、85％、90％、95％、98％或99％同一性的氨基酸序列。In some embodiments, any anti-transferrin receptor antibody provided herein comprises a light chain variable domain, which further comprises a light chain constant region. In some embodiments, the light chain constant region is a κ or λ light chain constant region. In some embodiments, the κ or λ light chain constant region is from a mammal, such as from a human, monkey, rat or mouse. In some embodiments, the light chain constant region is a human κ light chain constant region. In some embodiments, the light chain constant region is a human λ light chain constant region. It should be understood that any light chain constant region provided herein can be a variant of any light chain constant region provided herein. In some embodiments, the light chain constant region comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with any light chain constant region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2).
在一些实施方案中，抗转铁蛋白受体抗体是任何抗转铁蛋白受体抗体，例如选自表2的任一种抗转铁蛋白受体抗体。In some embodiments, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2.
在一些实施方案中，抗转铁蛋白受体抗体包含VL结构域，所述VL结构域包含任何抗转铁蛋白受体抗体(例如选自表2的任一种抗转铁蛋白受体抗体)的氨基酸序列，并且其中恒定区包含IgG、IgE、IgM、IgD、IgA或IgY免疫球蛋白分子或人IgG、IgE、IgM、IgD、IgA或IgY免疫球蛋白分子的恒定区的氨基酸序列。在一些实施方案中，抗转铁蛋白受体抗体包含任何VL结构域或VL结构域变体，以及任何VH结构域或VH结构域变体，其中VL和VH结构域或其变体来自同一抗体克隆，并且其中恒定区包含IgG、IgE、IgM、IgD、IgA或IgY免疫球蛋白分子或者免疫球蛋白分子的任何类别(例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或任何亚类(例如，IgG2a和IgG2b)的恒定区的氨基酸序列。人恒定区的非限制性实例在本领域中描述，例如参见上文Kabat E A et al.,(1991)。In some embodiments, the anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 2), and wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises any VL domain or VL domain variant, and any VH domain or VH domain variant, wherein the VL and VH domains or variants thereof are from the same antibody clone, and wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule or any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule. Non-limiting examples of human constant regions are described in the art, see, e.g., Kabat EA et al., (1991), supra.
在一些实施方案中，本公开内容的抗体可以以相对高的亲和力与靶抗原(例如，转铁蛋白受体)结合，例如KD小于10-6M、10-7M、10-8M、10-9M、10-10M、10-11M或更小。例如，抗转铁蛋白受体抗体可以5pM至500nM、例如50pM至100nM、例如500pM至50nM的亲和力与转铁蛋白受体蛋白(例如，人转铁蛋白受体)结合。本公开内容还包括与本文中所述的任何抗体竞争与转铁蛋白受体蛋白(例如，人转铁蛋白受体)的结合并且亲和力为50nM或更小(例如，20nM或更小、10nM或更小、500pM或更小、50pM或更小或者5pM或更小)的抗体。可使用任何合适的方法来测试抗转铁蛋白受体抗体的亲和力和结合动力学，所述方法包括但不限于生物传感器技术(例如，OCTET或BIACORE)。In some embodiments, the antibodies of the present disclosure can bind to a target antigen (e.g., transferrin receptor) with a relatively high affinity, e.g.,KD less than10-6 M,10-7 M, 10-8 M,10-9 M,10-10 M,10-11 M or less. For example, an anti-transferrin receptor antibody can bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity of 5pM to 500nM, e.g., 50pM to 100nM, e.g., 500pM to 50nM. The present disclosure also includes antibodies that compete with any antibody described herein for binding to a transferrin receptor protein (e.g., human transferrin receptor) and have an affinity of 50nM or less (e.g., 20nM or less, 10nM or less, 500pM or less, 50pM or less, or 5pM or less). The affinity and binding kinetics of anti-transferrin receptor antibodies can be tested using any suitable method including, but not limited to, biosensor technology (eg, OCTET or BIACORE).
在一些实施方案中，本公开内容的抗体可以以相对高的亲和力与靶抗原(例如，转铁蛋白受体)结合，例如KD小于10-6M、10-7M、10-8M、10-9M、10-10M、10-11M或更小。例如，抗转铁蛋白受体抗体可以5pM至500nM、例如50pM至100nM、例如500pM至50nM的亲和力与转铁蛋白受体蛋白(例如，人转铁蛋白受体)结合。本公开内容还包括与本文中所述的任何抗体竞争与转铁蛋白受体蛋白(例如，人转铁蛋白受体)的结合并且亲和力为50nM或更小(例如，20nM或更小、10nM或更小、500pM或更小、50pM或更小或者5pM或更小)的抗体。可使用任何合适的方法来测试抗转铁蛋白受体抗体的亲和力和结合动力学，所述方法包括但不限于生物传感器技术(例如，OCTET或BIACORE)。In some embodiments, the antibodies of the present disclosure can bind to a target antigen (e.g., transferrin receptor) with a relatively high affinity, e.g.,KD less than10-6 M,10-7 M, 10-8 M,10-9 M,10-10 M,10-11 M or less. For example, an anti-transferrin receptor antibody can bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity of 5pM to 500nM, e.g., 50pM to 100nM, e.g., 500pM to 50nM. The present disclosure also includes antibodies that compete with any antibody described herein for binding to a transferrin receptor protein (e.g., human transferrin receptor) and have an affinity of 50nM or less (e.g., 20nM or less, 10nM or less, 500pM or less, 50pM or less, or 5pM or less). The affinity and binding kinetics of anti-transferrin receptor antibodies can be tested using any suitable method including, but not limited to, biosensor technology (eg, OCTET or BIACORE).
在一些实施方案中，肌肉靶向剂是转铁蛋白受体抗体(例如，如国际申请公开WO2016/081643中所述的抗体及其变体，通过引用并入本文)。In some embodiments, the muscle targeting agent is a transferrin receptor antibody (eg, an antibody and variants thereof as described in International Application Publication No. WO 2016/081643, incorporated herein by reference).
在表3中提供了根据不同定义系统的抗体的重链和轻链CDR。已经描述了不同的定义系统，例如Kabat定义、Chothia定义和/或contact定义。参见例如(例如，Kabat,E.A.,etal.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242,Chothia et al.,(1989)Nature 342:877；Chothia,C.et al.(1987)J.Mol.Biol.196:901-917,Al-lazikani et al(1997)J.Molec.Biol.273:927-948；以及Almagro,J.Mol.Recognit.17:132-143(2004)。还参见hgmp.mrc.ac.uk和bioinf.org.uk/abs)。Heavy and light chain CDRs of antibodies according to different definition systems are provided in Table 3. Different definition systems have been described, such as the Kabat definition, the Chothia definition and/or the contact definition. See, e.g., (e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).
表3小鼠转铁蛋白受体抗体的重链和轻链CDR。Table 3 Heavy and light chain CDRs of mouse transferrin receptor antibodies.
还提供了重链可变结构域(VH)和轻链可变结构域序列：Also provided are heavy chain variable domain (VH) and light chain variable domain sequences:
VHVH
VLV L
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含与表3中所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2和CDR-H3。作为替代或补充，本公开内容的转铁蛋白受体抗体包含与表3中所示的CDR-L1、CDR-L2和CDR-L3相同的CDR-L1、CDR-L2和CDR-L3。In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2 and CDR-H3 identical to those shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise CDR-L1, CDR-L2 and CDR-L3 identical to those shown in Table 3.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含CDR-H1、CDR-H2和CDR-H3，与表3中所示的CDR-H1、CDR-H2和CDR-H3相比，其共同包含不超过5个氨基酸变异(例如，不超过5、4、3、2或1个氨基酸变异)。“共同”意指所有三个重链CDR中的氨基酸变异的总数均在限定的范围内。作为替代或补充，本公开内容的转铁蛋白受体抗体可包含CDR-L1、CDR-L2和CDR-L3，与表3中所示的CDR-L1、CDR-L2和CDR-L3相比，其共同包含不超过5个氨基酸变异(例如，不超过5、4、3、2或1个氨基酸变异)。In some embodiments, the transferrin receptor antibody of the present disclosure comprises CDR-H1, CDR-H2 and CDR-H3, which together comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variations) compared to the CDR-H1, CDR-H2 and CDR-H3 shown in Table 3. "Common" means that the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or in addition, the transferrin receptor antibody of the present disclosure may comprise CDR-L1, CDR-L2 and CDR-L3, which together comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variations) compared to the CDR-L1, CDR-L2 and CDR-L3 shown in Table 3.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含CDR-H1、CDR-H2和CDR-H3，其中至少一个与表3中所示的对应重链CDR相比包含不超过3个氨基酸变异(例如，不超过3、2或1个氨基酸变异)。作为替代或补充，本公开内容的转铁蛋白受体抗体可包含CDR-L1、CDR-L2和CDR-L3，其中至少一个与表3中所示的对应轻链CDR相比包含不超过3个氨基酸变异(例如，不超过3、2或1个氨基酸变异)。In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding heavy chain CDR shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding light chain CDR shown in Table 3.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含CDR-L3，其与表3中所示的CDR-L3相比包含不超过3个氨基酸变异(例如，不超过3、2或1个氨基酸变异)。在一些实施方案中，本公开内容的转铁蛋白受体抗体包含CDR-L3，其与表3中所示的CDR-L3相比包含1个氨基酸变异。在一些实施方案中，本公开内容的转铁蛋白受体抗体包含QHFAGTPLT(SEQID NO:31，根据Kabat和Chothia定义系统)或QHFAGTPLT(SEQ ID NO:32，根据Contact定义系统)的CDR-L3。在一些实施方案中，本公开内容的转铁蛋白受体抗体包含与表3中所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2、CDR-H3、CDR-L1和CDR-L2，并且包含QHFAGTPLT(SEQ ID NO:31，根据Kabat和Chothia定义系统)或QHFAGTPL(SEQ ID NO:32，根据Contact定义系统)的CDR-L3。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 comprising no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the CDR-L3 shown in Table 3. In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 comprising 1 amino acid variation compared to the CDR-L3 shown in Table 3. In some embodiments, the transferrin receptor antibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition system) or QHFAGTPLT (SEQ ID NO: 32, according to the Contact definition system). In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are identical to the CDR-H1, CDR-H2, and CDR-H3 shown in Table 3, and comprise a CDR-L3 of QHFAGTPLT (SEQ ID NO:31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO:32, according to the Contact definition system).
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含重链CDR，其共同与表3中所示的重链CDR具有至少80％(例如，80％、85％、90％、95％或98％)同一性。作为替代或补充，本公开内容的转铁蛋白受体抗体包含轻链CDR，其共同与表3中所示的轻链CDR具有至少80％(例如，80％、85％、90％、95％或98％)同一性。In some embodiments, the transferrin receptor antibodies of the present disclosure comprise heavy chain CDRs that collectively have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs shown in Table 3. Alternatively or additionally, the transferrin receptor antibodies of the present disclosure comprise light chain CDRs that collectively have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the light chain CDRs shown in Table 3.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含含有SEQ ID NO:33的氨基酸序列的VH。作为替代或补充，本公开内容的转铁蛋白受体抗体包含含有SEQ ID NO:34的氨基酸序列的VL。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 33. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:34.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含VH，所述VH与SEQ IDNO:33中所示的VH相比包含不超过20个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸变异)。作为替代或补充，本公开内容的转铁蛋白受体抗体包含VL，所述VL与SEQ ID NO:34中所示的VL相比包含不超过15个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、9、8、7、6、5、4、3、2或1个氨基酸变异)。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH shown in SEQ ID NO: 33. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO: 34.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含VH，所述VH包含与SEQID NO:33中所示的VH具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。作为替代或补充，本公开内容的转铁蛋白受体抗体包含VL，所述VL包含与SEQ ID NO:34中所示的VL具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VH shown in SEQ ID NO: 33. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VL shown in SEQ ID NO: 34.
在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化抗体(例如，抗体的人源化变体)。在一些实施方案中，本公开内容的转铁蛋白受体抗体包含与表3中所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2和CDR-L3，并且包含人源化重链可变区和/或人源化轻链可变区。In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3, and comprise humanized heavy chain variable regions and/or humanized light chain variable regions.
人源化抗体是人免疫球蛋白(接受体抗体)，其中来自接受体的互补决定区(CDR)的残基被来自具有期望特异性、亲和力和容量(capacity)的非人物种(供体抗体)例如小鼠、大鼠或兔的CDR的残基替代。在一些实施方案中，人免疫球蛋白的Fv框架区(FR)残基被相应的非人残基替代。此外，人源化抗体可包含在接受体抗体或者导入的CDR或框架序列中均未发现但被包含在内以进一步完善和优化抗体性能的残基。通常来说，人源化抗体将包含至少一个并且通常两个可变结构域的基本上全部，其中所有或基本上所有的CDR区对应于非人免疫球蛋白的那些，并且所有或基本上所有的FR区是人免疫球蛋白共有序列的那些。人源化抗体最佳地还将包含免疫球蛋白恒定区或结构域(Fc)(通常是人免疫球蛋白的那些)的至少一部分。抗体可具有如WO 99/58572中所述修饰的Fc区。人源化抗体的另一些形式具有相对于原始抗体改变的一个或更多个CDR(一个、两个、三个、四个、五个、六个)，其也称为源自来自原始抗体的一个或更多个CDR的一个或更多个CDR。人源化抗体还可能涉及亲和力成熟。Humanized antibodies are human immunoglobulins (acceptor antibodies), wherein the residues from the complementary determining regions (CDRs) of the acceptor are replaced by residues from non-human species (donor antibodies) such as mice, rats or rabbits with desired specificity, affinity and capacity. In some embodiments, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may be included in the acceptor antibodies or the CDRs or framework sequences imported that are not found but are included to further improve and optimize antibody performance. Generally speaking, humanized antibodies will include at least one and generally all of two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin consensus sequences. Humanized antibodies will also optimally include at least a portion of immunoglobulin constant regions or domains (Fc) (typically those of human immunoglobulins). Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, which is also referred to as one or more CDRs derived from one or more CDRs from the original antibody.Humanized antibodies may also involve affinity maturation.
在一些实施方案中，通过将CDR(例如，如表3中所示)接枝到IGKV1-NL1*01和IGHV1-3*01人可变结构域中来实现人源化。在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化变体，其与SEQ ID NO:34中所示的VL相比在第9、13、17、18、40、45和70位处包含一个或更多个氨基酸替换，和/或与SEQ ID NO:33中所示的VH相比在第1、5、7、11、12、20、38、40、44、66、75、81、83、87和108位包含一个或更多个氨基酸替换。在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化变体，其与SEQ ID NO:34中所示的VL相比在全部的第9、13、17、18、40、45和70位处包含氨基酸替换，和/或与SEQ ID NO:33中所示的VH相比在全部的第1、5、7、11、12、20、38、40、44、66、75、81、83、87和108位处包含氨基酸替换。In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 3) into IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL shown in SEQ ID NO: 34, and/or comprising one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID NO: 33. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL shown in SEQ ID NO:34, and/or comprising amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID NO:33.
在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化抗体，并且包含如SEQ ID NO:34中所示的VL的第43和48位处的残基。作为替代或补充，本公开内容的转铁蛋白受体抗体是人源化抗体，并且包含如SEQ ID NO:33中所示的VH的第48、67、69、71和73位处的残基。In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized antibody and comprises residues at positions 43 and 48 of VL as shown in SEQ ID NO: 34. Alternatively or additionally, the transferrin receptor antibody of the present disclosure is a humanized antibody and comprises residues at positions 48, 67, 69, 71, and 73 of VH as shown in SEQ ID NO: 33.
提供了可根据本公开内容使用的示例性人源化抗体的VH和VL氨基酸序列：The VH and VL amino acid sequences of exemplary humanized antibodies that can be used according to the present disclosure are provided:
人源化VHHumanized VH
人源化VLHumanized VL
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含含有SEQ ID NO:35的氨基酸序列的VH。作为替代或补充，本公开内容的转铁蛋白受体抗体包含含有SEQ ID NO:36的氨基酸序列的VL。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:36.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含VH，所述VH与SEQ IDNO:35中所示的VH相比包含不超过20个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸变异)。作为替代或补充，本公开内容的转铁蛋白受体抗体包含VL，所述VL与SEQ ID NO:36中所示的VL相比包含不超过15个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、9、8、7、6、5、4、3、2或1个氨基酸变异)。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH shown in SEQ ID NO: 35. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO: 36.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含VH，所述VH包含与SEQID NO:35中所示的VH具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。作为替代或补充，本公开内容的转铁蛋白受体抗体包含VL，所述VL包含与SEQ ID NO:36中所示的VL具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VH shown in SEQ ID NO: 35. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to the VL shown in SEQ ID NO: 36.
在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化变体，其与SEQ IDNO:34中所示的VL相比在第43和48位中的一个或更多个处包含氨基酸替换，和/或与SEQ IDNO:33中所示的VH相比在第48、67、69、71和73位中的一个或更多个处包含氨基酸替换。在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化变体，其与SEQ ID NO:34中所示的VL相比包含S43A和/或V48L突变，和/或与SEQ ID NO:33中所示的VH相比包含A67V、L69I、V71R和K73T突变中的一个或更多个。In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising an amino acid substitution at one or more of positions 43 and 48 compared to the VL shown in SEQ ID NO: 34, and/or an amino acid substitution at one or more of positions 48, 67, 69, 71, and 73 compared to the VH shown in SEQ ID NO: 33. In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising an S43A and/or V48L mutation compared to the VL shown in SEQ ID NO: 34, and/or comprising one or more of A67V, L69I, V71R, and K73T mutations compared to the VH shown in SEQ ID NO: 33.
在一些实施方案中，本公开内容的转铁蛋白受体抗体是人源化变体，其与SEQ IDNO:34中所示的VL相比在第9、13、17、18、40、43、48、45和70位中的一个或更多个处包含氨基酸替换，和/或与SEQ ID NO:33中所示的VH相比在第1、5、7、11、12、20、38、40、44、48、66、67、69、71、73、75、81、83、87和108位中的一个或更多个处包含氨基酸替换。In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising amino acid substitutions at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to the VL shown in SEQ ID NO:34, and/or comprising amino acid substitutions at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 compared to the VH shown in SEQ ID NO:33.
在一些实施方案中，本公开内容的转铁蛋白受体抗体是嵌合抗体，其可包含来自人抗体的重恒定区和轻恒定区。嵌合抗体是指具有来自第一物种的可变区或可变区的一部分以及来自第二物种的恒定区的抗体。通常来说，在这些嵌合抗体中，轻链和重链二者的可变区模拟源自一种哺乳动物(例如，非人哺乳动物，例如小鼠、兔和大鼠)的抗体的可变区，而恒定部分与源自另一种哺乳动物(例如人)的抗体中的序列同源。在一些实施方案中，可在可变区和/或恒定区中进行氨基酸修饰。In some embodiments, the transferrin receptor antibody of the present disclosure is a chimeric antibody, which may include a heavy constant region and a light constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region or a part of a variable region from a first species and a constant region from a second species. Generally speaking, in these chimeric antibodies, the variable regions of both light and heavy chains simulate the variable regions of antibodies derived from a mammal (e.g., non-human mammals, such as mice, rabbits and rats), while the constant portion is homologous to the sequence in an antibody derived from another mammal (e.g., people). In some embodiments, amino acid modifications may be performed in the variable region and/or constant region.
在一些实施方案中，本文中所述的转铁蛋白受体抗体是嵌合抗体，其可包含来自人抗体的重恒定区和轻恒定区。嵌合抗体是指具有来自第一物种的可变区或可变区的一部分以及来自第二物种的恒定区的抗体。通常来说，在这些嵌合抗体中，轻链和重链二者的可变区模拟源自一种哺乳动物(例如，非人哺乳动物，例如小鼠、兔和大鼠)的抗体的可变区，而恒定部分与源自另一种哺乳动物(例如人)的抗体中的序列同源。在一些实施方案中，可在可变区和/或恒定区中进行氨基酸修饰。In some embodiments, the transferrin receptor antibody described herein is a chimeric antibody, which may include a heavy constant region and a light constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Generally speaking, in these chimeric antibodies, the variable regions of both light and heavy chains simulate the variable regions of antibodies derived from a mammal (e.g., non-human mammals, such as mice, rabbits and rats), while the constant portion is homologous to the sequence in an antibody derived from another mammal (e.g., human). In some embodiments, amino acid modifications may be performed in the variable region and/or constant region.
在一些实施方案中，本文中所述的任何转铁蛋白受体抗体的重链可包含重链恒定区(CH)或其一部分(例如，CH1、CH2、CH3或其组合)。重链恒定区可具有任何合适的来源，例如人、小鼠、大鼠或兔。在一个特定的实例中，重链恒定区是来自人IgG例如IgG1、IgG2或IgG4的(γ重链)。下面给出了示例性人IgG1恒定区：In some embodiments, the heavy chain of any transferrin receptor antibody described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). Exemplary human IgG1 constant regions are given below:
在一些实施方案中，本文中所述的任何转铁蛋白受体抗体的轻链可还包含轻链恒定区(CL)，其可以是本领域已知的任何CL。在一些实例中，CL是κ轻链。在另一些实例中，CL是λ轻链。在一些实施方案中，CL是κ轻链，其序列提供如下：In some embodiments, the light chain of any transferrin receptor antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some instances, CL is a kappa light chain. In other instances, CL is a lambda light chain. In some embodiments, CL is a kappa light chain, the sequence of which is provided below:
其他抗体重链和轻链恒定区是本领域公知的，例如在IMGT数据库(www.imgt.org)或www.vbase2.org/vbstat.php.中提供的那些，二者都通过引用并入本文。Other antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org/vbstat.php., both of which are incorporated herein by reference.
下面提供了所述的转铁蛋白受体抗体的示例性重链和轻链氨基酸序列：Exemplary heavy and light chain amino acid sequences of the transferrin receptor antibodies are provided below:
重链(VH+人IgG1恒定区)Heavy chain (VH + human IgG1 constant region)
轻链(VL+κ轻链)Light chain (VL+κ light chain)
重链(人源化VH+人IgG1恒定区)Heavy chain (humanized VH + human IgG1 constant region)
轻链(人源化VL+κ轻链)Light chain (humanized VL + kappa light chain)
在一些实施方案中，本文中所述的转铁蛋白受体抗体包含重链，所述重链包含与SEQ ID NO:39具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。作为替代或补充，本文中所述的转铁蛋白受体抗体包含轻链，所述轻链包含与SEQ ID NO:40具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。在一些实施方案中，本文中所述的转铁蛋白受体抗体包含含有SEQ ID NO:39的氨基酸序列的重链。作为替代或补充，本文中所述的转铁蛋白受体抗体包含含有SEQ ID NO:40的氨基酸序列的轻链。In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to SEQ ID NO: 39. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity to SEQ ID NO: 40. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 39. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含重链，所述重链与SEQID NO:39中所示的重链相比包含不超过20个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸变异)。作为替代或补充，本公开内容的转铁蛋白受体抗体包含轻链，所述轻链与SEQ ID NO:40中所示的轻链相比包含不超过15个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、9、8、7、6、5、4、3、2或1个氨基酸变异)。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a heavy chain comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain shown in SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a light chain comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain shown in SEQ ID NO: 40.
在一些实施方案中，本文中所述的转铁蛋白受体抗体包含重链，所述重链包含与SEQ ID NO:41具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。作为替代或补充，本文中所述的转铁蛋白受体抗体包含轻链，所述轻链包含与SEQ ID NO:42具有至少80％(例如，80％、85％、90％、95％或98％)同一性的氨基酸序列。在一些实施方案中，本文中所述的转铁蛋白受体抗体包含含有SEQ ID NO:41的氨基酸序列的重链。作为替代或补充，本文中所述的转铁蛋白受体抗体包含含有SEQ ID NO:42的氨基酸序列的轻链。In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity with SEQ ID NO: 41. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identity with SEQ ID NO: 42. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 41. Alternatively or in addition, the transferrin receptor antibody described herein comprises a light chain comprising an amino acid sequence of SEQ ID NO: 42.
在一些实施方案中，本公开内容的转铁蛋白受体抗体包含重链，所述重链与SEQID NO:39中所示的人源化抗体的重链相比包含不超过20个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸变异)。作为替代或补充，本公开内容的转铁蛋白受体抗体包含轻链，所述轻链与SEQ ID NO:40中所示的人源化抗体的轻链相比包含不超过15个氨基酸变异(例如不超过20、19、18、17、16、15、14、13、12、11、9、8、7、6、5、4、3、2或1个氨基酸变异)。In some embodiments, the transferrin receptor antibody of the present disclosure comprises a heavy chain comprising no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain of the humanized antibody shown in SEQ ID NO: 39. Alternatively or additionally, the transferrin receptor antibody of the present disclosure comprises a light chain comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain of the humanized antibody shown in SEQ ID NO: 40.
在一些实施方案中，转铁蛋白受体抗体是完整抗体(全长抗体)的抗原结合片段(Fab)。完整抗体(全长抗体)的抗原结合片段可通过常规方法制备。例如，F(ab’)2片段可通过抗体分子的胃蛋白酶消化产生，并且Fab片段可通过还原F(ab’)2片段的二硫键而产生。下面提供了本文中所述的转铁蛋白受体抗体的示例性Fab氨基酸序列：In some embodiments, the transferrin receptor antibody is an antigen binding fragment (Fab) of a complete antibody (full-length antibody). The antigen binding fragment of a complete antibody (full-length antibody) can be prepared by conventional methods. For example, a F(ab')2 fragment can be produced by pepsin digestion of an antibody molecule, and a Fab fragment can be produced by reducing the disulfide bonds of the F(ab')2 fragment. An exemplary Fab amino acid sequence of a transferrin receptor antibody described herein is provided below:
重链FAB(VH+人IgG1恒定区的一部分)Heavy chain FAB (VH + part of the human IgG1 constant region)
重链FAB(人源化VH+人IgG1恒定区的一部分)Heavy chain FAB (humanized VH + part of human IgG1 constant region)
本文中所述的转铁蛋白受体抗体可以是任何抗体形式，包括但不限于完整(即，全长)抗体、其抗原结合片段(例如Fab、Fab’、F(ab’)2、Fv)、单链抗体、双特异性抗体或纳米抗体。在一些实施方案中，本文中所述的转铁蛋白受体抗体是scFv。在一些实施方案中，本文中所述的转铁蛋白受体抗体是scFv-Fab(例如，与恒定区的一部分融合的scFv)。在一些实施方案中，本文中所述的转铁蛋白受体抗体是与恒定区(例如，SEQ ID NO:39中所示的人IgG1恒定区)融合的scFv。The transferrin receptor antibodies described herein can be in any antibody form, including but not limited to complete (i.e., full-length) antibodies, antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the transferrin receptor antibodies described herein are scFv. In some embodiments, the transferrin receptor antibodies described herein are scFv-Fab (e.g., scFv fused to a portion of a constant region). In some embodiments, the transferrin receptor antibodies described herein are scFv fused to a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO:39).
b.其他肌肉靶向抗体b. Other muscle-targeted antibodies
在一些实施方案中，肌肉靶向抗体是特异性结合血幼素(hemojuvelin)、小窝蛋白-3、迪谢内肌营养不良肽、肌球蛋白Iib或CD63的抗体。在一些实施方案中，肌肉靶向抗体是特异性结合肌原性前体蛋白的抗体。示例性的肌原性前体蛋白包括但不限于ABCG2、M-钙黏着蛋白/钙黏着蛋白-15、小窝蛋白-1、CD34、FoxK1、整联蛋白α7、整联蛋白α7β1、MYF-5、MyoD、肌细胞生成蛋白、NCAM-1/CD56、Pax3、Pax7和Pax9。在一些实施方案中，肌肉靶向抗体是特异性结合骨骼肌蛋白的抗体。示例性的骨骼肌蛋白包括但不限于α-肌聚糖蛋白(alpha-Sarcoglycan)、β-肌聚糖蛋白、钙蛋白酶抑制剂、肌酸激酶MM/CKMM、eIF5A、烯醇化酶2/神经元特异性烯醇化酶、ε-肌聚糖蛋白、FABP3/H-FABP、GDF-8/肌生成抑制蛋白、GDF-11/GDF-8、整联蛋白α7、整联蛋白α7β1、整联蛋白β1/CD29、MCAM/CD146、MyoD、肌细胞生成蛋白、肌球蛋白轻链激酶抑制剂、NCAM-1/CD56和肌钙蛋白I。在一些实施方案中，肌肉靶向抗体是特异性结合平滑肌蛋白的抗体。示例性的平滑肌蛋白包括但不限于α-平滑肌肌动蛋白、VE-钙黏着蛋白、钙调蛋白结合蛋白/CALD1、钙调理蛋白1、结蛋白(Desmin)、组胺H2 R、胃动素R/GPR38、转凝蛋白/TAGLN、和波形蛋白。然而，应理解，针对其他靶标的抗体在本公开内容的范围内，并且本文中提供的靶标的示例性列表并不意图是限制性的。In some embodiments, the muscle targeting antibody is an antibody that specifically binds hemojuvelin, caveolin-3, dystrophin, myosin Iib or CD63. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to myogenic precursor proteins. Exemplary myogenic precursor proteins include but are not limited to ABCG2, M-cadherin/cadherin-15, caveolin-1, CD34, FoxK1, integrin α7, integrin α7β1, MYF-5, MyoD, myogenic protein, NCAM-1/CD56, Pax3, Pax7 and Pax9. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to skeletal muscle protein. Exemplary skeletal muscle proteins include, but are not limited to, alpha-Sarcoglycan, beta-Sarcoglycan, calpain inhibitor, creatine kinase MM/CKMM, eIF5A, enolase 2/neuron-specific enolase, epsilon-Sarcoglycan, FABP3/H-FABP, GDF-8/myostatin, GDF-11/GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1/CD29, MCAM/CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1/CD56, and troponin I. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, but are not limited to, α-smooth muscle actin, VE-cadherin, calmodulin binding protein/CALD1, calmodulin 1, desmin, histamine H2 R, motilin R/GPR38, transglutamin/TAGLN, and vimentin. However, it should be understood that antibodies to other targets are within the scope of the present disclosure, and the exemplary list of targets provided herein is not intended to be limiting.
c.抗体特征/改变c. Antibody characteristics/changes
在一些实施方案中，可在残基不太可能参与与靶抗原(例如，转铁蛋白受体)相互作用的位置(例如，如基于晶体结构确定的)处将保守突变引入抗体序列(例如，CDR或框架序列)中。在一些实施方案中，将一个、两个或更多个突变(例如，氨基酸替换)引入本文中所述的肌肉靶向抗体的Fc区(例如，在CH2结构域(人IgG1的第231至340位残基)和/或CH3结构域(人IgG1的第341至447位残基)和/或铰链区中，根据Kabat编号系统(例如，Kabat中的EU索引)编号)，以改变抗体的一种或更多种功能特性，例如血清半衰期、补体固定、Fc受体结合和/或抗原依赖性细胞毒性。In some embodiments, conservative mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in interacting with the target antigen (e.g., transferrin receptor) (e.g., as determined based on the crystal structure). In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and/or the CH3 domain (residues 341 to 447 of human IgG1) and/or the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular toxicity.
在一些实施方案中，将一个、两个或更多个突变(例如，氨基酸替换)引入Fc区(CH1结构域)的铰链区中，使得铰链区中的半胱氨酸残基的数目改变(例如，提高或降低)，如例如美国专利No.5,677,425中所述。可改变CH1结构域的铰链区中半胱氨酸残基的数目，例如以促进轻链和重链的组装，或改变(例如，提高或降低)抗体的稳定性或促进接头缀合。In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain may be altered, for example, to facilitate assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.
在一些实施方案中，将一个、两个或更多个突变(例如，氨基酸替换)引入本文中所述的肌肉靶向抗体的Fc区(例如，在CH2结构域(人IgG1的第231至340位残基)和/或CH3结构域(人IgG1的第341至447位残基)和/或铰链区中，根据Kabat编号系统(例如，Kabat中的EU索引)编号)，以提高或降低抗体对效应细胞表面上Fc受体(例如，激活的Fc受体)的亲和力。降低或提高抗体对Fc受体的亲和力的抗体Fc区中的突变以及将这样的突变引入Fc受体或其片段的技术是本领域技术人员已知的。可进行以改变抗体对Fc受体的亲和力的抗体Fc受体中的突变的实例描述于以下中：例如Smith P et al.,(2012)PNAS 109:6181-6186,美国专利No.6,737,056,以及国际公开No.WO 02/060919、WO 98/23289、和WO 97/34631，其通过引用并入本文。In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and/or the CH3 domain (residues 341 to 447 of human IgG1) and/or the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Examples of mutations in an antibody Fc receptor that can be made to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02/060919, WO 98/23289, and WO 97/34631, which are incorporated herein by reference.
在一些实施方案中，将一个、两个或更多个氨基酸突变(即，替换、插入或缺失)引入IgG恒定结构域或其FcRn结合片段(优选地，Fc或铰链-Fc结构域片段)中以改变(例如，降低或提高)体内抗体的半衰期。参见例如国际公开No.WO 02/060919、WO 98/23289和WO 97/34631，以及美国专利No.5,869,046、6,121,022、6,277,375和6,165,745，例如将改变(例如，降低或提高)体内抗体的半衰期的突变。In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain or an FcRn binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02/060919, WO 98/23289, and WO 97/34631, and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that will alter (e.g., reduce or increase) the half-life of an antibody in vivo.
在一些实施方案中，将一个、两个或更多个氨基酸突变(即，替换、插入或缺失)引入IgG恒定结构域或其FcRn结合片段(优选地，Fc或铰链-Fc结构域片段)中以降低体内抗转铁蛋白受体抗体的半衰期。在一些实施方案中，将一个、两个或更多个氨基酸突变(即，替换、插入或缺失)引入IgG恒定结构域或其FcRn结合片段(优选地，Fc或铰链-Fc结构域片段)中以提高体内抗体的半衰期。在一些实施方案中，抗体可在第二恒定(CH2)结构域(人IgG1的第231至340位残基)和/或第三恒定(CH3)结构域(人IgG1的第341至447位残基)(根据Kabat中的EU索引(Kabat E A et al.,(1991)同上)编号)中具有一个或更多个氨基酸突变(例如，替换)。在一些实施方案中，本文中所述的抗体的IgG1的恒定区包含在第252位处的甲硫氨酸(M)至酪氨酸(Y)替换，在第254位处的丝氨酸(S)至苏氨酸(T)替换，以及在第256位处的苏氨酸(T)至谷氨酸(E)替换，所述位置根据Kabat中的EU索引编号。参见美国专利No.7,658,921，其通过引用并入本文。这种类型的突变IgG(称为“YTE突变型”)已显示出与同一抗体的野生型形式相比半衰期提高4倍(参见Dall'Acqua W F et al.,(2006)J BiolChem 281:23514-24)。在一些实施方案中，抗体包含IgG恒定结构域，该结构域包含在第251至257、285至290、308至314、385至389和428至436位处的氨基酸残基的一个、两个、三个或更多个氨基酸替换，所述位置根据Kabat中的EU索引编号。In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231 to 340 of human IgG1) and/or the third constant (CH3) domain (residues 341 to 447 of human IgG1) (numbered according to the EU index in Kabat (Kabat EA et al., (1991) supra)). In some embodiments, the constant region of the IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, the positions being numbered according to the EU index in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to increase half-life by 4 times compared to the wild-type form of the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, as the positions are numbered according to the EU index as in Kabat.
在一些实施方案中，将一个、两个或更多个氨基酸替换引入IgG恒定结构域Fc区中，以改变抗转铁蛋白受体抗体的一种或更多种效应子功能。改变亲和力的效应配体可以是例如Fc受体或补体的C1组分。所述方法在美国专利No.5,624,821和5,648,260中有更详细的描述。在一些实施方案中，恒定区结构域的缺失或失活(通过点突变或其他方式)可降低循环抗体的Fc受体结合，从而提高肿瘤定位。对于使恒定结构域缺失或失活并且从而提高肿瘤定位的突变的描述，参见例如美国专利No.5,585,097和8,591,886。在一些实施方案中，可将一个或更多个氨基酸替换引入本文中所述的抗体的Fc区中，以去除Fc区上潜在的糖基化位点，这可降低Fc受体结合(参见，例如Shields R L et al.,(2001)J Biol Chem276:6591-604)。In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change one or more effector functions of the anti-transferrin receptor antibody. The effector ligand that changes affinity can be, for example, an Fc receptor or the C1 component of complement. The method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domain (by point mutation or other means) can reduce the Fc receptor binding of circulating antibodies, thereby improving tumor localization. For descriptions of mutations that delete or inactivate the constant domain and thereby improve tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions can be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604).
在一些实施方案中，可将本文中所述的肌肉靶向抗体的恒定区中的一个或更多个氨基替换为不同的氨基酸残基，使得抗体具有改变的Clq结合和/或者降低或消除的补体依赖性细胞毒性(complement dependent cytotoxicity，CDC)。这种方法在美国专利No.6,194,551(Idusogie等人)中有更详细的描述。在一些实施方案中，改变本文中所述的抗体的CH2结构域的N端区域中的一个或更多个氨基酸残基，从而改变抗体固定补体的能力。这种方法在国际公开No.WO 94/29351中有进一步描述。在一些实施方案中，对本文中所述的抗体的Fc区进行修饰以提高抗体介导抗体依赖性细胞毒性(antibody dependent cellularcytotoxicity，ADCC)的能力和/或提高抗体对Fcγ受体的亲和力。这种方法在国际公开No.WO 00/42072中有进一步描述。In some embodiments, one or more amino groups in the constant region of the muscle-targeted antibodies described herein can be replaced with different amino acid residues so that the antibody has altered Clq binding and/or reduced or eliminated complement dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibodies described herein are changed to change the ability of the antibody to fix complement. This method is further described in International Publication No. WO 94/29351. In some embodiments, the Fc region of the antibodies described herein is modified to improve the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or improve the affinity of the antibody for Fcγ receptors. This method is further described in International Publication No. WO 00/42072.
在一些实施方案中，本文中提供的抗体的重链和/或轻链可变结构域序列可用于产生例如CDR接枝、嵌合、人源化或复合的人抗体或抗原结合片段，如本文其他地方所述。如本领域普通技术人员所理解的，源自本文中提供的任何抗体的任何变体(CDR接枝、嵌合、人源化或复合的抗体)可用于本文中所述的组合物和方法中，并将保持特异性结合转铁蛋白受体的能力，从而使得相对于其所来源原始抗体，变体(CDR接枝、嵌合、人源化或复合的抗体)具有至少50％、至少60％、至少70％、至少80％、至少90％、至少95％或更多的与转铁蛋白受体的结合。In some embodiments, the heavy chain and/or light chain variable domain sequences of the antibodies provided herein can be used to produce, for example, CDR grafted, chimeric, humanized or composite human antibodies or antigen-binding fragments, as described elsewhere herein. As understood by those of ordinary skill in the art, any variant (CDR grafted, chimeric, humanized or composite antibody) derived from any antibody provided herein can be used in the compositions and methods described herein, and will maintain the ability to specifically bind to transferrin receptor, so that relative to the original antibody from which it is derived, the variant (CDR grafted, chimeric, humanized or composite antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor.
在一些实施方案中，本文中提供的抗体包含赋予抗体以期望性质的突变。例如，为了避免归因于已知与天然IgG4 mAb发生的Fab臂交换而引起的潜在并发症，本文中提供的抗体可包含稳定性‘Adair’突变(Angal S.,et al.,“A single amino acid substitutionabolishes the heterogeneity of chimeric mouse/human(IgG4)antibody,”MolImmunol 30,105-108；1993)，其中第228位(EU编号，根据Kabat编号为第241位残基)丝氨酸转化为脯氨酸，产生了IgG1样铰链序列。因此，任何抗体都可包含稳定性‘Adair’突变。In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to known Fab arm exchanges with natural IgG4 mAbs, the antibodies provided herein may include stability 'Adair' mutations (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), wherein the 228th (EU numbering, according to Kabat numbering, the 241st residue) serine is converted to proline, resulting in an IgG1-like hinge sequence. Therefore, any antibody may include stability 'Adair' mutations.
如本文所提供，本公开内容的抗体可任选地包含恒定区或其一部分。例如，VL结构域可在其C端连接至轻链恒定结构域，如Cκ或Cλ。类似地，VH结构域或其一部分可连接至重链的全部或一部分，所述重链例如IgA、IgD、IgE、IgG和IgM，以及任何同种型亚类。抗体可包括合适的恒定区(参见，例如，Kabat et al.,Sequences of Proteins of ImmunologicalInterest,No.91-3242,National Institutes of Health Publications,Bethesda,Md.(1991))。因此，在本公开内容范围内的抗体可包含与任何合适的恒定区组合的VH和VL结构域或其抗原结合部分。As provided herein, the antibodies of the present disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be connected to a light chain constant domain, such as Cκ or Cλ, at its C-terminus. Similarly, the VH domain or a portion thereof may be connected to all or a portion of a heavy chain, such as IgA, IgD, IgE, IgG and IgM, and any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of the present disclosure may include VH and VL domains or antigen binding portions thereof in combination with any suitable constant region.
ii.肌肉靶向肽ii. Muscle Targeting Peptides
本公开内容的一些方面提供了肌肉靶向肽作为肌肉靶向剂。已描述了与特定细胞类型结合的短肽序列(例如，长度为5至20个氨基酸的肽序列)。例如，细胞靶向肽已在以下中进行了描述：Vines e.,et al.,A.“Cell-penetrating and cell-targeting peptidesin drug delivery”Biochim Biophys Acta 2008,1786:126-38；Jarver P.,et al.,“Invivo biodistribution and efficacy of peptide mediated delivery”TrendsPharmacol Sci 2010；31:528-35；Samoylova T.I.,et al.,“Elucidation of muscle-binding peptides by phage display screening”Muscle Nerve 1999；22:460-6；美国专利No.6,329,501，其于2001年12月11日授权，标题为“METHODS AND COMPOSITIONS FORTARGETING COMPOUNDS TO MUSCLE”；以及Samoylov A.M.,et al.,“Recognition of cell-specific binding of phage display derived peptides using an acoustic wavesensor.”Biomol Eng 2002；18:269-72；其各自的全部内容均通过引用并入本文。通过设计肽与特定的细胞表面抗原(例如，受体)相互作用，可实现对所期望组织例如肌肉的选择性。已研究了骨骼肌靶向并且能够递送一系列分子载荷。这些方法可对肌肉组织具有高度选择性，而没有大的抗体或病毒颗粒的许多实际缺点。因此，在一些实施方案中，肌肉靶向剂是长度为4至50个氨基酸的肌肉靶向肽。在一些实施方案中，肌肉靶向肽的长度为4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个氨基酸。可使用数种方法中的任一种(例如噬菌体展示)来产生肌肉靶向肽。Some aspects of the present disclosure provide muscle targeting peptides as muscle targeting agents.Short peptide sequences (eg, peptide sequences 5 to 20 amino acids in length) that bind to specific cell types have been described. For example, cell targeting peptides have been described in: Vines E., et al., A. "Cell-penetrating and cell-targeting peptides in drug delivery" Biochim Biophys Acta 2008, 1786: 126-38; Jarver P., et al., "Invivo biodistribution and efficacy of peptide mediated delivery" Trends Pharmacol Sci 2010; 31: 528-35; Samoylova T.I., et al., "Elucidation of muscle-binding peptides by phage display screening" Muscle Nerve 1999; 22: 460-6; U.S. Patent No. 6,329,501, which was issued on December 11, 2001, and is entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov A.M., et al., "Recognition of cell-specific binding of phage display derived peptides using an acoustic wavesensor." Biomol Eng 2002; 18: 269-72; the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for desired tissues such as muscle can be achieved. Skeletal muscle targeting has been studied and is capable of delivering a range of molecular payloads. These methods can be highly selective for muscle tissue without many of the practical disadvantages of large antibodies or viral particles. Therefore, in some embodiments, the muscle targeting agent is a muscle targeting peptide with a length of 4 to 50 amino acids. In some embodiments, the muscle targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Any of several methods, such as phage display, can be used to generate muscle targeting peptides.
在一些实施方案中，肌肉靶向肽可与和某些其他细胞相比在肌细胞中过表达或相对高地表达的内化细胞表面受体，例如转铁蛋白受体结合。在一些实施方案中，肌肉靶向肽可靶向转铁蛋白受体(例如，与之结合)。在一些实施方案中，靶向转铁蛋白受体的肽可包含天然配体(例如转铁蛋白)的片段。在一些实施方案中，靶向转铁蛋白受体的肽如2000年11月30日提交的美国专利No.6,743,893，“RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THATBIND THE HUMAN TRANSFERRIN RECEPTOR”中所述。在一些实施方案中，靶向转铁蛋白受体的肽如Kawamoto,M.et al,“A novel transferrin receptor-targeted hybrid peptidedisintegrates cancer cell membrane to induce rapid killing of cancer cells.”BMC Cancer.2011Aug 18；11:359中所述。在一些实施方案中，靶向转铁蛋白受体的肽如2011年5月20日提交的美国专利No.8,399,653，“TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY”中所述。In some embodiments, the muscle targeting peptide can bind to an internalized cell surface receptor that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells, such as the transferrin receptor. In some embodiments, the muscle targeting peptide can target the transferrin receptor (e.g., bind to it). In some embodiments, the peptide targeting the transferrin receptor can include a fragment of a natural ligand (e.g., transferrin). In some embodiments, the peptide targeting the transferrin receptor is as described in U.S. Patent No. 6,743,893, filed on November 30, 2000, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THATBIND THE HUMAN TRANSFERRIN RECEPTOR". In some embodiments, the peptide targeting the transferrin receptor is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011Aug 18; 11: 359. In some embodiments, the peptide targeting transferrin receptor is as described in U.S. Patent No. 8,399,653, filed May 20, 2011, “TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY.”
如上所述，已报道了肌肉靶向肽的一些实例。例如，使用呈递表面七肽的噬菌体展示文库鉴定了肌肉特异性肽。作为一个实例，具有氨基酸序列ASSLNIA(SEQ ID NO:6)的肽在体外与C2C12鼠肌管结合，并且在体内与小鼠肌肉组织结合。因此，在一些实施方案中，肌肉靶向剂包含氨基酸序列ASSLNIA(SEQ ID NO:6)。该肽在小鼠中进行静脉内注射之后展示出提高了与心脏和骨骼肌组织结合的特异性，以及降低了与肝、肾和脑的结合。使用噬菌体展示已鉴定了另外的肌肉特异性肽。例如，通过噬菌体展示文库鉴定了12个氨基酸肽用于在DMD治疗的情况下进行肌肉靶向。参见Yoshida D.,et al.,“Targeting of salicylateto skin and muscle following topical injections in rats.”Int J Pharm 2002；231:177-84；其全部内容在此通过引用并入。在此，鉴定了具有序列SKTFNTHPQSTP(SEQ IDNO:7)的12个氨基酸肽，并且该肌肉靶向肽相对于ASSLNIA(SEQ ID NO:6)肽显示出提高了与C2C12细胞的结合。As described above, some examples of muscle targeting peptides have been reported. For example, muscle-specific peptides were identified using a phage display library presenting surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 6) binds to C2C12 mouse myotubes in vitro and binds to mouse muscle tissue in vivo. Therefore, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 6). The peptide exhibits increased specificity for binding to cardiac and skeletal muscle tissue after intravenous injection in mice, as well as reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, a 12 amino acid peptide was identified by a phage display library for muscle targeting in the context of DMD treatment. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats." Int J Pharm 2002; 231: 177-84; the entire contents of which are incorporated herein by reference. Here, a 12 amino acid peptide with the sequence SKTFNTHPQSTP (SEQ ID NO:7) was identified and this muscle-targeted peptide showed improved binding to C2C12 cells relative to the ASSLNIA (SEQ ID NO:6) peptide.
用于鉴定相对于其他细胞类型对肌肉(例如，骨骼肌)具有选择性的肽的另一方法包括体外选择，这已在Ghosh D.,et al.,“Selection of muscle-binding peptides fromcontext-specific peptide-presenting phage libraries for adenoviral vectortargeting”J Virol 2005；79:13667-72中进了描述；其全部内容通过引用并入本文。通过将随机的12聚体(12-mer)肽噬菌体展示文库与非肌细胞类型的混合物预孵育选择出了非特异性细胞结合物。在数轮选择之后，12个氨基酸肽TARGEHKEEELI(SEQ ID NO:8)最频繁地出现。因此，在一些实施方案中，肌肉靶向剂包含氨基酸序列TARGEHKEEELI(SEQ ID NO:8)。Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types includes in vitro selection, which has been described in Ghosh D., et al., "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting" J Virol 2005; 79: 13667-72; the entire contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After several rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 8) appeared most frequently. Therefore, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 8).
肌肉靶向剂可以是含氨基酸的分子或肽。肌肉靶向肽可对应于优先与肌细胞中发现的蛋白质受体结合的蛋白质序列。在一些实施方案中，肌肉靶向肽包含高倾向性的疏水性氨基酸，例如缬氨酸，使得该肽优先靶向肌细胞。在一些实施方案中，先前未表征或公开过肌肉靶向肽。可使用数种方法中的任一种(例如噬菌体展示肽文库、单珠单化合物肽文库或位置扫描合成肽组合文库)来构思、产生、合成和/或衍生这些肽。示例性方法已在本领域中表征并通过引用并入(Gray,B.P.and Brown,K.C.“Combinatorial Peptide Libraries:Mining for Cell-Binding Peptides”Chem Rev.2014,114:2,1020–1081.；Samoylova,T.I.and Smith,B.F.“Elucidation of muscle-binding peptides by phage displayscreening.”Muscle Nerve,1999,22:4.460-6.)。在一些实施方案中，先前已公开了肌肉靶向肽(参见，例如Writer M.J.et al.“Targeted gene delivery to human airwayepithelial cells with synthetic vectors incorporating novel targetingpeptides selected by phage display.”J.Drug Targeting.2004；12:185；Cai,D.“BDNF-mediated enhancement of inflammation and injury in the aging heart.”PhysiolGenomics.2006,24:3,191-7.；Zhang,L.“Molecular profiling of heart endothelialcells.”Circulation,2005,112:11,1601-11.；McGuire,M.J.et al.“In vitro selectionof a peptide with high selectivity for cardiomyocytes in vivo.”J MolBiol.2004,342:1,171-82.)。示例性的肌肉靶向肽包含以下组的氨基酸序列：CQAQGQLVC(SEQ ID NO:9)、CSERSMNFC(SEQ ID NO:10)、CPKTRRVPC(SEQ ID NO:11)、WLSEAGPVVTVRALRGTGSW(SEQ ID NO:12)、ASSLNIA(SEQ ID NO:6)、CMQHSMRVC(SEQ ID NO:13)和DDTRHWG(SEQ ID NO:14)。在一些实施方案中，肌肉靶向肽可包含约2至25个氨基酸、约2至20个氨基酸、约2至15个氨基酸、约2至10个氨基酸或约2至5个氨基酸。肌肉靶向肽可包含天然氨基酸例如半胱氨酸、丙氨酸或者非天然存在或经修饰氨基酸。非天然氨基酸包括β-氨基酸、高氨基酸(homo-amino acid)、脯氨酸衍生物、3-经取代的丙氨酸衍生物、线性核心氨基酸、N-甲基氨基酸和本领域中已知的其他。在一些实施方案中，肌肉靶向肽可以是线性的；在另一些实施方案中，肌肉靶向肽可以是环状的，例如双环的(参见，例如Silvana,M.G.et al.Mol.Therapy,2018,26:1,132–147.)。肌肉靶向剂可以是适配体，例如肽适配体，其相对于其他细胞类型优先靶向肌细胞。The muscle targeting agent can be an amino acid-containing molecule or peptide. The muscle targeting peptide can correspond to a protein sequence that preferentially binds to a protein receptor found in muscle cells. In some embodiments, the muscle targeting peptide comprises a highly propensity hydrophobic amino acid, such as valine, such that the peptide preferentially targets muscle cells. In some embodiments, muscle targeting peptides have not been previously characterized or disclosed. These peptides can be conceived, generated, synthesized, and/or derived using any of several methods, such as phage display peptide libraries, single bead single compound peptide libraries, or position scanning synthetic peptide combinatorial libraries. Exemplary methods have been characterized in the art and incorporated by reference (Gray, B.P. and Brown, K.C. "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, T.I. and Smith, B.F. "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4.460-6.). In some embodiments, muscle targeting peptides have been previously disclosed (see, e.g., Writer M.J. et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004; 12: 185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006, 24: 3, 191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005, 112: 11, 1601-11.; McGuire, M.J. et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004, 342: 1, 171-82.). Exemplary muscle targeting peptides include amino acid sequences of the following groups: CQAQGQLVC (SEQ ID NO: 9), CSERSMNFC (SEQ ID NO: 10), CPKTRRVPC (SEQ ID NO: 11), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 12), ASSLNIA (SEQ ID NO: 6), CMQHSMRVC (SEQ ID NO: 13), and DDTRHWG (SEQ ID NO: 14). In some embodiments, the muscle targeting peptide may include about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The muscle targeting peptide may include natural amino acids such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-natural amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art. In some embodiments, the muscle targeting peptide can be linear; in other embodiments, the muscle targeting peptide can be cyclic, such as bicyclic (see, e.g., Silvana, M.G. et al. Mol. Therapy, 2018, 26: 1, 132–147.). The muscle targeting agent can be an aptamer, such as a peptide aptamer, which preferentially targets muscle cells relative to other cell types.
iii.肌肉靶向受体配体iii. Muscle-targeted receptor ligands
肌肉靶向剂可以是配体，例如与受体蛋白结合的配体。肌肉靶向配体可以是蛋白质，例如与由肌细胞表达的内化细胞表面受体结合的转铁蛋白。因此，在一些实施方案中，肌肉靶向剂是转铁蛋白或其与转铁蛋白受体结合的衍生物。肌肉靶向配体可替代地是小分子，例如相对于其他细胞类型优先靶向肌细胞的亲脂性小分子。可靶向肌细胞的一些示例性亲脂性小分子包括包含以下的化合物：胆固醇、胆固醇基、硬脂酸、棕榈酸、油酸、油烯基、亚麻烯(linolene)、亚油酸、肉豆蔻酸、甾醇类、二氢睾酮、睾酮衍生物、甘油、烷基链、三苯甲基类和烷氧基酸。The muscle targeting agent can be a ligand, such as a ligand that binds to a receptor protein. The muscle targeting ligand can be a protein, such as transferrin that binds to an internalized cell surface receptor expressed by a muscle cell. Therefore, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to a transferrin receptor. The muscle targeting ligand can alternatively be a small molecule, such as a lipophilic small molecule that preferentially targets muscle cells relative to other cell types. Some exemplary lipophilic small molecules that can target muscle cells include compounds comprising: cholesterol, cholesterol radicals, stearic acid, palmitic acid, oleic acid, oleyl, linolene, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, trityl groups, and alkoxy acids.
iv.其他肌肉靶向剂iv. Other muscle targeting agents
用于靶向肌细胞(例如，骨骼肌细胞)的一种策略是使用肌转运体蛋白(例如在肌膜上表达的转运体蛋白)的底物。在一些实施方案中，肌肉靶向剂是对肌肉组织具有特异性的流入转运体的底物。在一些实施方案中，流入转运体对骨骼肌组织具有特异性。两类主要的转运体在骨骼肌肌膜上表达：(1)三磷酸腺苷(ATP)结合盒(ABC)超家族，其促进从骨骼肌组织流出和(2)溶质运载体(SLC)超家族，其可促进底物流入骨骼肌中。在一些实施方案中，肌肉靶向剂是与转运体的ABC超家族或SLC超家族结合的底物。在一些实施方案中，与转运体的ABC或SLC超家族结合的底物是天然底物。在一些实施方案中，与转运体的ABC或SLC超家族结合的底物是非天然底物，例如，其与转运体的ABC或SLC超家族结合的合成衍生物。One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates of muscle transporter proteins (e.g., transporter proteins expressed on the sarcolemma). In some embodiments, a muscle targeting agent is a substrate of an influx transporter that is specific for muscle tissue. In some embodiments, the influx transporter is specific for skeletal muscle tissue. Two major classes of transporters are expressed on the skeletal muscle sarcolemma: (1) the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue and (2) the solute carrier (SLC) superfamily, which facilitates substrate influx into skeletal muscle. In some embodiments, a muscle targeting agent is a substrate that binds to the ABC superfamily or the SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a natural substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-natural substrate, for example, a synthetic derivative that binds to the ABC or SLC superfamily of transporters.
在一些实施方案中，肌肉靶向剂是靶向SLC转运蛋白超家族的本文中所述的任何肌肉靶向剂(例如，抗体、核酸、小分子、肽、适配体、脂质、糖部分)。在一些实施方案中，肌肉靶向剂可以靶向转运蛋白(例如，可以是底物)。SLC转运体是平衡型的，或者使用跨膜产生的质子或钠离子梯度来驱动底物的转运。具有高骨骼肌表达的示例性SLC转运体包括但不限于SATT转运体(ASCT1；SLC1A4)、GLUT4转运体(SLC2A4)、GLUT7转运体(GLUT7；SLC2A7)、ATRC2转运体(CAT-2；SLC7A2)、LAT3转运体(KIAA0245；SLC7A6)、PHT1转运体(PTR4；SLC15A4)、OATP-J转运体(OATP5A1；SLC21A15)、OCT3转运体(EMT；SLC22A3)、OCTN2转运体(FLJ46769；SLC22A5)、ENT转运体(ENT1；SLC29A1和ENT2；SLC29A2)、PAT2转运体(SLC36A2)和SAT2转运体(KIAA1382；SLC38A2)。这些转运体可促进底物流入骨骼肌中，为肌肉靶向提供机会。In some embodiments, the muscle targeting agent is any muscle targeting agent described herein (e.g., an antibody, nucleic acid, small molecule, peptide, aptamer, lipid, sugar moiety) that targets the SLC transporter superfamily. In some embodiments, the muscle targeting agent can target a transporter (e.g., can be a substrate). SLC transporters are equilibrium-type or use a proton or sodium ion gradient generated across a membrane to drive transport of a substrate. Exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can facilitate substrate influx into skeletal muscle, providing an opportunity for muscle targeting.
在一些实施方案中，肌肉靶向剂是平衡型核苷转运体2(equilibrativenucleoside transporter 2，ENT2)转运体的底物。相对于其他转运体，ENT2在骨骼肌中具有最高的mRNA表达之一。虽然人ENT2(hENT2)在大多数身体器官例如脑、心脏、胎盘、胸腺、胰腺、前列腺和肾中表达，但其在骨骼肌中特别丰富。人ENT2根据其浓度梯度促进其底物的吸收。ENT2通过转运广泛的嘌呤和嘧啶核苷碱基在维持核苷稳态中发挥作用。hENT2转运体对除肌苷之外的所有核苷(腺苷、鸟苷、尿苷、胸苷和胞苷)均具有低的亲和力。因此，在一些实施方案中，肌肉靶向剂是ENT2底物。示例性的ENT2底物包括但不限于肌苷、2’,3’-二脱氧肌苷和氯法拉滨(calofarabine)。在一些实施方案中，本文中提供的任何肌肉靶向剂均与分子载荷(例如，寡核苷酸载荷)相关。在一些实施方案中，肌肉靶向剂与分子载荷共价连接。在一些实施方案中，肌肉靶向剂与分子载荷非共价连接。In some embodiments, the muscle targeting agent is a substrate of the equilibrative nucleoside transporter 2 (ENT2) transporter. ENT2 has one of the highest mRNA expressions in skeletal muscle relative to other transporters. Although human ENT2 (hENT2) is expressed in most body organs such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, it is particularly abundant in skeletal muscle. Human ENT2 promotes the absorption of its substrate according to its concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleoside bases. The hENT2 transporter has a low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. Therefore, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2', 3'-dideoxyinosine, and calofarabine. In some embodiments, any muscle targeting agent provided herein is associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular cargo. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular cargo.
在一些实施方案中，肌肉靶向剂是有机阳离子/肉碱转运体(OCTN2)的底物，所述有机阳离子/肉碱转运体是钠离子依赖性的高亲和力肉碱转运体。在一些实施方案中，肌肉靶向剂是肉碱、米屈肼(mildronate)、乙酰肉碱或其与OCTN2结合的任意衍生物。在一些实施方案中，肉碱、米屈肼、乙酰肉碱或其衍生物与分子载荷(例如，寡核苷酸载荷)共价连接。肌肉靶向剂可以是蛋白质，该蛋白质是以靶向肌细胞的至少一种可溶性形式存在的蛋白质。在一些实施方案中，肌肉靶向蛋白可以是血幼素(也称为排斥性导向分子C或血色素沉着症2型蛋白)，所述血幼素是参与铁超负荷和稳态的蛋白。在一些实施方案中，血幼素可以是全长或片段，或者与功能性血幼素蛋白具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％序列同一性的突变体。在一些实施方案中，血幼素突变体可以是可溶性片段，可缺乏N端信号传导，和/或缺乏C端锚定结构域。在一些实施方案中，血幼素可以以GenBank RefSeq登录号NM_001316767.1，NM_145277.4，NM_202004.3，NM_213652.3，或NM_213653.3进行注释。应理解，血幼素可以是人、非人灵长类或啮齿动物来源的。In some embodiments, the muscle targeting agent is a substrate of the organic cation/carnitine transporter (OCTN2), which is a sodium ion-dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, meldonate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, meldonate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload). The muscle targeting agent can be a protein that is present in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein can be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), which is a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin can be full length or a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack N-terminal signaling, and/or lack a C-terminal anchoring domain. In some embodiments, hemojuvelin may be annotated with GenBank RefSeq accession number NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be understood that hemojuvelin may be of human, non-human primate or rodent origin.
B.分子载荷B. Molecular Loading
本公开内容的一些方面提供了分子载荷，例如，用于调节生物学结局，例如，DNA序列的转录、蛋白质的表达或蛋白质的活性。在一些实施方案中，分子载荷与肌肉靶向剂共价连接或以其他方式相关联。应理解，根据本公开内容可使用多种类型的肌肉靶向剂。例如，分子载荷可包含以下或由以下组成：寡核苷酸(例如，反义寡核苷酸)、肽(例如，结合肌细胞中与疾病相关的核酸或蛋白质的肽)、蛋白质(例如，结合肌细胞中与疾病相关的核酸或蛋白质的蛋白质)或小分子(例如，调节肌细胞中与疾病相关的核酸或蛋白质之功能的小分子)。在一些实施方案中，这样的分子载荷能够靶向肌细胞，例如在通过相关肌肉靶向剂递送至肌细胞之后通过与肌细胞中的核酸或蛋白质特异性结合。在一些实施方案中，分子载荷是包含具有与表1中提供的基因互补的区域的链的寡核苷酸。本文中进一步详细描述了示例性分子载荷，然而，应理解，本文中提供的示例性分子载荷并不意味着是限制性的。Some aspects of the present disclosure provide molecular payloads, for example, for regulating biological outcomes, such as transcription of a DNA sequence, expression of a protein, or activity of a protein. In some embodiments, the molecular payload is covalently linked to or otherwise associated with a muscle targeting agent. It should be understood that various types of muscle targeting agents can be used according to the present disclosure. For example, the molecular payload may comprise or consist of an oligonucleotide (e.g., an antisense oligonucleotide), a peptide (e.g., a peptide that binds to a disease-associated nucleic acid or protein in a muscle cell), a protein (e.g., a protein that binds to a disease-associated nucleic acid or protein in a muscle cell), or a small molecule (e.g., a small molecule that regulates the function of a disease-associated nucleic acid or protein in a muscle cell). In some embodiments, such a molecular payload is capable of targeting a muscle cell, for example, by specifically binding to a nucleic acid or protein in a muscle cell after being delivered to the muscle cell by a relevant muscle targeting agent. In some embodiments, the molecular payload is an oligonucleotide comprising a chain having a region complementary to a gene provided in Table 1. Exemplary molecular payloads are described in further detail herein, however, it should be understood that the exemplary molecular payloads provided herein are not meant to be limiting.
在一些实施方案中，至少一个(例如，至少2个、至少3个、至少4个、至少5个、至少10个)分子载荷(例如寡核苷酸)与肌肉靶向剂共价连接。在一些实施方案中，与肌肉靶向剂连接的所有分子载荷是相同的，例如，靶向相同的基因。在一些实施方案中，与肌肉靶向剂连接的所有分子载荷是不同的，例如分子载荷可靶向相同靶基因的不同部分，或者分子载荷可靶向至少两种不同的靶基因。在一些实施方案中，肌肉靶向剂可与一些相同的分子载荷或一些不同的分子载荷连接。In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) molecular payloads (e.g., oligonucleotides) are covalently linked to a muscle targeting agent. In some embodiments, all molecular payloads linked to a muscle targeting agent are identical, e.g., target the same gene. In some embodiments, all molecular payloads linked to a muscle targeting agent are different, e.g., the molecular payloads can target different portions of the same target gene, or the molecular payloads can target at least two different target genes. In some embodiments, a muscle targeting agent can be linked to some of the same molecular payloads or some different molecular payloads.
本公开内容还提供了包含多个复合物的组合物，其中至少80％(例如，至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％)的复合物包含与相同数目的分子载荷(例如，寡核苷酸)共价连接的分子靶向剂。The present disclosure also provides compositions comprising a plurality of complexes, wherein at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the complexes comprise a molecular targeting agent covalently linked to the same number of molecular payloads (e.g., oligonucleotides).
i.寡核苷酸i. Oligonucleotides
如本文中所述的，任何合适的寡核苷酸均可用作分子载荷。在一些实施方案中，寡核苷酸可被设计为引起mRNA的降解(例如，寡核苷酸可以是引起降解的间隔聚体、siRNA、核酶或适配体)。在一些实施方案中，寡核苷酸可被设计为阻断mRNA的翻译(例如，寡核苷酸可以是阻断翻译的混合聚体、siRNA或适配体)。在一些实施方案中，寡核苷酸可被设计为引起mRNA的降解和阻断翻译。在一些实施方案中，寡核苷酸可以是用于指导酶(例如，基因编辑酶)活性的指导核酸(例如，指导RNA)。本文中提供了寡核苷酸的另一些实例。应理解，在一些实施方案中，通过将功能序列(例如，反义链序列)从一种格式并入另一种格式，一种格式的寡核苷酸(例如，反义寡核苷酸)可适当地适应于另一种格式(例如，siRNA寡核苷酸)。As described herein, any suitable oligonucleotide can be used as a molecular payload. In some embodiments, oligonucleotides can be designed to cause degradation of mRNA (for example, oligonucleotides can be spaced polymers, siRNA, ribozymes or aptamers that cause degradation). In some embodiments, oligonucleotides can be designed to block translation of mRNA (for example, oligonucleotides can be mixed polymers, siRNA or aptamers that block translation). In some embodiments, oligonucleotides can be designed to cause degradation of mRNA and block translation. In some embodiments, oligonucleotides can be guide nucleic acids (for example, guide RNA) for guiding enzyme (for example, gene editing enzyme) activity. Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, by incorporating a functional sequence (for example, an antisense strand sequence) from one format into another format, an oligonucleotide of one format (for example, an antisense oligonucleotide) can be appropriately adapted to another format (for example, siRNA oligonucleotides).
在一些实施方案中，寡核苷酸可包含与表1中提供的靶基因互补的区域。下面为表1的选择的基因提供了进一步的非限制性实例。In some embodiments, an oligonucleotide may comprise a region complementary to a target gene provided in Table 1. Further non-limiting examples for selected genes of Table 1 are provided below.
DMPK/DM1DMPK/DM1
在一些实施方案中，可用于靶向DMPK(例如用于治疗DM1)的寡核苷酸的一些实例提供在以下中：美国专利申请公开20100016215A1，其于2010年1月1日公开，标题为Compound And Method For Treating Myotonic Dystrophy；美国专利申请公开20130237585A1，其于2010年7月19日公开，Modulation Of Dystrophia Myotonica-Protein Kinase(DMPK)Expression；美国专利申请公开20150064181A1，其于2015年3月5日公开，标题为“Antisense Conjugates For Decreasing Expression Of Dmpk”；美国专利申请公开20150238627A1，其于2015年8月27日公开，标题为“Peptide-Linked MorpholinoAntisense Oligonucleotides For Treatment Of Myotonic Dystrophy”；Pandey,S.K.etal.“Identification and Characterization of Modified AntisenseOligonucleotides Targeting DMPK in Mice and Nonhuman Primates for theTreatment of Myotonic Dystrophy Type 1”J.of Pharmacol Exp Ther,2015,355:329-340.；Langlois,M.et al.“Cytoplasmic and Nuclear Retained DMPK mRNAs AreTargets for RNA Interference in Myotonic Dystrophy Cells”J.BiologicalChemistry,2005,280:17,16949-16954.；Jauvin,D.et al.“Targeting DMPK withAntisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type1Mice”,Mol.Ther:Nucleic Acids,2017,7:465-474.；Mulders,S.A.et al.“Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonicdystrophy”PNAS,2009,106:33,13915-13920.；Wheeler,T.M.et al.,“Targeting nuclearRNA for in vivo correction of myotonic dystrophy”Nature,2012,488(7409):111-115.；和美国专利申请公开20160304877A1，其于2016年10月20日公开，标题为“CompoundsAnd Methods For Modulation Of Dystrophia Myotonica-Protein Kinase(Dmpk)Expression”，其各自的内容通过引用整体并入本文。In some embodiments, some examples of oligonucleotides that can be used to target DMPK (e.g., for the treatment of DM1) are provided in the following: U.S. Patent Application Publication 20100016215A1, published on January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication 20130237585A1, published on July 19, 2010, Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication 20150064181A1, published on March 5, 2015, entitled “Antisense Conjugates For Decreasing Expression Of Dmpk”; U.S. Patent Application Publication 20150238627A1, published on August 27, 2015, entitled “Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy"; Pandey, S.K. et al. "Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for theTreatment of Myotonic Dystrophy Type 1" J. of Pharmacol Exp Ther, 2015, 355:329-340.; Langlois, M. et al. "Cytoplasmic and Nuclear Retained DMPK mRNAs AreTargets for RNA Interference in Myotonic Dystrophy Cells" J. Biological Chemistry, 2005, 280: 17, 16949-16954.; Jauuclevin, D. et al. "Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice", Mol. Ther: N Acids, 2017, 7:465-474.；Mulders, S.A. et al. "Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy" PNAS, 2009, 106:33, 13915-13920.；Wheeler, T.M. et al., "Targeting nuclear RNA for in vivo correction of myotonic dystrophy" Nature, 2012, 488(7409): 111-115.；And U.S. Patent Application Publication No. 20160304877A1, published on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression", the contents of each of which are incorporated herein by reference in their entirety.
用于促进DMPK基因编辑的寡核苷酸的一些实例包括美国专利申请公开20170088819A1，其于2017年3月3日公开，标题为“Genetic Correction Of MyotonicDystrophy Type 1”；和国际专利申请公开WO18002812A1，其于2018年4月1日公开，标题为“Materials And Methods For Treatment Of Myotonic Dystrophy Type 1(DM1)AndOther Related Disorders”，其各自的内容通过引用整体并入本文。Some examples of oligonucleotides for promoting DMPK gene editing include U.S. Patent Application Publication No. 20170088819A1, published on March 3, 2017, entitled “Genetic Correction Of Myotonic Dystrophy Type 1”; and International Patent Application Publication No. WO18002812A1, published on April 1, 2018, entitled “Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM1) And Other Related Disorders”, the contents of each of which are incorporated herein by reference in their entirety.
在一些实施方案中，寡核苷酸可具有与DMPK的突变体形式例如，如在以下中报道的突变体形式互补的区域：Botta A.et al.“The CTG repeat expansion sizecorrelates with the splicing defects observed in muscles from myotonicdystrophy type 1patients.”J Med Genet.2008Oct；45(10):639-46.；和Machuca-TziliL.et al.“Clinical and molecular aspects of the myotonic dystrophies:areview.”Muscle Nerve.2005Jul；32(1):1-18.；其各自的内容通过引用整体并入本文。In some embodiments, the oligonucleotide may have a region complementary to a mutant form of DMPK, for example, as reported in: Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients." J Med Genet. 2008 Oct; 45(10): 639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review." Muscle Nerve. 2005 Jul; 32(1): 1-18.; the contents of each of which are incorporated herein by reference in their entirety.
在一些实施方案中，本文中提供的寡核苷酸是靶向DMPK的反义寡核苷酸。在一些实施方案中，靶向的寡核苷酸是靶向DMPK的任一种反义寡核苷酸(例如，间隔聚体)，如美国专利申请公开US20160304877A1中所述，其于2016年10月20日公开，标题为“Compounds AndMethods For Modulation Of Dystrophia Myotonica-Protein Kinase(DMPK)Expression,”其通过引用并入本文)。在一些实施方案中，DMPK靶向寡核苷酸靶向如Genbank登录No.NM_001081560.2中所示或如Genbank登录No.NG_009784.1中所示的DMPK基因序列的区域。In some embodiments, the oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK. In some embodiments, the oligonucleotide targeted is any antisense oligonucleotide targeting DMPK (e.g., spaced polymers), as described in U.S. Patent Application Publication US20160304877A1, which is disclosed on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," which is incorporated herein by reference). In some embodiments, the DMPK targeting oligonucleotide targets the region of the DMPK gene sequence as shown in Genbank Accession No.NM_001081560.2 or as shown in Genbank Accession No.NG_009784.1.
在一些实施方案中，DMPK靶向寡核苷酸包含含有与靶区域互补的区域的核苷酸序列，所述靶区域是Genbank登录No.NM_001081560.2中的至少10个连续核苷酸(例如，至少10个、至少12个、至少14个、至少16个或更多个连续核苷酸)。In some embodiments, the DMPK targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region, the target region being at least 10 consecutive nucleotides (e.g., at least 10, at least 12, at least 14, at least 16, or more consecutive nucleotides) in Genbank Accession No. NM_001081560.2.
在一些实施方案中，DMPK靶向寡核苷酸包含间隔聚体基序。“间隔聚体”意指其中具有多个支持RNA酶H切割的核苷酸的内部区域位于具有一个或更多个核苷酸的外部区域之间的嵌合反义化合物，其中包含内部区域的核苷酸在化学上不同于包含外部区域的一个或更多个核苷酸。内部区域可被称为“间隔区段”并且外部区域可被称为“翼区段”。在一些实施方案中，DMPK靶向寡核苷酸包含一个或更多个经修饰核苷酸，和/或一个或更多个经修饰核苷酸间键联。在一些实施方案中，核苷酸间键联是硫代磷酸酯键联。在一些实施方案中，寡核苷酸包含完整的硫代磷酸酯主链。在一些实施方案中，寡核苷酸是具有cET末端的DNA间隔聚体(例如，3-10-3；cET-DNA-cET)。在一些实施方案中，DMPK靶向寡核苷酸包含一个或更多个6’-(S)-CH3二环核苷酸、一个或更多个β-D-2’-脱氧核糖核苷酸和/或者一个或更多个5-甲基胞嘧啶核苷酸。In some embodiments, the DMPK targeting oligonucleotide comprises a spacer polymer motif. "Spacer polymer" means a chimeric antisense compound in which an internal region having a plurality of nucleotides supporting RNA enzyme H cleavage is located between an external region having one or more nucleotides, wherein the nucleotides comprising the internal region are chemically different from one or more nucleotides comprising the external region. The internal region may be referred to as a "spacer segment" and the external region may be referred to as a "wing segment". In some embodiments, the DMPK targeting oligonucleotide comprises one or more modified nucleotides, and/or one or more modified internucleotide linkages. In some embodiments, the internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA spacer polymer (e.g., 3-10-3; cET-DNA-cET) with a cET end. In some embodiments, the DMPK targeting oligonucleotide comprises one or more 6'-(S)-CH3 bicyclic nucleotides, one or more β-D-2'-deoxyribonucleotides and/or one or more 5-methylcytosine nucleotides.
DUX4/FSHDDUX4/FSHD
在一些实施方案中，可用于靶向DUX4(例如用于治疗FSHD)的寡核苷酸的实例提供在以下中：美国专利号9,988,628，其于2017年2月2日公开，标题为“AGENTS USEFUL INTREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”；美国专利号9,469,851，其于2014年10月30日公开，标题为“RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITINGEXPRESSION OF DUX4”；美国专利申请公开20120225034，其于2012年9月6日公开，标题为“AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”；PCT专利申请公开号WO 2013/120038，其于2013年8月15日公开，标题为“MORPHOLINO TARGETINGDUX4FOR TREATING FSHD”；Chen et al.,“Morpholino-mediated Knockdown of DUX4Toward Facioscapulohumeral Muscular Dystrophy Therapeutics,”MolecularTherapy,2016,24:8,1405-1411.；以及Ansseau et al.,“Antisense OligonucleotidesUsed to Target the DUX4 mRNAas Therapeutic Approaches in FacioscapulohumeralMuscular Dystrophy(FSHD),”Genes,2017,8,93.，其各自的内容已其整体并入本文。在一些实施方案中，寡核苷酸是与靶DUX4基因或mRNA杂交的反义寡核苷酸、吗啉代、siRNA、shRNA或其他核苷酸。In some embodiments, examples of oligonucleotides that can be used to target DUX4 (e.g., for the treatment of FSHD) are provided in the following: U.S. Patent No. 9,988,628, which was published on February 2, 2017, and is entitled “AGENTS USEFUL INTREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; U.S. Patent No. 9,469,851, which was published on October 30, 2014, and is entitled “RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4”; U.S. Patent Application Publication No. 20120225034, which was published on September 6, 2012, and is entitled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; PCT Patent Application Publication No. WO 2013/120038, published on August 15, 2013, entitled "MORPHOLINO TARGETING DUX4 FOR TREATING FSHD"; Chen et al., "Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics," Molecular Therapy, 2016, 24:8, 1405-1411.; and Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes, 2017, 8, 93., the contents of each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA or other nucleotide that hybridizes to the target DUX4 gene or mRNA.
在一些实施方案中，例如用于治疗FSHD，寡核苷酸可具有与低甲基化的紧凑的D4Z4重复互补的区域，如以下中所述：Daxinger,et al.,“Genetic and EpigeneticContributors to FSHD,”2015年在Curr Opin Genet Dev中公开；Lim J-W,et al.,DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenoussiRNAsuggests mechanisms and therapies for FSHD Hum Mol Genet.2015Sep 1；24(17):4817–4828，其各自的内容以其整体并入本文。In some embodiments, for example for the treatment of FSHD, the oligonucleotide may have a region complementary to the hypomethylated compact D4Z4 repeats, as described in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," published in Curr Opin Genet Dev in 2015; Lim J-W, et al., DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817–4828, the contents of each of which are incorporated herein in their entirety.
DNM2/CNMDNM2/CNM
在一些实施方案中，可用于靶向DNM2(例如用于治疗CNM)的寡核苷酸的实例提供在以下中：美国专利申请公开号20180142008，其于2018年5月24日公开，标题为“DYNAMIN2INHIBITOR FOR THE TREATMENT OF DUCHENNE’S MUSCULAR DYSTROPHY”，以及PCT申请公开号WO 2018/100010A1，其于2018年6月7日公开，标题为“ALLELE-SPECIFIC SILENCINGTHERAPY FOR DYNAMIN 2-RELATED DISEASES”。例如，在一些实施方案中，寡核苷酸是特异性干扰DNM2表达的RNAi、反义核酸、siRNA或核酶。可用于靶向DNM2的寡核苷酸的另一些实例提供在以下中：Tasfaout,et al.,“Single Intramuscular Injection of AAV-shRNAReduces DNM2 and Prevents Myotubular Myopathy in Mice,”，2018年4月4日在Mol.Ther.中公开，以及Tasfaout,et al.,“Antisense oligonucleotide-mediated Dnm2knockdown prevents and reverts myotubular myopathy in mice,”NatureCommunications volume 8,Article number:15661(2017)。在一些实施方案中，寡核苷酸是有效靶向DNM2 mRNA的shRNA或吗啉代。在一些实施方案中，寡核苷酸编码对miR-133活性具有抗性的野生型DNM2，如以下中所述：Todaka,et al.“Overexpression of NF90-NF45Represses Myogenic MicroRNA Biogenesis,Resulting in Development of SkeletalMuscle Atrophy and Centronuclear Muscle Fibers,”，2015年7月在Mol.Cell Biol.中公开。可用于靶向DNM2的寡核苷酸的另一些实例提供在以下中：Gibbs,et al.,“TwoDynamin-2Genes are Required for Normal Zebrafish Development”，2013年在PLoSOne中公开，其各自的内容以其整体并入本文。In some embodiments, examples of oligonucleotides that can be used to target DNM2 (e.g., for the treatment of CNM) are provided in U.S. Patent Application Publication No. 20180142008, published on May 24, 2018, entitled "DYNAMIN2 INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY," and PCT Application Publication No. WO 2018/100010A1, published on June 7, 2018, entitled "ALLELE-SPECIFIC SILENCING THERAPY FOR DYNAMIN 2-RELATED DISEASES." For example, in some embodiments, the oligonucleotide is an RNAi, antisense nucleic acid, siRNA, or ribozyme that specifically interferes with the expression of DNM2. Other examples of oligonucleotides that can be used to target DNM2 are provided in: Tasfaout, et al., "Single Intramuscular Injection of AAV-shRNAReduces DNM2 and Prevents Myotubular Myopathy in Mice," published in Mol. Ther. on April 4, 2018, and Tasfaout, et al., "Antisense oligonucleotide-mediated Dnm2knockdown prevents and reverts myotubular myopathy in mice," Nature Communications volume 8, Article number: 15661 (2017). In some embodiments, the oligonucleotide is an shRNA or morpholino that effectively targets DNM2 mRNA. In some embodiments, the oligonucleotide encodes a wild-type DNM2 that is resistant to miR-133 activity, as described in Todaka, et al. "Overexpression of NF90-NF45 Represses Myogenic MicroRNA Biogenesis, Resulting in Development of Skeletal Muscle Atrophy and Centronuclear Muscle Fibers," published in Mol. Cell Biol. in July 2015. Other examples of oligonucleotides that can be used to target DNM2 are provided in Gibbs, et al., "Two Dynamin-2 Genes are Required for Normal Zebrafish Development," published in PLoS One in 2013, the contents of each of which are incorporated herein in their entirety.
在一些实施方案中，例如用于治疗CNM，寡核苷酸可具有与CNM相关的DNM2中的突变体互补的区域，如以下中所述：et al,“Mutation Spectrum in the LargeGTPase Dynamin 2,and Genotype-Phenotype Correlation in Autosomal DominantCentronuclear Myopathy,”，如在2012年在Hum.Mutat.中公开，其内容以其整体并入本文。In some embodiments, for example for the treatment of CNM, the oligonucleotide may have a region complementary to a mutant in DNM2 associated with CNM, as described below: et al, "Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy," as published in Hum. Mutat., 2012, the contents of which are incorporated herein in their entirety.
庞贝病Pompe disease
在一些实施方案中，例如用于治疗庞贝病，寡核苷酸介导在GAA疾病等位基因中包含外显子2，如van der Wal,et al.,“GAA Deficiency in Pompe Disease is Alleviatedby Exon Inclusion in iPSC-Derived Skeletal Muscle Cells,”Mol Ther NucleicAcids.2017Jun 16；7:101–115，其内容通过引用并入本文。因此，在一些实施方案中，寡核苷酸可具有与GAA疾病等位基因互补的区域。In some embodiments, for example for the treatment of Pompe disease, the oligonucleotide mediates the inclusion of exon 2 in the GAA disease allele, as described in van der Wal, et al., "GAA Deficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells," Mol Ther Nucleic Acids. 2017 Jun 16; 7: 101–115, the contents of which are incorporated herein by reference. Thus, in some embodiments, the oligonucleotide may have a region complementary to the GAA disease allele.
在一些实施方案中，例如用于治疗庞贝病，利用寡核苷酸(例如RNAi或反义寡核苷酸)来抑制肌细胞中野生型GYS1的表达，如例如以下中报道的：Clayton,et al.,“Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase1Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease,”2017年在Mol Ther Nucleic Acids中公开，或美国专利申请公开号2017182189，其于2017年6月29日公开，标题为“INHIBITING OR DOWNREGULATING GLYCOGEN SYNTHASE BYCREATING PREMATURE STOP CODONS USING ANTISENSE OLIGONUCLEOTIDES”，其内容通过引用并入本文。因此，在一些实施方案中，寡核苷酸可具有反义链，该反义链具有与对应于RefSeq号NM_002103.4的人GYS1序列和/或对应于RefSeq号NM_030678.3的小鼠GYS1序列的序列互补的区域。In some embodiments, for example, for the treatment of Pompe disease, oligonucleotides (e.g., RNAi or antisense oligonucleotides) are used to inhibit the expression of wild-type GYS1 in muscle cells, as reported, for example, in Clayton, et al., “Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease,” published in Mol Ther Nucleic Acids in 2017, or U.S. Patent Application Publication No. 2017182189, published on June 29, 2017, entitled “INHIBITING OR DOWNREGULATING GLYCOGEN SYNTHASE BY CREATING PREMATURE STOP CODONS USING ANTISENSE OLIGONUCLEOTIDES,” the contents of which are incorporated herein by reference. Thus, in some embodiments, an oligonucleotide can have an antisense strand having a region complementary to a sequence corresponding to the human GYS1 sequence of RefSeq No. NM_002103.4 and/or a mouse GYS1 sequence corresponding to RefSeq No. NM_030678.3.
ACVR1/FOPACVR1/FOP
在一些实施方案中，可用于靶向ACVR1(例如用于治疗FOP)的寡核苷酸的实例提供在以下中：美国专利申请2009/0253132，2009年10月8日公开，“Mutated ACVR1 fordiagnosis and treatment of fibrodysplasia ossificans progressiva(FOP)”；WO2015/152183，2015年10月8日公开，“Prophylactic agent and therapeutic agent forfibrodysplasia ossificans progressive”；Lowery,J.W.et al,“Allele-specific RNAInterference in FOP-Silencing the FOP gene”,GENE THERAPY,vol.19,2012,pages701–702；Takahashi,M.et al.“Disease-causing allele-specific silencing againstthe ALK2 mutants,R206H and G356D,in fibrodysplasia ossificans progressiva”Gene Therapy(2012)19,781–785；Shi,S.et al.“Antisense-Oligonucleotide MediatedExon Skipping in Activin-Receptor-Like Kinase 2:Inhibiting the Receptor ThatIs Overactive in Fibrodysplasia Ossificans Progressiva”Plos One,July 2013,Vol8:7,e69096.；美国专利申请2017/0159056，2017年6月8日公开，“Antisenseoligonucleotides and methods of use thereof”；美国专利No.8,859,752，2014年10月4日授权，“SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva(FOP)”；WO2004/094636，2004年11月4日公开，“Effective sirna knock-down constructs”，其各自的内容以其整体并入本文。In some embodiments, examples of oligonucleotides that can be used to target ACVR1 (e.g., for the treatment of FOP) are provided in the following: U.S. Patent Application 2009/0253132, published on October 8, 2009, "Mutated ACVR1 for diagnosis and treatment of fibrodysplasia ossificans progressiva (FOP)"; WO2015/152183, published on October 8, 2015, "Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive"; Lowery, J.W. et al, "Allele-specific RNA Interference in FOP-Silencing the FOP gene", GENE THERAPY, vol. 19, 2012, pages 701-702; Takahashi, M. et al. "Disease-causing allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva” Gene Therapy (2012) 19, 781–785; Shi, S. et al. “Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor ThatIs Overactive in Fibrodysplasia Ossificans Progressiva” Plos One, July 2013, Vol8:7, e69096.; U.S. Patent Application 2017/0159056, published on June 8, 2017, “Antisense oligonucleotides and methods of use thereof”; U.S. Patent No. 8,859,752, issued on October 4, 2014, “SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)”; WO2004/094636, published on November 4, 2004, “Effective siRNA knock-down constructs", the contents of each of which are incorporated herein in their entirety.
FXN/弗里德赖希共济失调FXN/Friedreich's Ataxia
在一些实施方案中，可用于靶向FXN和/或以其他方式补偿共济蛋白缺乏(例如用于治疗弗里德赖希共济失调)的寡核苷酸的实例提供在以下中：Li,L.et al“Activatingfrataxin expression by repeat-targeted nucleic acids”Nat.Comm.2016,7:10606.；WO 2016/094374，2016年6月16日公开，“Compositions and methods for treatment offriedreich's ataxia.”；WO 2015/020993，2015年2月12日公开，“RNAi COMPOSITIONS ANDMETHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA”；WO 2017/186815，2017年11月2日公开，“Antisense oligonucleotides for enhanced expression of frataxin”；WO 2008/018795，2008年2月14日公开，“Methods and means for treating dna repeatinstability associated genetic disorders”；美国专利申请2018/0028557，2018年2月1日公开,“Hybrid oligonucleotides and uses thereof”；WO 2015/023975，2015年2月19日公开，“Compositions and methods for modulating RNA”；WO 2015/023939，2015年2月19日公开，“Compositions and methods for modulating expression of frataxin”；美国专利申请2017/0281643，2017年10月5日公开，“Compounds and methods formodulating frataxin expression”；Li L.et al.,“Activating frataxin expressionby repeat-targeted nucleic acids”Nature Communications,2016年2月4日公开；以及Li L.et al.“Activation of Frataxin Protein Expression by AntisenseOligonucleotides Targeting the Mutant Expanded Repeat”Nucleic AcidTher.2018Feb；28(1):23-33.，其各自的内容以其整体并入本文。In some embodiments, examples of oligonucleotides that can be used to target FXN and/or otherwise compensate for frataxin deficiency (e.g., for the treatment of Friedreich's ataxia) are provided in the following: Li, L. et al "Activating frataxin expression by repeat-targeted nucleic acids" Nat. Comm. 2016, 7: 10606.; WO 2016/094374, published on June 16, 2016, "Compositions and methods for treatment of offriedreich's ataxia."; WO 2015/020993, published on February 12, 2015, "RNAi COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA"; WO 2017/186815, published on November 2, 2017, "Antisense oligonucleotides for enhanced expression of frataxin"; WO 2008/018795, published on February 14, 2008, “Methods and means for treating DNA repeat instability associated genetic disorders”; U.S. patent application 2018/0028557, published on February 1, 2018, “Hybrid oligonucleotides and uses thereof”; WO 2015/023975, published on February 19, 2015, “Compositions and methods for modulating RNA”; WO 2015/023939, published on February 19, 2015, “Compositions and methods for modulating expression of frataxin”; U.S. patent application 2017/0281643, published on October 5, 2017, “Compounds and methods for modulating frataxin expression”; Li L. et al., “Activating frataxin expression by repeat-targeted nucleic acids” Nature Communications, published on February 4, 2016; and Li L.et al. "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat" Nucleic Acid Ther. 2018 Feb; 28(1): 23-33., the contents of each of which are incorporated herein in their entirety.
在一些实施方案中，寡核苷酸载荷被配置成(例如，作为间隔聚体或RNAi寡核苷酸)用于抑制天然反义转录物的表达，所述转录物抑制FXN表达例如如以下中公开的：美国专利No.9,593,330，2011年6月9日提交，“Treatment of frataxin(FXN)related diseasesby inhibition of natural antisense transcript to FXN”，其内容通过引用整体并入本文。In some embodiments, the oligonucleotide payload is configured (e.g., as a spacer or RNAi oligonucleotide) to inhibit expression of a natural antisense transcript that inhibits FXN expression, such as disclosed in U.S. Pat. No. 9,593,330, filed June 9, 2011, “Treatment of frataxin (FXN) related diseases by inhibition of natural antisense transcript to FXN,” the contents of which are incorporated herein by reference in their entirety.
用于促进FXN基因编辑的寡核苷酸的一些实例包括WO 2016/094845，2016年6月16日公开，“Compositions and methods for editing nucleic acids in cells utilizingoligonucleotides”；WO 2015/089354，2015年6月18日公开，“Compositions and methodsof use of CRISPR-Cas systems in nucleotide repeat disorders”；WO 2015/139139，2015年9月24日公开，“CRISPR-based methods and products for increasing frataxinlevels and uses thereof”，以及WO 2018/002783，2018年1月4日公开，“Materials andmethods for treatment of Friedreich ataxia and other related disorders”，其各自的内容以其整体并入本文。Some examples of oligonucleotides for facilitating FXN gene editing include WO 2016/094845, published on June 16, 2016, “Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides”; WO 2015/089354, published on June 18, 2015, “Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders”; WO 2015/139139, published on September 24, 2015, “CRISPR-based methods and products for increasing frataxin levels and uses thereof”, and WO 2018/002783, published on January 4, 2018, “Materials and methods for treatment of Friedreich ataxia and other related disorders”, the contents of each of which are incorporated herein in their entirety.
用于通过靶向非FXN基因(例如FXN的表观遗传调节物)来促进FXN基因表达的寡核苷酸的实例包括WO 2015/023938，2015年2月19日公开，“Epigenetic regulators offrataxin”，其内容以其整体并入本文。Examples of oligonucleotides for promoting FXN gene expression by targeting non-FXN genes (eg, epigenetic regulators of FXN) include WO 2015/023938, published February 19, 2015, "Epigenetic regulators of frataxin," the contents of which are incorporated herein in their entirety.
在一些实施方案中，寡核苷酸可具有与如下所示序列互补的区域：来自人的FXN基因(基因ID 2395；NC_000009.12)和/或来自小鼠的FXN基因(基因ID 14297；NC_000085.6)。在一些实施方案中，寡核苷酸可具有与FXN的突变形式互补的区域，例如如以下中报道的：例如Montermini,L.et al.“The Friedreich ataxia GAA triplet repeat:premutationand normal alleles.”Hum.Molec.Genet.,1997,6:1261-1266.；Filla,A.et al.“Therelationship between trinucleotide(GAA)repeat length and clinical features inFriedreich ataxia.”Am.J.Hum.Genet.1996,59:554-560.；Pandolfo,M.Friedreichataxia:the clinical picture.J.Neurol.2009,256,3–8.，其各自的内容通过引用整体并入本文。In some embodiments, the oligonucleotide may have a region complementary to a sequence such as the FXN gene from human (Gene ID 2395; NC_000009.12) and/or the FXN gene from mouse (Gene ID 14297; NC_000085.6). In some embodiments, the oligonucleotide may have a region complementary to a mutant form of FXN, such as reported in, for example, Montemini, L. et al. "The Friedreich ataxia GAA triplet repeat: premutation and normal alleles." Hum. Molec. Genet., 1997, 6: 1261-1266.; Filla, A. et al. "The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia." Am. J. Hum. Genet. 1996, 59: 554-560.; Pandolfo, M. Friedreich ataxia: the clinical picture. J. Neurol. 2009, 256, 3–8., the contents of each of which are incorporated herein by reference in their entirety.
DMD/肌养蛋白病(Dystrophinopathy)DMD/Dystrophinopathy
可用于靶向DMD的寡核苷酸的实例提供在以下中：美国专利申请公开US20100130591A1，其于2010年5月27日公开，标题为“MULTIPLE EXON SKIPPINGCOMPOSITIONS FOR DMD”；美国专利No.8,361,979，其于2013年1月29日授权，标题为“MEANSAND METHOD FOR INDUCING EXON-SKIPPING”；美国专利申请公开20120059042，其于2012年3月8日公开，标题为“METHOD FOR EFFICIENT EXON(44)SKIPPING IN DUCHENNE MUSCULARDYSTROPHY AND ASSOCIATED MEANS；美国专利申请公开20140329881，其于2014年11月6日公开，标题为“EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY”；美国专利No.8,232,384，其于2012年7月31日授权，标题为“ANTISENSE OLIGONUCLEOTIDES FORINDUCING EXON SKIPPING AND METHODS OF USE THEREOF”；美国专利申请公开20120022134A1，其于2012年1月26日公开，标题为“METHODS AND MEANS FOR EFFICIENTSKIPPING OF EXON 45IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA；美国专利申请公开20120077860，其于2012年3月29日公开，标题为“ADENO-ASSOCIATED VIRAL VECTOR FOREXON SKIPPING IN A GENE ENCODING ADISPENSABLE DOMAN PROTEIN”；美国专利No.8,324,371，其于2012年12月4日授权，标题为“OLIGOMERS”；美国专利No.9,078,911，其于2015年7月14日授权，标题为“ANTISENSE OLIGONUCLEOTIDES”；美国专利No.9,079,934，其于2015年7月14日授权，标题为“ANTISENSE NUCLEIC ACIDS”；美国专利No.9,034,838，其于2015年5月19日授权，标题为“MIR-31IN DUCHENNE MUSCULAR DYSTROPHY THERAPY”；以及国际专利公开WO2017062862A3，其于2017年4月13日公开，标题为“OLIGONUCLEOTIDECOMPOSITIONS AND METHODS THEREOF”；其各自的内容以其整体并入本文。Examples of oligonucleotides that can be used to target DMD are provided in the following: U.S. Patent Application Publication US20100130591A1, which was published on May 27, 2010, and is entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD”; U.S. Patent No. 8,361,979, which was issued on January 29, 2013, and is entitled “MEANS AND METHOD FOR INDUCING EXON-SKIPPING”; U.S. Patent Application Publication 20120059042, which was published on March 8, 2012, and is entitled “METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULARDYSTROPHY AND ASSOCIATED MEANS; U.S. Patent Application Publication 20140329881, which was published on November 6, 2014, and is entitled “EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY”; U.S. Patent No. 8,232,384, issued on July 31, 2012, entitled “ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF”; U.S. Patent Application Publication 20120022134A1, published on January 26, 2012, entitled “METHODS AND MEANS FOR EFFICIENTSKIPPING OF EXON 45IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA”; U.S. Patent Application Publication 20120077860, published on March 29, 2012, entitled “ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING ADISPENSABLE DOMAN U.S. Patent No. 8,324,371, issued on December 4, 2012, entitled “OLIGOMERS”; U.S. Patent No. 9,078,911, issued on July 14, 2015, entitled “ANTISENSE OLIGONUCLEOTIDES”; U.S. Patent No. 9,079,934, issued on July 14, 2015, entitled “ANTISENSE NUCLEIC ACIDS”; U.S. Patent No. 9,034,838, issued on May 19, 2015, entitled “MIR-31IN DUCHENNE MUSCULAR DYSTROPHY THERAPY”; and International Patent Publication WO2017062862A3, published on April 13, 2017, entitled “OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; the contents of each of which are incorporated herein in their entirety.
用于促进DMD基因编辑的寡核苷酸的实例包括国际专利公开WO2018053632A1，其于2018年3月29日公开，标题为“METHODS OF MODIFYING THE DYSTROPHIN GENE ANDRESTORING DYSTROPHIN EXPRESSION AND USES THEREOF”；国际专利公开WO2017049407A1，其于2017年3月30日公开，标题为“MODIFICATION OF THE DYSTROPHIN GENE AND USESTHEREOF”；国际专利公开WO2016161380A1，其于2016年10月6日公开，标题为“CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY ANDBECKER MUSCULAR DYSTROPHY”；国际专利公开WO2017095967，其于2017年6月8日公开，标题为“THERAPEUTIC TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BYGENE EDITING AND METHODS OF USE”；国际专利公开WO2017072590A1，其于2017年5月4日公开，标题为“MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULARDYSTROPHY”；国际专利公开WO2018098480A1，其于2018年5月31日公开，标题为“PREVENTIONOF MUSCULAR DYSTROPHY BY CRISPR/CPF1-MEDIATED GENE EDITING”；美国专利申请公开US20170266320A1，其于2017年9月21日公开，标题为“RNA-Guided Systems for In VivoGene Editing”；国际专利公开WO2016025469A1，其于2016年2月18日公开，标题为“PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR/CAS9-MEDIATED GENE EDITING”；美国专利申请公开2016/0201089，其于2016年7月14日公开，标题为“RNA-GUIDED GENE EDITINGAND GENE REGULATION”；以及美国专利申请公开2013/0145487，其于2013年6月6日公开，标题为“MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHNGENE AND USES THEREOF”，其各自的内容以其整体并入本文。在一些实施方案中，寡核苷酸可具有与多种物种(例如，选自人、小鼠和非人物种)的DMD基因序列互补的区域。Examples of oligonucleotides for promoting DMD gene editing include International Patent Publication WO2018053632A1, published on March 29, 2018, entitled “METHODS OF MODIFYING THE DYSTROPHIN GENE ANDRESTORING DYSTROPHIN EXPRESSION AND USES THEREOF”; International Patent Publication WO2017049407A1, published on March 30, 2017, entitled “MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF”; International Patent Publication WO2016161380A1, published on October 6, 2016, entitled “CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY”; International Patent Publication WO2017095967, which was published on June 8, 2017, and is entitled “THERAPEUTIC TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE”; International Patent Publication WO2017072590A1, which was published on May 4, 2017, and is entitled “MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULARDYSTROPHY”; International Patent Publication WO2018098480A1, which was published on May 31, 2018, and is entitled “PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR/CPF1-MEDIATED GENE U.S. Patent Application Publication No. US20170266320A1, published on September 21, 2017, entitled “RNA-Guided Systems for In Vivo Gene Editing”; International Patent Publication No. WO2016025469A1, published on February 18, 2016, entitled “PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR/CAS9-MEDIATED GENE EDITING”; U.S. Patent Application Publication No. 2016/0201089, published on July 14, 2016, entitled “RNA-GUIDED GENE EDITING AND GENE REGULATION”; and U.S. Patent Application Publication No. 2013/0145487, published on June 6, 2013, entitled “MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF", the contents of each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotides may have regions complementary to DMD gene sequences of multiple species (e.g., selected from human, mouse, and non-human species).
在一些实施方案中，寡核苷酸可具有与突变DMD等位基因互补的区域，所述突变DMD等位基因例如在人DMD的外显子1至79中的任何一个中具有至少一个突变的DMD等位基因，其导致移码和不正确的RNA剪接/加工。In some embodiments, the oligonucleotide may have a region complementary to a mutant DMD allele, such as a DMD allele having at least one mutation in any one of exons 1 to 79 of human DMD that results in a frameshift and improper RNA splicing/processing.
MYH7/肥厚型心肌病MYH7/Hypertrophic Cardiomyopathy
可用作载荷(例如用于靶向MYH7)的寡核苷酸的实例提供在以下中：美国专利申请公开20180094262，其于2018年4月5日公开，标题为Inhibitors of MYH7B and UsesThereof；美国专利申请公开20160348103，其于2016年12月1日公开，标题为Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNAInterference；美国专利申请公开20160237430，其于2016年8月18日公开，标题为“Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy”；美国专利申请公开20160032286，其于2016年2月4日公开，标题为“Inhibitors of MYH7BandUses Thereof”；美国专利申请公开20140187603，其于2014年7月3日公开，标题为“MicroRNA Inhibitors Comprising Locked Nucleotides”；美国专利申请公开20140179764，其于2014年6月26日公开，标题为“Dual Targeting of miR-208and miR-499in the Treatment of Cardiac Disorders”；美国专利申请公开20120114744，其于2012年5月10日公开，标题为“Compositions and Methods to Treat Muscular andCardiovascular Disorders”；其各自的内容以其整体并入本文。Examples of oligonucleotides that can be used as payloads (e.g., for targeting MYH7) are provided in the following: U.S. Patent Application Publication 20180094262, published on April 5, 2018, entitled Inhibitors of MYH7B and Uses Thereof; U.S. Patent Application Publication 20160348103, published on December 1, 2016, entitled Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference; U.S. Patent Application Publication 20160237430, published on August 18, 2016, entitled “Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy”; U.S. Patent Application Publication 20160032286, published on February 4, 2016, entitled “Inhibitors of MYH7B and Uses Thereof”. Thereof”; U.S. Patent Application Publication 20140187603, published on July 3, 2014, entitled “MicroRNA Inhibitors Comprising Locked Nucleotides”; U.S. Patent Application Publication 20140179764, published on June 26, 2014, entitled “Dual Targeting of miR-208and miR-499in the Treatment of Cardiac Disorders”; U.S. Patent Application Publication 20120114744, published on May 10, 2012, entitled “Compositions and Methods to Treat Muscular and Cardiovascular Disorders”; the contents of each of which are incorporated herein in their entirety.
在一些实施方案中，寡核苷酸可靶向lncRNA或mRNA，例如用于降解。在一些实施方案中，寡核苷酸可靶向(例如用于降解)编码参与错配修复途径的蛋白质(例如MSH2、MutLalpha、MutSbeta、MutLalpha)的核酸。参与错配修复途径的蛋白质(其中编码这样的蛋白质的mRNA可被本文中所述的寡核苷酸靶向)描述在以下中：Iyer,R.R.et al.,“DNAtriplet repeat expansion and mismatch repair”Annu Rev Biochem.2015；84:199-226.；以及Schmidt M.H.and Pearson C.E.,“Disease-associated repeat instabilityand mismatch repair”DNA Repair(Amst).2016Feb；38:117-26。In some embodiments, the oligonucleotides may target lncRNA or mRNA, for example, for degradation. In some embodiments, the oligonucleotides may target (e.g., for degradation) nucleic acids encoding proteins (e.g., MSH2, MutLalpha, MutSbeta, MutLalpha) involved in the mismatch repair pathway. Proteins involved in the mismatch repair pathway (wherein mRNA encoding such proteins may be targeted by the oligonucleotides described herein) are described in the following: Iyer, R.R.et al., "DNAtriplet repeat expansion and mismatch repair" Annu Rev Biochem. 2015; 84: 199-226.; and Schmidt M.H. and Pearson C.E., "Disease-associated repeat instability and mismatch repair" DNA Repair (Amst). 2016 Feb; 38: 117-26.
a.寡核苷酸尺寸/序列a. Oligonucleotide size/sequence
寡核苷酸可具有多种不同的长度，例如，取决于格式。在一些实施方案中，寡核苷酸的长度为7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、75或更多个核苷酸。在一些实施方案中，寡核苷酸的长度为8至50个核苷酸、长度为8至40个核苷酸、长度为8至30个核苷酸、长度为10至15个核苷酸、长度为10至20个核苷酸、长度为15至25个核苷酸、长度为21至23个核苷酸，等。Oligonucleotide can have multiple different lengths, for example, depending on format. In some embodiments, the length of oligonucleotide is 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,75 or more nucleotides. In some embodiments, the length of oligonucleotide is 8 to 50 nucleotides, the length is 8 to 40 nucleotides, the length is 8 to 30 nucleotides, the length is 10 to 15 nucleotides, the length is 10 to 20 nucleotides, the length is 15 to 25 nucleotides, the length is 21 to 23 nucleotides, etc.
在一些实施方案中，当寡核苷酸的互补核酸序列与靶分子(例如，mRNA)的结合干扰靶标(例如，mRNA)的正常功能导致缺乏活性(例如，抑制翻译)或表达(例如，降解靶mRNA)，并且具有足够程度的互补性以避免在以下情况下的序列与非靶标的非特异性结合时，出于本公开内容的目的，寡核苷酸的互补核酸序列可与靶核酸特异性杂交或对靶核酸具有特异性：在其中期望避免非特异性结合的条件下，例如在生理条件下在体内测定或治疗性处理的情况下，以及在体外测定的情况下，在其中在合适的严格条件下进行测定的条件下。因此，在一些实施方案中，寡核苷酸可与靶核酸的连续核苷酸至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％互补。在一些实施方案中，互补核苷酸序列不需要与其所靶向的100％互补来特异性地杂交或对靶核酸具有特异性。In some embodiments, when the binding of the complementary nucleic acid sequence of the oligonucleotide to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) resulting in a lack of activity (e.g., inhibition of translation) or expression (e.g., degradation of the target mRNA), and has a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-targets under the following conditions, for the purposes of the present disclosure, the complementary nucleic acid sequence of the oligonucleotide can specifically hybridize with the target nucleic acid or have specificity for the target nucleic acid: under conditions where it is desired to avoid non-specific binding, such as under physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays, under conditions where the assay is performed under suitable stringent conditions. Thus, in some embodiments, the oligonucleotide can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the contiguous nucleotides of the target nucleic acid. In some embodiments, a complementary nucleotide sequence need not be 100% complementary to its targeted sequence to specifically hybridize or be specific to a target nucleic acid.
在一些实施方案中，寡核苷酸包含与靶核酸互补的区域，所述区域的长度为8至15、8至30、8至40或10至50、或5至50或5至40个核苷酸。在一些实施方案中，寡核苷酸与靶核酸的互补区的长度为5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个核苷酸。在一些实施方案中，互补区与靶核酸的至少8个连续核苷酸互补。在一些实施方案中，与靶核酸的连续核苷酸部分相比，寡核苷酸可包含1、2或3个碱基错配。在一些实施方案中，寡核苷酸在15个碱基上可具有多至3个错配，或在10个碱基上具有多至2个错配。In some embodiments, the oligonucleotide comprises a region complementary to the target nucleic acid, and the length of the region is 8 to 15, 8 to 30, 8 to 40 or 10 to 50 or 5 to 50 or 5 to 40 nucleotides. In some embodiments, the length of the complementary region of the oligonucleotide and the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In some embodiments, the complementary region is complementary to at least 8 continuous nucleotides of the target nucleic acid. In some embodiments, compared with the continuous nucleotide portion of the target nucleic acid, the oligonucleotide can include 1, 2 or 3 base mispairings. In some embodiments, an oligonucleotide can have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
在一些实施方案中，寡核苷酸与本文中提供的任一寡核苷酸的靶序列互补(例如，至少85％、至少90％、至少95％或100％)。在一些实施方案中，这样的靶序列与本文中所述的寡核苷酸100％互补。In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to a target sequence of any of the oligonucleotides provided herein. In some embodiments, such a target sequence is 100% complementary to an oligonucleotide described herein.
在一些实施方案中，应理解在C5位置处核碱基尿嘧啶的甲基化形成胸腺嘧啶。因此，在一些实施方案中，具有C5甲基化尿嘧啶(或5-甲基尿嘧啶)的核苷酸或核苷可以等同地鉴定为胸腺嘧啶核苷酸或核苷。In some embodiments, it is understood that methylation of the nucleobase uracil at the C5 position forms thymine. Therefore, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyluracil) can be equivalently identified as a thymine nucleotide or nucleoside.
在一些实施方案中，本文中提供的任一寡核苷酸中的任一个或更多个胸腺嘧啶碱基(thymine bases，T’s)可独立地且任选地为尿嘧啶碱基(uracil bases，U’s)，和/或本文中提供的寡核苷酸中的任一个或更多个U’s可独立地且任选地为T’s。In some embodiments, any one or more thymine bases (T's) in any of the oligonucleotides provided herein may independently and optionally be uracil bases (U's), and/or any one or more U's in the oligonucleotides provided herein may independently and optionally be T's.
b.寡核苷酸修饰：b. Oligonucleotide modification:
本文中所述的寡核苷酸可被修饰，例如，包含经修饰糖部分、经修饰核苷间键联、经修饰核苷酸和/或其组合。另外，在一些实施方案中，寡核苷酸可表现出一种或更多种以下特性：不介导选择性剪接；不是免疫刺激性的；具有核酸酶抗性；与未经修饰寡核苷酸相比具有提高的细胞摄取；对细胞或哺乳动物无毒；提高了在细胞中内部排出内体；使TLR刺激最小化；或避免模式识别受体。本文中所述的寡核苷酸的任何经修饰化学或格式可彼此组合。例如，同一寡核苷酸内可包括一种、两种、三种、四种、五种或更多种不同类型的修饰。The oligonucleotides described herein may be modified, for example, to include a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide, and/or a combination thereof. In addition, in some embodiments, the oligonucleotide may exhibit one or more of the following properties: not mediating alternative splicing; not immunostimulatory; having nuclease resistance; having increased cellular uptake compared to unmodified oligonucleotides; being nontoxic to cells or mammals; increasing internal expulsion of endosomes in cells; minimizing TLR stimulation; or avoiding pattern recognition receptors. Any modified chemistry or format of the oligonucleotides described herein may be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included in the same oligonucleotide.
在一些实施方案中，可使用某些核苷酸修饰，所述核苷酸修饰使并入它们的寡核苷酸比天然寡脱氧核苷酸或寡核糖核苷酸分子对核酸酶消化更具抗性；这些经修饰寡核苷酸比未经修饰寡核苷酸完整存活更长的时间。经修饰寡核苷酸的一些具体实例包括含有经修饰主链的那些，例如经修饰核苷间键联，例如硫代磷酸酯键联、磷酸三酯键联、甲基膦酸酯键联、短链烷基键联或环烷基糖间键联或短链杂原子键联或杂环糖间键联。因此，本公开内容的寡核苷酸可例如通过并入修饰(例如核苷酸修饰)而稳定化以抵抗溶核降解。In some embodiments, certain nucleotide modifications may be used that render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact longer than unmodified oligonucleotides. Some specific examples of modified oligonucleotides include those containing modified backbones, such as modified internucleoside linkages, such as phosphorothioate linkages, phosphotriester linkages, methylphosphonate linkages, short-chain alkyl linkages or cycloalkyl sugar inter-linkages or short-chain heteroatom linkages or heterocyclic sugar inter-linkages. Thus, the oligonucleotides of the present disclosure may be stabilized, for example, by incorporating modifications (e.g., nucleotide modifications) to resist nucleolytic degradation.
在一些实施方案中，寡核苷酸的长度可以是多至50个或多至100个核苷酸，其中寡核苷酸的2至10、2至15、2至16、2至17、2至18、2至19、2至20、2至25、2至30、2至40、2至45或更多个核苷酸是经修饰核苷酸。寡核苷酸的长度可以是8至30个核苷酸，其中寡核苷酸的2至10、2至15、2至16、2至17、2至18、2至19、2至20、2至25、2至30个核苷酸是经修饰核苷酸。寡核苷酸的长度可以是8至15个核苷酸，其中寡核苷酸的2至4、2至5、2至6、2至7、2至8、2至9、2至10、2至11、2至12、2至13、2至14个核苷酸是经修饰核苷酸。任选地，寡核苷酸可具有除1、2、3、4、5、6、7、8、9或10个经修饰核苷酸之外的每个核苷酸。寡核苷酸修饰在本文中进一步描述。In some embodiments, the length of the oligonucleotide can be up to 50 or up to 100 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45 or more nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide can be 8 to 30 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide can be 8 to 15 nucleotides, wherein 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides. Optionally, the oligonucleotide can have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified nucleotides. Oligonucleotide modifications are further described herein.
c.经修饰核苷酸c. Modified nucleotides
在一些实施方案中，寡核苷酸包含2’-经修饰核苷酸，例如2’-脱氧、2’-脱氧-2’-氟、2’-O-甲基、2’-O-甲氧基乙基(2’-O-MOE)、2’-O-氨基丙基(2’-O-AP)、2’-O-二甲基氨基乙基(2’-O-DMAOE)、2’-O-二甲基氨基丙基(2’-O-DMAP)、2’-O-二甲基氨基乙氧基乙基(2’-O-DMAEOE)或2’-O--N-甲基乙酰胺基(2’-O--NMA)。In some embodiments, the oligonucleotide comprises 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-O--N-methylacetamido (2'-O--NMA).
在一些实施方案中，寡核苷酸可包含至少一个2’-O-甲基-修饰的核苷酸，并且在一些实施方案中，所有核苷酸均包含2’-O-甲基修饰。在一些实施方案中，寡核苷酸包含经修饰核苷酸，其中核糖环包含连接环中的两个原子例如连接2’-O原子与4’-C原子的桥部分。在一些实施方案中，寡核苷酸是“锁定的”，例如包含其中核糖环被连接2’-O原子和4’-C原子的亚甲基桥“锁定”的经修饰核苷酸。LNA的一些实例描述于国际专利申请公开WO/2008/043753中，其于2008年4月17日公开，并且标题为“RNA Antagonist Compounds ForThe Modulation Of PCSK9”，其内容通过引用整体并入本文。In some embodiments, the oligonucleotide may comprise at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all nucleotides comprise a 2'-O-methyl modification. In some embodiments, the oligonucleotide comprises a modified nucleotide in which the ribose ring comprises a bridge portion connecting two atoms in the ring, such as a 2'-O atom and a 4'-C atom. In some embodiments, the oligonucleotide is "locked", for example comprising a modified nucleotide in which the ribose ring is "locked" by a methylene bridge connecting a 2'-O atom and a 4'-C atom. Some examples of LNAs are described in International Patent Application Publication WO/2008/043753, which was published on April 17, 2008, and is entitled "RNA Antagonist Compounds For The Modulation Of PCSK9", the contents of which are incorporated herein by reference in their entirety.
可用于本文中公开的寡核苷酸的其他修饰包括亚乙基桥联核酸(ENA)。ENA包括但不限于2’-O、4’-C-亚乙基桥联核酸。ENA的一些实例提供在以下中：国际专利公开No.WO2005/042777，其于2005年5月12日公开，并且标题为“APP/ENA Antisense”；Morita etal.,Nucleic Acid Res.,Suppl 1:241-242,2001；Surono et al.,Hum.Gene Ther.,15:749-757,2004；Koizumi,Curr.Opin.Mol.Ther.,8:144-149,2006和Horie et al.,NucleicAcids Symp.Ser(Oxf),49:171-172,2005；其公开内容通过引用整体并入本文。Other modifications that can be used for the oligonucleotides disclosed herein include ethylene bridged nucleic acids (ENAs). ENAs include, but are not limited to, 2'-O, 4'-C-ethylene bridged nucleic acids. Some examples of ENAs are provided in: International Patent Publication No. WO2005/042777, published on May 12, 2005, and entitled "APP/ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1: 241-242, 2001; Surono et al., Hum. Gene Ther., 15: 749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49: 171-172, 2005; the disclosures of which are incorporated herein by reference in their entirety.
在一些实施方案中，寡核苷酸可包含桥联核苷酸，例如锁核酸(locked nucleicacid，LNA)核苷酸、约束性乙基(constrained ethyl，cEt)核苷酸或亚乙基桥联核酸(ENA)核苷酸。在一些实施方案中，寡核苷酸包含在以下美国专利或专利申请公开之一中公开的经修饰核苷酸：美国专利7,399,845，其于2008年7月15日授权，并且标题为“6-ModifiedBicyclic Nucleic Acid Analogs”；美国专利7,741,457，其于2010年6月22日授权，并且标题为“6-Modified Bicyclic Nucleic Acid Analogs”；美国专利8,022,193，其于2011年9月20日授权，并且标题为“6-Modified Bicyclic Nucleic Acid Analogs”；美国专利7,569,686，其于2009年8月4日授权，并且标题为“Compounds And Methods For SynthesisOf Bicyclic Nucleic Acid Analogs”；美国专利7,335,765，其于2008年2月26日授权，并且标题为“Novel Nucleoside And Oligonucleotide Analogues”；美国专利7,314,923，其于2008年1月1日授权，并且标题为“Novel Nucleoside And OligonucleotideAnalogues”；美国专利7,816,333，其于2010年10月19日授权，并且标题为“Oligonucleotide Analogues And Methods Utilizing The Same”和美国公开号2011/0009471，现在为美国专利8,957,201，其于2015年2月17日授权，并且标题为“Oligonucleotide Analogues And Methods Utilizing The Same”，其各自的全部内容出于所有目的通过引用并入本文。In some embodiments, an oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. In some embodiments, the oligonucleotide comprises a modified nucleotide disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, which was issued on July 15, 2008, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,741,457, which was issued on June 22, 2010, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 8,022,193, which was issued on September 20, 2011, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,569,686, which was issued on August 4, 2009, and is entitled “Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,335,765, which was issued on February 26, 2008, and is entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,314,923, issued on January 1, 2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,816,333, issued on October 19, 2010, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same” and U.S. Publication No. 2011/0009471, now U.S. Patent 8,957,201, issued on February 17, 2015, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same”, the entire contents of each of which are incorporated herein by reference for all purposes.
在一些实施方案中，寡核苷酸包含在糖的2’位被修饰的至少一个核苷酸，优选2’-O-烷基、2’-O-烷基-O-烷基或2’-氟修饰的核苷酸。在另一些优选实施方案中，RNA修饰包括在RNA的3’端的嘧啶、无碱基残基或倒置碱基的核糖上的2’-氟、2’-氨基和2’O-甲基修饰。In some embodiments, the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-fluoro modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2'-O-methyl modifications on the ribose of pyrimidines, abasic residues or inverted bases at the 3' end of the RNA.
在一些实施方案中，寡核苷酸可具有至少一个经修饰核苷酸，其导致与不具有至少一个经修饰核苷酸的寡核苷酸相比，寡核苷酸的Tm提高1℃、2℃、3℃、4℃或5℃。寡核苷酸可具有多个经修饰核苷酸，其导致与不具有经修饰核苷酸的寡核苷酸相比，寡核苷酸的Tm总体提高2℃、3℃、4℃、5℃、6℃、7℃、8℃、9℃、10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃或更高。In some embodiments, the oligonucleotide may have at least one modified nucleotide that results in an increase in the Tm of the oligonucleotide by 1° C., 2° C., 3° C., 4° C., or 5° C. compared to an oligonucleotide that does not have at least one modified nucleotide. The oligonucleotide may have multiple modified nucleotides that result in an overall increase in the Tm of the oligonucleotide by 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or more compared to an oligonucleotide that does not have a modified nucleotide.
寡核苷酸可包含不同种类的替代核苷酸。例如，寡核苷酸可包含替代脱氧核糖核苷酸或核糖核苷酸和2’-氟-脱氧核糖核苷酸。寡核苷酸可包含替代脱氧核糖核苷酸或核糖核苷酸和2’-O-甲基核苷酸。寡核苷酸可包含替代2’-氟核苷酸和2’-O-甲基核苷酸。寡核苷酸可包含替代桥联核苷酸和2’-氟或2’-O-甲基核苷酸。The oligonucleotide may comprise different types of alternative nucleotides. For example, the oligonucleotide may comprise alternative deoxyribonucleotides or ribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternative deoxyribonucleotides or ribonucleotides and 2'-O-methyl nucleotides. The oligonucleotide may comprise alternative 2'-fluoro nucleotides and 2'-O-methyl nucleotides. The oligonucleotide may comprise alternative bridging nucleotides and 2'-fluoro or 2'-O-methyl nucleotides.
d.核苷酸间键联/主链d. Internucleotide linkage/backbone
在一些实施方案中，寡核苷酸可包含硫代磷酸酯键联或其他经修饰核苷酸间键联。在一些实施方案中，寡核苷酸包含硫代磷酸酯核苷间键联。在一些实施方案中，寡核苷酸在至少两个核苷酸之间包含硫代磷酸酯核苷间键联。在一些实施方案中，寡核苷酸在所有核苷酸之间包含硫代磷酸酯核苷间键联。例如，在一些实施方案中，寡核苷酸在核苷酸序列的5’或3’端的第一、第二和/或第三核苷酸间键联处包含经修饰核苷酸间键联。In some embodiments, the oligonucleotide may comprise a phosphorothioate linkage or other modified internucleotide linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage between all nucleotides. For example, in some embodiments, the oligonucleotide comprises a modified internucleotide linkage at the first, second, and/or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.
可使用的含磷的键联包括但不限于：硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基磷酸三酯、甲基膦酸酯和包含3’亚烷基膦酸酯和手性膦酸酯的其他烷基膦酸酯、次膦酸酯、包含3’氨基磷酸酯和氨基烷基磷酰胺酯的磷酰胺酯、硫羰基磷酰胺酯、硫羰基烷基膦酸酯、硫羰基烷基磷酸三酯和具有正常3’-5’键联的硼烷磷酸酯、这些的2’-5’连接类似物、以及具有倒置极性的那些，其中相邻的核苷单元对连接3’-5’与5’-3’或2’-5’与5’-2’；参见美国专利no.Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3' alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' phosphoramidates and aminoalkylphosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with inverted polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. No.
在一些实施方案中，寡核苷酸可具有杂原子主链，例如亚甲基(甲基亚氨基)或MMI主链；酰胺主链(参见De Mesmaeker et al.Ace.Chem.Res.1995,28:366-374)；吗啉代主链(参见Summerton and Weller，美国专利No.5,034,506)；或肽核酸(PNA)主链(其中寡核苷酸的磷酸二酯主链被聚酰胺主链替换，核苷酸与聚酰胺主链的氮杂氮原子直接或间接结合，参见Nielsen et al.,Science 1991,254,1497)。In some embodiments, the oligonucleotide may have a heteroatom backbone, such as a methylene (methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28: 366-374); a morpholino backbone (see Summerton and Weller, U.S. Pat. No. 5,034,506); or a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotides are directly or indirectly bound to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).
e.立体特异性寡核苷酸e. Stereospecific oligonucleotides
在一些实施方案中，寡核苷酸的核苷酸间磷原子是手性的，并且基于手性磷原子的构型调节寡核苷酸的特性。在一些实施方案中，可使用适当方法来以立体控制的方式合成P-手性寡核苷酸类似物(例如，如Oka N,Wada T,Stereocontrolled synthesis ofoligonucleotide analogs containing chiral internucleotidic phosphorusatoms.Chem Soc Rev.2011Dec；40(12):5829-43中所述)。在一些实施方案中，提供了含硫代磷酸酯的寡核苷酸，其包含通过基本上所有的Sp或基本上所有的Rp硫代磷酸酯糖间键联连接在一起的核苷单元。在一些实施方案中，具有基本上手性纯糖间键联的这样的硫代磷酸酯寡核苷酸通过酶或化学合成制备，如例如于1996年12月12日授权的美国专利5,587,261中所述，其内容通过引用整体并入本文。在一些实施方案中，手性控制的寡核苷酸提供了靶核酸的选择性切割模式。例如，在一些实施方案中，手性控制的寡核苷酸在核酸的互补序列内提供了单个位点切割，如例如美国专利申请公开20170037399A1中所述，其于2017年2月2日公开，标题为“CHIRAL DESIGN”，其内容通过引用整体并入本文。In some embodiments, the internucleotide phosphorus atom of the oligonucleotide is chiral, and the characteristics of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, appropriate methods can be used to synthesize P-chiral oligonucleotide analogs in a stereo controlled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorusatoms. Chem Soc Rev. 2011 Dec; 40 (12): 5829-43). In some embodiments, oligonucleotides containing thiophosphates are provided, which include nucleoside units connected together by substantially all Sp or substantially all Rp thiophosphate sugar inter-linkages. In some embodiments, such thiophosphate oligonucleotides with substantially chiral pure sugar inter-linkages are prepared by enzyme or chemical synthesis, as described in, for example, U.S. Patent No. 5,587,261, authorized on December 12, 1996, the contents of which are incorporated herein by reference as a whole. In some embodiments, the oligonucleotides controlled by chirality provide a selective cleavage pattern of a target nucleic acid. For example, in some embodiments, chirality-controlled oligonucleotides provide single-site cleavage within the complementary sequence of a nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399A1, published on February 2, 2017, entitled “CHIRAL DESIGN,” the contents of which are incorporated herein by reference in their entirety.
f.吗啉代f. Morpholino
在一些实施方案中，寡核苷酸可以是基于吗啉代的化合物。基于吗啉代的寡聚化合物描述于Dwaine A.Braasch and David R.Corey,Biochemistry,2002,41(14),4503-4510)；Genesis,volume 30,issue 3,2001；Heasman,J.,Dev.Biol.,2002,243,209-214；Nasevicius et al.,Nat.Genet.,2000,26,216-220；Lacerra et al.,Proc.Natl.Acad.Sci.,2000,97,9591-9596；和1991年7月23日授权的美国专利No.5,034,506。在一些实施方案中，基于吗啉代的寡聚化合物是磷酸二酰胺吗啉代寡聚物(PMO)(例如，如Iverson,Curr.Opin.Mol.Ther.,3:235-238,2001；和Wang et al.,J.Gene Med.,12:354-364,2010中所述；其公开内容通过引用整体并入本文)。In some embodiments, the oligonucleotide can be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41 (14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
g.肽核酸(PNA)g. Peptide nucleic acid (PNA)
在一些实施方案中，寡核苷酸的核苷酸单元的糖和核苷间键联(主链)二者均被新的基团替换。在一些实施方案中，维持碱基单元用于与适当的核酸靶化合物杂交。一种这样的寡聚化合物(已显示具有优异的杂交特性的寡核苷酸模拟物)被称为肽核酸(PNA)。在PNA化合物中，寡核苷酸的糖-主链被含酰胺的主链(例如，氨基乙基甘氨酸主链)替换。核碱基被保留并且与主链酰胺部分的氮杂氮原子直接或间接结合。报道制备PNA化合物的代表性出版物包括但不限于美国专利no.5,539,082；5,714,331；和5,719,262，其各自通过引用并入本文。PNA化合物的进一步教导可在Nielsen et al.,Science,1991,254,1497-1500中发现。In some embodiments, both the sugar and the internucleoside linkage (main chain) of the nucleotide unit of the oligonucleotide are replaced by new groups. In some embodiments, the base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound (an oligonucleotide mimetic that has been shown to have excellent hybridization properties) is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-main chain of the oligonucleotide is replaced by an amide-containing main chain (e.g., an aminoethylglycine main chain). The core base is retained and is directly or indirectly bound to the aza nitrogen atom of the main chain amide portion. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patents no. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
h.间隔聚体h. Gap polymer
在一些实施方案中，寡核苷酸是间隔聚体。间隔聚体寡核苷酸通常具有式5’-X-Y-Z-3’，其中X和Z作为围绕间隔区Y的侧翼区。在一些实施方案中，Y区域是核苷酸的连续延伸，例如，至少6个DNA核苷酸的区域，其能够募集RNA酶(例如RNA酶H)。在一些实施方案中，间隔聚体与靶核酸结合，在该点处RNA酶被募集并随后可切割靶核酸。在一些实施方案中，Y区5’和3’二者的侧翼是包含高亲和力经修饰核苷酸，例如1至6个经修饰核苷酸的X和Z区。经修饰核苷酸的一些实例包括但不限于2’MOE或2’OMe或锁核酸碱基(LNA)。在一些实施方案中，侧翼序列X和Z的长度可以是1至20个核苷酸、1至8个核苷酸或1至5个核苷酸。侧翼序列X和Z可具有相似长度或不同长度。在一些实施方案中，间隔区段Y可以是5至20个核苷酸的核苷酸序列，长度尺寸为12个核苷酸或6至10个核苷酸。In some embodiments, oligonucleotide is a spacer. Spacer oligonucleotides generally have the formula 5'-X-Y-Z-3', wherein X and Z are used as flanking regions around spacer Y. In some embodiments, the Y region is a continuous extension of nucleotides, for example, a region of at least 6 DNA nucleotides, which can recruit RNA enzymes (such as RNA enzyme H). In some embodiments, the spacer is bound to the target nucleic acid, at which point the RNA enzyme is recruited and can subsequently cut the target nucleic acid. In some embodiments, the flanks of both 5' and 3' of the Y region are X and Z regions comprising high-affinity modified nucleotides, for example, 1 to 6 modified nucleotides. Some examples of modified nucleotides include but are not limited to 2'MOE or 2'OMe or locked nucleic acid bases (LNA). In some embodiments, the length of the flanking sequences X and Z can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides. Flanking sequences X and Z can have similar lengths or different lengths. In some embodiments, the spacer segment Y can be a nucleotide sequence of 5 to 20 nucleotides, with a length size of 12 nucleotides or 6 to 10 nucleotides.
在一些实施方案中，除DNA核苷酸之外，间隔聚体寡核苷酸的间隔区可包含已知可被接受用于有效的RNA酶H作用的经修饰核苷酸，例如C4’-取代的核苷酸、无环核苷酸和阿拉伯糖(arabino)构型的核苷酸。在一些实施方案中，间隔区包含一个或更多个未经修饰间核苷。在一些实施方案中，一个或两个侧翼区在至少两个、至少三个、至少四个、至少五个或更多个核苷酸之间各自独立地包含一个或更多个硫代磷酸酯核苷间键联(例如，硫代磷酸酯核苷间键联或其他键联)。在一些实施方案中，间隔区和两个侧翼区在至少两个、至少三个、至少四个、至少五个或更多个核苷酸之间各自独立地包含经修饰核苷间键联(例如，硫代磷酸酯核苷间键联或其他键联)。In some embodiments, in addition to DNA nucleotides, the spacer of the spacer oligonucleotide may include modified nucleotides known to be acceptable for effective RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabinose (arabino) configuration nucleotides. In some embodiments, the spacer comprises one or more unmodified internucleosides. In some embodiments, one or two flanking regions each independently comprise one or more thiophosphate nucleoside interlinkages (e.g., thiophosphate nucleoside interlinkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the spacer and two flanking regions each independently comprise modified nucleoside interlinkages (e.g., thiophosphate nucleoside interlinkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
可使用适当的方法产生间隔聚体。教导制备间隔聚体的代表性美国专利、美国专利出版物和PCT出版物包括但不限于美国专利No.5,013,830；5,149,797；5,220,007；5,256,775；The gapmers can be produced using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;
5,366,878；5,403,711；5,491,133；5,565,350；5,623,065；5,652,355；5,652,356；5,700,922；5,898,031；7,432,250；和7,683,036；and 7,683,036;
美国专利公开No.US20090286969、US20100197762和US20110112170；以及PCT公开No.WO2008049085和WO2009090182，其各自通过引用整体并入本文。U.S. Patent Publication Nos. US20090286969, US20100197762, and US20110112170; and PCT Publication Nos. WO2008049085 and WO2009090182, each of which is herein incorporated by reference in its entirety.
i.混合聚体i. Mixed polymers
在一些实施方案中，本文中所述的寡核苷酸可以是混合聚体或者包含混合聚体序列模式。通常来说，混合聚体是包含天然和非天然核苷酸二者的寡核苷酸或者通常以替代模式包含两种不同类型的非天然核苷酸的寡核苷酸。混合聚体通常比未经修饰寡核苷酸具有更高的结合亲和力，并且可用于特异性结合靶分子，例如，来阻断靶分子上的结合位点。通常来说，混合聚体不向靶分子募集RNA酶并且因此不促进靶分子的切割。已描述了不能募集RNA酶H的这样的寡核苷酸，例如，参见WO2007/112754或WO2007/112753。In some embodiments, oligonucleotides described herein can be mixed polymers or comprise mixed polymer sequence patterns. Generally speaking, mixed polymers are oligonucleotides comprising both natural and non-natural nucleotides or oligonucleotides comprising two different types of non-natural nucleotides with alternative patterns generally. Mixed polymers have higher binding affinity than unmodified oligonucleotides generally, and can be used for specific binding target molecules, for example, to block the binding site on the target molecule. Generally speaking, mixed polymers do not raise RNA enzymes to the target molecule and therefore do not promote the cutting of the target molecule. Such oligonucleotides that can not raise RNA enzyme H have been described, for example, referring to WO2007/112754 or WO2007/112753.
在一些实施方案中，混合聚体包含重复模式的核苷酸类似物和天然核苷酸、或一种类型的核苷酸类似物和第二种类型的核苷酸类似物，或者由其组成。然而，混合聚体不需要包含重复模式，并且可替代地包含经修饰核苷酸和天然存在核苷酸的任何排列，或一种经修饰核苷酸和第二种经修饰核苷酸的任何排列。重复模式可以是例如每第二个或每第三个核苷酸是经修饰核苷酸(例如LNA)，并且剩余核苷酸是天然核苷酸(例如DNA)或是2’经取代的核苷酸类似物，例如2’MOE或2’氟类似物，或者本文中所述的任何其他经修饰核苷酸。公认的是，经修饰核苷酸的重复模式，例如LNA单元，可在固定位置例如在5’或3’端与经修饰核苷酸组合。In some embodiments, the mixed polymer comprises or consists of a repeating pattern of nucleotide analogs and natural nucleotides, or one type of nucleotide analogs and a second type of nucleotide analogs. However, the mixed polymer need not comprise a repeating pattern, and may alternatively comprise any arrangement of modified nucleotides and naturally occurring nucleotides, or any arrangement of one modified nucleotide and a second modified nucleotide. The repeating pattern may be, for example, that every second or every third nucleotide is a modified nucleotide (e.g., LNA), and the remaining nucleotides are natural nucleotides (e.g., DNA) or are 2' substituted nucleotide analogs, such as 2'MOE or 2' fluoro analogs, or any other modified nucleotides described herein. It is recognized that a repeating pattern of modified nucleotides, such as LNA units, may be combined with modified nucleotides at fixed positions, such as at the 5' or 3' end.
在一些实施方案中，混合聚体不包含多于5个、多于4个、多于3个或多于2个连续的天然核苷酸(例如DNA核苷酸)的区域。在一些实施方案中，混合聚体包含至少一个由至少两个连续的经修饰核苷酸，例如至少两个连续的LNA组成的区域。在一些实施方案中，混合聚体包含至少一个由至少三个连续的经修饰核苷酸单元，例如至少三个连续的LNA组成的区域。In some embodiments, the mixed polymer does not contain a region of more than 5, more than 4, more than 3, or more than 2 consecutive natural nucleotides (e.g., DNA nucleotides). In some embodiments, the mixed polymer contains at least one region consisting of at least two consecutive modified nucleotides, such as at least two consecutive LNAs. In some embodiments, the mixed polymer contains at least one region consisting of at least three consecutive modified nucleotide units, such as at least three consecutive LNAs.
在一些实施方案中，混合聚体不包含多于7个、多于6个、多于5个、多于4个、多于3个或多于2个连续的核苷酸类似物例如LNA的区域。在一些实施方案中，LNA单元可被其他核苷酸类似物例如本文中提及的那些替换。In some embodiments, the mixed polymer does not contain regions of more than 7, more than 6, more than 5, more than 4, more than 3 or more than 2 consecutive nucleotide analogs such as LNA. In some embodiments, the LNA units may be replaced by other nucleotide analogs such as those mentioned herein.
混合聚体可被设计为包含亲和力增强的经修饰核苷酸(例如在非限制性实例中LNA核苷酸和2’-O-甲基核苷酸)的混合物。在一些实施方案中，混合聚体在至少两个、至少三个、至少四个、至少五个或更多个核苷酸之间包含经修饰核苷间键联(例如，硫代磷酸酯核苷间键联或其他键联)。Mixed polymers can be designed to include a mixture of modified nucleotides with enhanced affinity (e.g., LNA nucleotides and 2'-O-methyl nucleotides in a non-limiting example). In some embodiments, the mixed polymers include modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
可使用任何合适的方法产生混合聚体。教导制备混合聚体的代表性美国专利、美国专利出版物和PCT出版物包括美国专利公开No.US20060128646、US20090209748、US20090298916、US20110077288和US20120322851以及美国专利No.7687617。Any suitable method may be used to produce the mixed polymers. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixed polymers include U.S. Patent Publication Nos. US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851 and U.S. Patent No. 7,687,617.
在一些实施方案中，混合聚体包含一个或更多个吗啉代核苷酸。例如，在一些实施方案中，混合聚体可包含与一个或更多个其他核苷酸(例如，DNA、RNA核苷酸)或经修饰核苷酸(例如，LNA、2’-O-甲基核苷酸)混合(例如，以交替方式混合)的吗啉代核苷酸。In some embodiments, the mixed polymer comprises one or more morpholino nucleotides. For example, in some embodiments, the mixed polymer may comprise morpholino nucleotides mixed (e.g., mixed in an alternating manner) with one or more other nucleotides (e.g., DNA, RNA nucleotides) or modified nucleotides (e.g., LNA, 2'-O-methyl nucleotides).
在一些实施方案中，混合聚体可用于剪接纠正或外显子跳读，例如，如以下中报道的：Touznik A.,et al.,LNA/DNA mixmer-based antisense oligonucleotides correctalternative splicing of the SMN2 gene and restore SMN protein expression intype 1SMA fibroblasts Scientific Reports,volume7,Article number:3672(2017)，Chen S.et al.,Synthesis of a Morpholino Nucleic Acid(MNA)-UridinePhosphoramidite,and Exon Skipping Using MNA/2’-O-Methyl Mixmer AntisenseOligonucleotide,Molecules 2016,21,1582，其各自的内容通过引用并入本文。In some embodiments, the mixed polymer can be used for splicing correction or exon skipping, for example, as reported in: Touznik A., et al., LNA/DNA mixmer-based antisense oligonucleotides correctalternative splicing of the SMN2 gene and restore SMN protein expression intype 1SMA fibroblasts Scientific Reports, volume 7, Article number: 3672 (2017), Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA) -Uridine Phosphoramidite, and Exon Skipping Using MNA/2'-O-Methyl Mixmer Antisense Oligonucleotide, Molecules 2016, 21, 1582, the contents of each of which are incorporated herein by reference.
j.RNA干扰(RNAi)j. RNA interference (RNAi)
在一些实施方案中，本文中提供的寡核苷酸可以是小干扰RNA(smallinterfering RNA，siRNA，也称为短干扰RNA或沉默RNA)的形式。siRNA是一类双链RNA分子，通常长度为约20至25个碱基对，其通过细胞中的RNA干扰(RNAi)途径靶向核酸(例如，mRNA)用于降解。siRNA分子的特异性可通过分子反义链与其靶RNA的结合来确定。尽管更长的siRNA也可能是有效的，但有效的siRNA分子的长度通常少于30至35个碱基对，以防止通过干扰素应答触发细胞中的非特异性RNA干扰途径。In some embodiments, the oligonucleotides provided herein can be in the form of small interfering RNA (small interfering RNA, siRNA, also referred to as short interfering RNA or silencing RNA). siRNA is a type of double-stranded RNA molecule, typically about 20 to 25 base pairs in length, which targets nucleic acids (e.g., mRNA) for degradation by RNA interference (RNAi) pathways in cells. The specificity of siRNA molecules can be determined by the binding of the antisense strand of the molecule to its target RNA. Although longer siRNAs may also be effective, the length of effective siRNA molecules is typically less than 30 to 35 base pairs to prevent nonspecific RNA interference pathways in cells from being triggered by interferon responses.
在选择适当的靶RNA序列之后，可使用适当的方法设计和制备包含与全部或部分靶序列互补的核苷酸序列(即反义序列)的siRNA分子(参见，例如PCT公开号WO 2004/016735；以及美国专利公开No.2004/0077574和2008/0081791)。After selecting an appropriate target RNA sequence, siRNA molecules comprising a nucleotide sequence complementary to all or part of the target sequence (ie, an antisense sequence) can be designed and prepared using appropriate methods (see, e.g., PCT Publication No. WO 2004/016735; and U.S. Patent Publication Nos. 2004/0077574 and 2008/0081791).
siRNA分子可以是双链的(即，包含反义链和互补有义链的dsRNA分子)或单链的(即，仅包含反义链的ssRNA分子)。siRNA分子可包含具有自身互补的有义链和反义链的双链体(duplex)、不对称双链体、发夹或不对称发夹二级结构。siRNA molecules can be double-stranded (i.e., dsRNA molecules comprising an antisense strand and a complementary sense strand) or single-stranded (i.e., ssRNA molecules comprising only an antisense strand). siRNA molecules can comprise a duplex, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure having a self-complementary sense strand and an antisense strand.
双链siRNA可包含相同长度或不同长度的RNA链。双链siRNA分子也可由单个寡核苷酸组装成茎-环结构，其中siRNA分子的自身互补有义和反义区通过以下连接：一个或更多个基于核酸或基于非核酸的接头，以及环状单链RNA，其具有两个或更多个环结构和包含自身互补有义链和反义链的茎，其中环状RNA可在体内或体外加工以产生能够介导RNAi的活性siRNA分子。因此，本文中也考虑了小发夹RNA(small hairpin RNA，shRNA)分子。除通常由间隔区或环序列隔开的反向互补(有义)序列之外，这些分子还包含特定的反义序列。间隔区或环的切割提供了单链RNA分子及其反向互补物，使得它们可进行退火以形成dsRNA分子(任选地，具有可导致从任一或两条链的3’端和/或5’端添加或去除一个、两个、三个或更多个核苷酸的另外的加工步骤)。间隔区可具有足够的长度以允许反义和有义序列在切割间隔区(和任选地，可导致从任一或两条链的3’端和/或5’端添加或去除一个、两个、三个、四个或更多个核苷酸的后续加工步骤)之前进行退火并形成双链结构(或茎)。间隔区序列可以是位于两个互补核苷酸序列区域之间的不相关核苷酸序列，当退火成双链核酸时其包含shRNA。Double-stranded siRNA can comprise RNA chains of the same length or different lengths. Double-stranded siRNA molecules can also be assembled into stem-loop structures by single oligonucleotides, wherein the self-complementary sense and antisense regions of siRNA molecules are connected by the following: one or more nucleic acid-based or non-nucleic acid-based joints, and circular single-stranded RNA, which has two or more loop structures and a stem comprising self-complementary sense strands and antisense strands, wherein circular RNA can be processed in vivo or in vitro to produce active siRNA molecules that can mediate RNAi. Therefore, small hairpin RNA (small hairpin RNA, shRNA) molecules are also considered herein. Except the reverse complementary (sense) sequence separated by spacer or loop sequence usually, these molecules also comprise specific antisense sequences. The cutting of spacer or loop provides single-stranded RNA molecules and their reverse complements, so that they can be annealed to form dsRNA molecules (optionally, with the other processing steps that can cause the 3' end and/or 5' end of any one or two chains to add or remove one, two, three or more nucleotides). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally, subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3' and/or 5' ends of either or both strands). The spacer sequence may be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, when annealed into a double-stranded nucleic acid, comprises the shRNA.
siRNA分子的总长度可根据所设计siRNA分子的类型从约14至约100个核苷酸变化。通常来说，这些核苷酸中的约14至约50个与RNA靶序列互补，即构成siRNA分子的特异性反义序列。例如，当siRNA是双链siRNA或单链siRNA时，长度可从约14至约50个核苷酸变化，而当siRNA是shRNA或环状分子时，长度可从约40个核苷酸至约100个核苷酸变化。The total length of the siRNA molecule can vary from about 14 to about 100 nucleotides depending on the type of siRNA molecule being designed. Generally speaking, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double-stranded siRNA or a single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, and when the siRNA is a shRNA or a circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.
siRNA分子可在分子的一个末端包含3’突出端，另一末端可以是平末端或也具有突出端(5’或3’)。当siRNA分子在分子的两个末端均包含突出端时，突出端的长度可相同或不同。在一个实施方案中，本公开内容的siRNA分子在分子的两个末端包含约1至约3个核苷酸的3’突出端。The siRNA molecule may comprise a 3' overhang at one end of the molecule, and the other end may be a blunt end or also have an overhang (5' or 3'). When the siRNA molecule comprises overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one embodiment, the siRNA molecule of the present disclosure comprises a 3' overhang of about 1 to about 3 nucleotides at both ends of the molecule.
k.微RNA(miRNA)k. MicroRNA (miRNA)
在一些实施方案中，寡核苷酸可以是微RNA(miRNA)。微RNA(称为“miRNA”)是小的非编码RNA，属于通过与靶RNA转录物上的互补位点结合来控制基因表达的一类调节分子。通常来说，miRNA由大的RNA前体(称为初级miRNA(pri-miRNA))产生，所述RNA前体在细胞核中加工成约70个核苷酸前体miRNA，前体miRNA折叠成不完美的茎-环结构。这些前体miRNA通常在胞质内经历另外的加工步骤，在此长度为18至25个核苷酸的成熟miRNA被RNA酶III酶Dicer从前体miRNA发夹的一侧切离。In some embodiments, oligonucleotides can be microRNA (miRNA). MicroRNA (referred to as "miRNA") is a small non-coding RNA, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Generally speaking, miRNA is produced by large RNA precursors (referred to as primary miRNA (pri-miRNA)), which are processed into about 70 nucleotide precursor miRNAs in the nucleus, and the precursor miRNAs are folded into imperfect stem-loop structures. These precursor miRNAs are usually subjected to additional processing steps in the cytoplasm, and the mature miRNAs of 18 to 25 nucleotides in length are cut off from one side of the precursor miRNA hairpin by the RNase III enzyme Dicer.
本文中使用的miRNA包括初级miRNA、前体miRNA、成熟miRNA或保留成熟miRNA的生物学活性的其变体的片段。在一个实施方案中，miRNA的尺寸范围可以是21个核苷酸至170个核苷酸。在一个实施方案中，miRNA的尺寸范围是长度为70至170个核苷酸。在另一个实施方案中，可使用长度为21至25个核苷酸的成熟miRNA。The miRNA used herein includes a fragment of a variant thereof that is a primary miRNA, a precursor miRNA, a mature miRNA or retains the biological activity of a mature miRNA. In one embodiment, the size range of the miRNA can be 21 nucleotides to 170 nucleotides. In one embodiment, the size range of the miRNA is a length of 70 to 170 nucleotides. In another embodiment, a mature miRNA of 21 to 25 nucleotides can be used.
l.适配体1. Aptamers
在一些实施方案中，本文中提供的寡核苷酸可以是适配体的形式。通常来说，在分子载荷的情况下，适配体是与靶标(例如细胞中的小分子、蛋白质、核酸)特异性结合的任何核酸。在一些实施方案中，适配体是DNA适配体或RNA适配体。在一些实施方案中，核酸适配体是单链DNA或RNA(ssDNA或ssRNA)。应理解，单链核酸适配体可形成螺旋和/或环结构。形成核酸适配体的核酸可包含天然核苷酸、经修饰核苷酸、具有插入在一个或更多个核苷酸之间的烃接头(例如，亚烷基)或聚醚接头(例如，PEG接头)的天然核苷酸、具有插入在一个或更多个核苷酸之间的烃或PEG接头的经修饰核苷酸，或其组合。描述适配体和制备适配体的方法的示例性出版物和专利包括，例如，Lorsch and Szostak,1996；Jayasena,1999；美国专利No.5,270,163；5,567,588；5,650,275；5,670,637；5,683,867；5,696,249；5,789,157；5,843,653；5,864,026；5,989,823；6,569,630；8,318,438和PCT申请WO 99/31275，其各自通过引用并入本文。In some embodiments, the oligonucleotide provided herein can be in the form of an aptamer. Generally speaking, in the case of molecular load, an aptamer is any nucleic acid that specifically binds to a target (e.g., a small molecule, protein, nucleic acid in a cell). In some embodiments, an aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, a nucleic acid aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA). It should be understood that a single-stranded nucleic acid aptamer can form a spiral and/or ring structure. The nucleic acid forming a nucleic acid aptamer can include natural nucleotides, modified nucleotides, natural nucleotides with hydrocarbon joints (e.g., alkylene) or polyether joints (e.g., PEG joints) inserted between one or more nucleotides, modified nucleotides with hydrocarbons or PEG joints inserted between one or more nucleotides, or a combination thereof. Exemplary publications and patents describing aptamers and methods of making aptamers include, for example, Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT Application WO 99/31275, each of which is incorporated herein by reference.
m.核酶m. Ribozyme
在一些实施方案中，本文中提供的寡核苷酸可以是核酶的形式。核酶(核糖核酸酶)是能够进行特定的生化反应，类似于蛋白质酶作用的分子，通常是RNA分子。核酶是具有催化活性，包括在与其杂交的RNA分子(例如，mRNA、含RNA的底物、lncRNA和核酶本身)中的特定磷酸二酯键联处进行切割的能力的分子。In some embodiments, the oligonucleotides provided herein can be in the form of ribozymes. Ribozymes (ribonucleases) are molecules that can perform specific biochemical reactions, similar to the action of protein enzymes, typically RNA molecules. Ribozymes are molecules with catalytic activity, including the ability to cut at specific phosphodiester bonds in RNA molecules (e.g., mRNA, RNA-containing substrates, lncRNA, and ribozymes themselves) hybridized therewith.
核酶可采用数种物理结构之一，其中之一被称为“锤头状的”。锤头状核酶由包含9个保守碱基的催化核心、双链茎和环结构(茎-环II)以及与靶RNA侧翼区催化核心互补的两个区域构成。通过形成双链茎I和III，侧翼区能够使核酶与靶RNA特异性结合。通过3’,5’-磷酸二酯至2’,3’-环状磷酸二酯的酯交换反应，在特定核糖核苷酸三联体旁以顺式(即，切割包含锤头状基序的同一RNA分子)或以反式(切割除包含核酶的RNA底物之外的RNA底物)发生切割。不希望受理论束缚，认为这种催化活性需要在核酶的催化区域中存在特定的、高度保守的序列。Ribozymes can adopt one of several physical structures, one of which is called "hammerhead". Hammerhead ribozymes consist of a catalytic core containing 9 conserved bases, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the catalytic core of the target RNA flanking regions. The flanking regions enable the ribozyme to specifically bind to the target RNA by forming double-stranded stems I and III. Cleavage occurs next to a specific ribonucleotide triplet in cis (i.e., cleaving the same RNA molecule containing the hammerhead motif) or in trans (cleaving an RNA substrate other than the RNA substrate containing the ribozyme) by transesterification of 3',5'-phosphodiester to 2',3'-cyclic phosphodiester. Without wishing to be bound by theory, it is believed that this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
核酶结构中的修饰还包括用非核苷酸分子替换或替换分子的多个非核心部分。例如，Benseler et al.(J.Am.Chem.Soc.(1993)115:8483-8484)公开了锤头状样分子，其中茎II的两个碱基对和环II的所有四个核苷酸被基于六乙二醇、丙二醇、双(三乙二醇)磷酸酯、三(丙二醇)二磷酸酯或双(丙二醇)磷酸酯的非核苷接头替换。Ma et al.(Biochem.(1993)32:1751-1758；Nucleic Acids Res.(1993)21:2585-2589)用非核苷酸的乙二醇相关的接头替换了TAR核酶发夹的六个核苷酸环。Thomson et al.(Nucleic Acids Res.(1993)21:5600-5603)用长度为13、17和19个原子的线性非核苷酸接头替换了环II。Modification in ribozyme structure also comprises replacing or replacing multiple non-core parts of molecule with non-nucleotide molecule.For example, Benseler et al. (J.Am.Chem.Soc. (1993) 115:8483-8484) disclose hammerhead-like molecule, wherein two base pairs of stem II and all four nucleotides of ring II are replaced by non-nucleoside linkers based on hexaethylene glycol, propylene glycol, bis(triethylene glycol) phosphate, tri(propylene glycol) diphosphate or bis(propylene glycol) phosphate. Ma et al. (Biochem. (1993) 32:1751-1758; Nucleic Acids Res. (1993) 21:2585-2589) replaced six nucleotide rings of TAR ribozyme hairpin with the relevant linker of ethylene glycol of non-nucleotide. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear non-nucleotidic linkers of 13, 17, and 19 atoms in length.
核酶寡核苷酸可使用公知的方法(参见，例如，PCT公开WO9118624；WO9413688；WO9201806；和WO 92/07065；以及美国专利5436143和5650502)制备，或者可从商业来源(例如，US Biochemicals)购买，而且如果需要的话，可并入核苷酸类似物来提高寡核苷酸对细胞中通过核酸酶降解的抗性。可以任何已知的方式，例如通过使用例如由AppliedBiosystems,Inc.或Milligen产生的市售合成仪来合成核酶。也可通过常规手段在重组载体中产生核酶。参见Molecular Cloning:A Laboratory Manual,Cold Spring HarborLaboratory(现行版)。核酶RNA序列可常规地合成，例如通过使用RNA聚合酶例如T7或SP6。Ribozyme oligonucleotides can be prepared using known methods (see, e.g., PCT Publications WO9118624; WO9413688; WO9201806; and WO 92/07065; and U.S. Pat. Nos. 5,436,143 and 5,650,502), or can be purchased from commercial sources (e.g., US Biochemicals), and, if desired, nucleotide analogs can be incorporated to increase the resistance of the oligonucleotide to degradation by nucleases in cells. Ribozymes can be synthesized in any known manner, such as by using commercially available synthesizers produced by, for example, Applied Biosystems, Inc. or Milligen. Ribozymes can also be produced in recombinant vectors by conventional means. See Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (current edition). Ribozyme RNA sequences can be conventionally synthesized, such as by using RNA polymerases such as T7 or SP6.
n.指导核酸(guide nucleic acid)n.Guide nucleic acid
在一些实施方案中，寡核苷酸是指导核酸，例如，指导RNA(gRNA)分子。通常来说，指导RNA是由以下构成的短的合成RNA：(1)与核酸可编程DNA结合蛋白(napDNAbp)(例如，Cas9)结合的支架序列，和(2)核苷酸间隔区部分，其定义了与gRNA结合的DNA靶序列(例如，基因组DNA靶标)，以将核酸可编程DNA结合蛋白引至接近DNA靶序列。在一些实施方案中，napDNAbp是与一种或更多种RNA形成复合物(例如，结合或缔合)的核酸可编程蛋白，所述RNA将核酸可编程蛋白靶向靶DNA序列(例如，靶基因组DNA序列)。在一些实施方案中，当与RNA复合时，核酸可编程核酸酶可被称为核酸酶:RNA复合物。指导RNA可作为两种或更多种RNA的复合体，或者作为单个RNA分子存在。In some embodiments, the oligonucleotide is a guide nucleic acid, for example, a guide RNA (gRNA) molecule. Generally speaking, a guide RNA is a short synthetic RNA consisting of: (1) a scaffold sequence that binds to a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., Cas9), and (2) a nucleotide spacer portion that defines a DNA target sequence (e.g., a genomic DNA target) that binds to the gRNA to direct the nucleic acid programmable DNA binding protein to a proximity DNA target sequence. In some embodiments, napDNAbp is a nucleic acid programmable protein that forms a complex (e.g., binds or associates) with one or more RNAs, and the RNA targets the nucleic acid programmable protein to a target DNA sequence (e.g., a target genomic DNA sequence). In some embodiments, when compounded with RNA, a nucleic acid programmable nuclease may be referred to as a nuclease: RNA complex. The guide RNA may exist as a complex of two or more RNAs, or as a single RNA molecule.
作为单个RNA分子存在的指导RNA(gRNA)可被称为单指导RNA(single-guide RNA，sgRNA)，尽管gRNA也被用于指代作为单个分子或者作为两种或更多种分子的复合物存在的指导RNA。通常来说，作为单个RNA种类存在的gRNA包含两个结构域：(1)与靶核酸享有同源性的结构域(即，指导Cas9复合物与靶标的结合)；以及(2)结合Cas9蛋白的结构域。在一些实施方案中，结构域(2)对应于被称为tracrRNA的序列，并且包含茎-环结构。在一些实施方案中，结构域(2)与如Jinek et al.,Science 337:816-821(2012)(其全部内容通过引用并入本文)中提供的tracrRNA相同或同源。A guide RNA (gRNA) that exists as a single RNA molecule may be referred to as a single-guide RNA (sgRNA), although gRNA is also used to refer to a guide RNA that exists as a single molecule or as a complex of two or more molecules. Generally speaking, a gRNA that exists as a single RNA species comprises two domains: (1) a domain that shares homology with the target nucleic acid (i.e., guides the binding of the Cas9 complex to the target); and (2) a domain that binds the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence referred to as tracrRNA and comprises a stem-loop structure. In some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821 (2012) (the entire contents of which are incorporated herein by reference).
在一些实施方案中，gRNA包含结构域(1)和(2)中的两个或更多个，并且可被称为扩增gRNA(extended gRNA)。例如，如本文中所述的，扩增gRNA将结合两个或更多个Cas9蛋白并且在两个或更多个不同的区域结合靶核酸。gRNA包含与靶位点互补的核苷酸序列，其介导核酸酶/RNA复合物与所述靶位点的结合，提供核酸酶:RNA复合物的序列特异性。在一些实施方案中，RNA可编程核酸酶是(CRISPR相关系统)Cas9核酸内切酶，例如来自酿脓链球菌(Streptococcus pyogenes)的Cas9(Csn1)(参见，例如，In some embodiments, the gRNA comprises two or more of domains (1) and (2) and may be referred to as an extended gRNA. For example, as described herein, an extended gRNA will bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more different regions. The gRNA comprises a nucleotide sequence complementary to a target site that mediates binding of the nuclease/RNA complex to the target site, providing sequence specificity of the nuclease: RNA complex. In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, such as Cas9 (Csn1) from Streptococcus pyogenes (see, e.g.,
其各自的全部内容通过引用并入本文。The entire contents of each are incorporated herein by reference.
o.剪接改变寡核苷酸o. Splice-altering oligonucleotides
在一些实施方案中，本公开内容的寡核苷酸(例如，包含吗啉代的反义寡核苷酸)靶向剪接。在一些实施方案中，寡核苷酸通过在基因中诱导外显子跳读和恢复读码框来靶向剪接。作为非限制性实例，寡核苷酸可诱导编码移码突变的外显子和/或编码过早终止密码子的外显子的跳读。在一些实施方案中，寡核苷酸可通过阻断剪接体对剪接位点的识别来诱导外显子跳读。在一些实施方案中，与参考蛋白质相比，外显子跳读导致截短但具有功能性的蛋白质(例如，如下所述的截短但具有功能性的DMD蛋白)。在一些实施方案中，寡核苷酸促进包含特定外显子(例如，如下所述的SMN2基因的外显子7)。在一些实施方案中，寡核苷酸可通过靶向剪接位点抑制序列来诱导包含外显子。RNA剪接涉及肌肉疾病，包括迪谢内肌营养不良(DMD)和脊髓性肌萎缩(spinal muscular atrophy，SMA)。In some embodiments, oligonucleotides of the present disclosure (e.g., antisense oligonucleotides comprising morpholinos) target splicing. In some embodiments, oligonucleotides target splicing by inducing exon skipping and restoring reading frames in genes. As non-limiting examples, oligonucleotides can induce exon skipping of exons encoding frameshift mutations and/or exons encoding premature stop codons. In some embodiments, oligonucleotides can induce exon skipping by blocking the recognition of spliceosomes to splice sites. In some embodiments, compared with reference proteins, exon skipping leads to truncated but functional proteins (e.g., truncated but functional DMD proteins as described below). In some embodiments, oligonucleotides promote the inclusion of specific exons (e.g., exon 7 of SMN2 genes as described below). In some embodiments, oligonucleotides can induce the inclusion of exons by targeting splice site inhibitory sequences. RNA splicing is related to muscle diseases, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (spinal muscular atrophy, SMA).
编码肌养蛋白(DMD)的基因中的改变(例如，缺失、点突变和重复)引起DMD。这些改变可导致移码突变和/或无义突变。在一些实施方案中，本公开内容的寡核苷酸促进一个或更多个DMD外显子(例如，外显子8、外显子43、外显子44、外显子45、外显子50、外显子51、外显子52、外显子53和/或外显子55)的跳读，并产生功能性的截短蛋白质。参见，例如，2013年7月16日公开的美国专利No.8,486,907和2014年9月18日公开的U.S.20140275212。Changes (e.g., deletions, point mutations, and duplications) in the gene encoding dystrophin (DMD) cause DMD. These changes can result in frameshift mutations and/or nonsense mutations. In some embodiments, the oligonucleotides of the present disclosure promote the skipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and/or exon 55) and produce functional truncated proteins. See, e.g., U.S. Patent No. 8,486,907, published on July 16, 2013, and U.S. 20140275212, published on September 18, 2014.
在SMA中，存在功能性SMN1的丢失。尽管SMN2基因是SMN1的旁系同源物，但SMN2基因的选择性剪接主要导致外显子7的跳读和随后产生无法补偿SMN1丢失的截短的SMN蛋白。在一些实施方案中，本公开内容的寡核苷酸促进包含SMN2外显子7。在一些实施方案中，寡核苷酸是靶向SMN2剪接位点抑制序列的反义寡核苷酸(参见，例如2010年11月23日公开的美国专利号7,838,657)。In SMA, there is a loss of functional SMN1. Although the SMN2 gene is a paralog of SMN1, alternative splicing of the SMN2 gene primarily results in the skipping of exon 7 and the subsequent production of a truncated SMN protein that cannot compensate for the loss of SMN1. In some embodiments, the oligonucleotides of the present disclosure promote the inclusion of SMN2 exon 7. In some embodiments, the oligonucleotide is an antisense oligonucleotide targeted to an SMN2 splice site inhibitory sequence (see, e.g., U.S. Pat. No. 7,838,657, published Nov. 23, 2010).
p.多聚体p. Polymer
在一些实施方案中，分子载荷可包含通过接头连接的2个或更多个寡核苷酸的多聚体(例如，串联体)。在一些实施方案中，以这种方式，复合物/缀合物的寡核苷酸负载可提高到超过靶向剂上的可用连接位点(例如，抗体上的可用硫醇位点)，或者以其他方式调节以实现特定的载荷负载量。多聚体中的寡核苷酸可相同或不同(例如，靶向不同基因或同一基因上的不同位点或者其产物)。In some embodiments, the molecular payload may comprise a polymer of two or more oligonucleotides connected by a linker (e.g., a concatemer). In some embodiments, in this manner, the oligonucleotide loading of the complex/conjugate may be increased to exceed the available attachment sites on the targeting agent (e.g., available thiol sites on an antibody), or otherwise adjusted to achieve a specific payload loading. The oligonucleotides in the polymer may be the same or different (e.g., targeting different genes or different sites on the same gene or its products).
在一些实施方案中，多聚体包含通过可切割接头连接在一起的2个或更多个寡核苷酸。然而，在一些实施方案中，多聚体包含通过不可切割接头连接在一起的2个或更多个寡核苷酸。在一些实施方案中，多聚体包含2、3、4、5、6、7、8、9、10个或更多个连接在一起的寡核苷酸。在一些实施方案中，多聚体包含2至5、2至10或4至20个连接在一起的寡核苷酸。In some embodiments, the polymer comprises 2 or more oligonucleotides connected together by a cleavable joint. However, in some embodiments, the polymer comprises 2 or more oligonucleotides connected together by a non-cleavable joint. In some embodiments, the polymer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides connected together. In some embodiments, the polymer comprises 2 to 5, 2 to 10 or 4 to 20 oligonucleotides connected together.
在一些实施方案中，多聚体包含2个或更多个首尾相连(以线性排列)的寡核苷酸。在一些实施方案中，多聚体包含2个或更多个通过基于寡核苷酸的接头(例如，聚-dT接头，一种无碱基接头)首尾相连的寡核苷酸。在一些实施方案中，多聚体包含一个寡核苷酸的5’端，其与另一个寡核苷酸的3’端连接。在一些实施方案中，多聚体包含一个寡核苷酸的3’端，其与另一个寡核苷酸的3’端连接。在一些实施方案中，多聚体包含一个寡核苷酸的5’端，其与另一个寡核苷酸的5’端连接。尽管如此，在一些实施方案中，多聚体可包含分支结构，所述分支结构包含通过分支接头连接在一起的多个寡核苷酸。In some embodiments, a polymer comprises 2 or more oligonucleotides connected end to end (in a linear arrangement). In some embodiments, a polymer comprises 2 or more oligonucleotides connected end to end by a joint based on oligonucleotides (e.g., a poly-dT joint, a baseless joint). In some embodiments, a polymer comprises the 5' end of an oligonucleotide, which is connected to the 3' end of another oligonucleotide. In some embodiments, a polymer comprises the 3' end of an oligonucleotide, which is connected to the 3' end of another oligonucleotide. In some embodiments, a polymer comprises the 5' end of an oligonucleotide, which is connected to the 5' end of another oligonucleotide. Nevertheless, in some embodiments, a polymer may comprise a branched structure comprising a plurality of oligonucleotides connected together by a branched joint.
可用于本文中提供的复合物中的多聚体的另一些实例在以下中公开：例如，美国专利申请号2015/0315588A1，标题为Methods of delivering multiple targetingoligonucleotides to a cell using cleavable linkers，其于2015年11月5日公开；美国专利申请号2015/0247141A1，标题为Multimeric Oligonucleotide Compounds，其于2015年9月3日公开；美国专利申请号US 2011/0158937A1，标题为ImmunostimulatoryOligonucleotide Multimers，其于2011年6月30日公开；和美国专利号5,693,773，标题为Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers TargetingNucleic Acids Comprising Mixed Sequences Of Purines And Pyrimidines，其于1997年12月2日授权，其各自的内容通过引用整体并入本文。Other examples of multimers that can be used in the complexes provided herein are disclosed in, for example, U.S. Patent Application No. 2015/0315588A1, entitled Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers, published on November 5, 2015; U.S. Patent Application No. 2015/0247141A1, entitled Multimeric Oligonucleotide Compounds, published on September 3, 2015; U.S. Patent Application No. US 2011/0158937A1, entitled Immunostimulatory Oligonucleotide Multimers, published on June 30, 2011; and U.S. Patent No. 5,693,773, entitled Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And Pyrimidines, issued December 2, 1997, the contents of each of which are incorporated herein by reference in their entirety.
C.接头C. Connector
本文中所述的复合物通常包含连接本文中所述的肌肉靶向剂(例如，抗TfR抗体)中任一种与分子载荷的接头。接头包含至少一个共价键。在一些实施方案中，接头可以是连接肌肉靶向剂(例如，抗TfR抗体)与分子载荷的单键，例如二硫键或二硫桥。然而，在一些实施方案中，接头可通过多个共价键连接本文中所述的肌肉靶向剂(例如，抗TfR抗体)中的任一种与分子。在一些实施方案中，接头可以是可切割接头。然而，在一些实施方案中，接头可以是不可切割接头。接头通常在体外和体内是稳定的，并且在某些细胞环境中可以是稳定的。另外，通常来说接头不负面影响抗TfR抗体或分子载荷的功能特性。接头合成的一些实例和方法是本领域中已知的(参见，例如Kline,T.et al.“Methods to Make HomogenousAntibody Drug Conjugates.”Pharmaceutical Research,2015,32:11,3480–3493.；Jain,N.et al.“Current ADC Linker Chemistry”Pharm Res.2015,32:11,3526–3540.；McCombs,J.R.and Owen,S.C.“Antibody Drug Conjugates:Design and Selection ofLinker,Payload and Conjugation Chemistry”AAPS J.2015,17:2,339–351.)。The complex described herein generally comprises a joint connecting any one of the muscle targeting agents described herein (e.g., anti-TfR antibodies) and a molecular payload. The joint comprises at least one covalent bond. In some embodiments, the joint can be a single bond, such as a disulfide bond or a disulfide bridge, connecting the muscle targeting agent (e.g., anti-TfR antibody) and the molecular payload. However, in some embodiments, the joint can connect any one of the muscle targeting agents described herein (e.g., anti-TfR antibodies) to the molecule by multiple covalent bonds. In some embodiments, the joint can be a cleavable joint. However, in some embodiments, the joint can be a non-cleavable joint. The joint is generally stable in vitro and in vivo, and can be stable in certain cellular environments. In addition, generally speaking, the joint does not negatively affect the functional properties of the anti-TfR antibody or the molecular payload. Some examples and methods of linker synthesis are known in the art (see, e.g., Kline, T. et al. "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32: 11, 3480-3493.; Jain, N. et al. "Current ADC Linker Chemistry" Pharm Res. 2015, 32: 11, 3526-3540.; McCombs, J. R. and Owen, S. C. "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry" AAPS J. 2015, 17: 2, 339-351.).
接头的前体通常将包含允许与抗TfR抗体和分子载荷二者连接的两种不同的反应性物质。在一些实施方案中，两种不同的反应性物质可以是亲核体和/或(例如，和)亲电体。在一些实施方案中，接头通过与抗TfR抗体的赖氨酸残基或半胱氨酸残基缀合而与肌肉靶向剂(例如，抗TfR抗体)连接。在一些实施方案中，接头通过含马来酰亚胺接头与肌肉靶向剂(例如，抗TfR抗体)的半胱氨酸残基连接，其中任选地，含有马来酰亚胺接头包含马来酰亚胺己酰基或马来酰亚胺基甲基环己烷-1-羧酸酯基团。在一些实施方案中，接头通过3-芳基丙腈官能团与肌肉靶向剂(例如，抗TfR抗体)的半胱氨酸残基或巯基官能化的分子载荷连接。在一些实施方案中，接头与肌肉靶向剂(例如，抗TfR抗体)的赖氨酸残基连接。在一些实施方案中，接头通过酰胺键、氨基甲酸酯键、酰肼、三唑、硫醚或二硫键与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接。The precursor of the joint will generally include two different reactive substances that allow connection to both anti-TfR antibodies and molecular payloads. In some embodiments, two different reactive substances can be nucleophiles and/or (for example, and) electrophiles. In some embodiments, the joint is connected to a muscle targeting agent (for example, an anti-TfR antibody) by being conjugated to a lysine residue or a cysteine residue of an anti-TfR antibody. In some embodiments, the joint is connected to a cysteine residue of a muscle targeting agent (for example, an anti-TfR antibody) by a maleimide joint containing a maleimide joint, wherein optionally, a maleimidohexanoyl or maleimidomethylcyclohexane-1-carboxylate group is included in the maleimide joint. In some embodiments, the joint is connected to a cysteine residue or a sulfhydryl-functionalized molecular payload of a muscle targeting agent (for example, an anti-TfR antibody) by a 3-aryl propionitrile functional group. In some embodiments, the joint is connected to a lysine residue of a muscle targeting agent (for example, an anti-TfR antibody). In some embodiments, the linker is attached to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular payload via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, or a disulfide bond.
i.可切割接头i. Cleavable Linkers
可切割接头可以是蛋白酶敏感性接头、pH敏感性接头或谷胱甘肽敏感性接头。这些接头通常仅在胞内可切割，并且优选在胞外环境，例如肌细胞胞外中稳定。The cleavable linker can be a protease-sensitive linker, a pH-sensitive linker or a glutathione-sensitive linker. These linkers are usually only cleavable intracellularly and are preferably stable in an extracellular environment, such as outside a muscle cell.
蛋白酶敏感性接头可通过蛋白酶酶活性切割。这些接头通常包含肽序列，并且长度可为2至10个氨基酸、约2至5个氨基酸、约5至10个氨基酸、约10个氨基酸、约5个氨基酸、约3个氨基酸或约2个氨基酸。在一些实施方案中，肽序列可包含天然氨基酸例如半胱氨酸、丙氨酸或者非天然存在或经修饰氨基酸。非天然氨基酸包括β-氨基酸、高氨基酸、脯氨酸衍生物、3-经取代的丙氨酸衍生物、线性核心氨基酸、N-甲基氨基酸和本领域已知的其他氨基酸。在一些实施方案中，蛋白酶敏感性接头包含缬氨酸-瓜氨酸或丙氨酸-瓜氨酸序列。在一些实施方案中，蛋白酶敏感性接头可被溶酶体蛋白酶(例如组织蛋白酶B(cathepsin B))和/或(例如，和)内体蛋白酶切割。Protease sensitive joints can be cut by protease enzyme activity. These joints usually contain peptide sequences, and the length can be 2 to 10 amino acids, about 2 to 5 amino acids, about 5 to 10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids or about 2 amino acids. In some embodiments, the peptide sequence may include natural amino acids such as cysteine, alanine or non-natural or modified amino acids. Non-natural amino acids include β-amino acids, high amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids and other amino acids known in the art. In some embodiments, protease sensitive joints include valine-citrulline or alanine-citrulline sequences. In some embodiments, protease sensitive joints can be cut by lysosomal proteases (e.g., cathepsin B) and/or (e.g., and) endosomal proteases.
pH敏感性接头是在高或低pH环境中容易降解的共价键联。在一些实施方案中，pH敏感性接头可在4至6的pH下被切割。在一些实施方案中，pH敏感性接头包含腙或环缩醛。在一些实施方案中，pH敏感性接头在内体或溶酶体内被切割。A pH sensitive linker is a covalent linkage that is easily degraded in a high or low pH environment. In some embodiments, the pH sensitive linker can be cleaved at a pH of 4 to 6. In some embodiments, the pH sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH sensitive linker is cleaved within an endosome or lysosome.
在一些实施方案中，谷胱甘肽敏感性接头包含二硫化物部分。在一些实施方案中，谷胱甘肽敏感性接头通过与细胞内的谷胱甘肽物质进行二硫化物交换反应来切割。在一些实施方案中，二硫化物部分还包含至少一种氨基酸，例如半胱氨酸残基。In some embodiments, the glutathione-sensitive linker comprises a disulfide portion. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with an intracellular glutathione substance. In some embodiments, the disulfide portion further comprises at least one amino acid, such as a cysteine residue.
在一些实施方案中，接头是Val-cit接头(例如，如美国专利6,214,345中所述，其通过引用并入本文)。在一些实施方案中，在缀合之前，val-cit接头具有以下结构：In some embodiments, the linker is a Val-cit linker (e.g., as described in U.S. Pat. No. 6,214,345, which is incorporated herein by reference). In some embodiments, prior to conjugation, the val-cit linker has the following structure:
在一些实施方案中，在缀合之后，val-cit接头具有以下结构：In some embodiments, after conjugation, the val-cit linker has the following structure:
在一些实施方案中，Val-cit接头与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接。在一些实施方案中，在点击化学缀合之前，与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接的val-cit接头具有以下结构：In some embodiments, the Val-cit linker is connected to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation). In some embodiments, prior to click chemistry conjugation, the val-cit linker connected to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the following structure:
其中n为0至15中的任意数字。在一些实施方案中，n为3。wherein n is any number from 0 to 15. In some embodiments, n is 3.
在一些实施方案中，与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接的val-cit接头(例如，经由不同的化学部分)与分子载荷(例如，寡核苷酸)缀合。在一些实施方案中，与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接并且与分子载荷(例如，寡核苷酸)缀合的val-cit接头具有(在点击化学缀合之前)的结构：In some embodiments, a val-cit linker connected to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated (e.g., via a different chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, a val-cit linker connected to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to a molecular payload (e.g., an oligonucleotide) has a structure (before click chemistry conjugation):
其中n为0至15中的任意数字。在一些实施方案中，n为3。wherein n is any number from 0 to 15. In some embodiments, n is 3.
在一些实施方案中，在与分子载荷(例如，寡核苷酸)缀合之后，val-cit接头包含以下结构：In some embodiments, after conjugation to a molecular payload (e.g., an oligonucleotide), the val-cit linker comprises the following structure:
其中n为0至15中的任意数字，并且其中m为0至15中的任意数字。在一些实施方案中，n为3并且m为4。wherein n is any number from 0 to 15, and wherein m is any number from 0 to 15. In some embodiments, n is 3 and m is 4.
ii.不可切割接头ii. Non-cleavable linker
在一些实施方案中，可使用不可切割接头。通常来说，不可切割接头在细胞或生理环境中不容易被降解。在一些实施方案中，不可切割接头包含任选经取代的烷基，其中所述取代可包括卤素、羟基、氧物质和其他常见的取代。在一些实施方案中，接头可包含任选经取代的烷基、任选经取代的亚烷基、任选经取代的亚芳基、杂亚芳基、包含至少一种非天然氨基酸的肽序列、截短的聚糖、不能被酶降解的一种或更多种糖、叠氮化物、炔-叠氮化物、包含LPXT序列的肽序列、硫醚、生物素、联苯、聚乙二醇或等同化合物的重复单元、酸酯、酰胺、磺酰胺和/或(例如，和)烷氧基-胺接头。在一些实施方案中，分选酶介导的连接将用于共价连接包含LPXT序列的肌肉靶向剂(例如，抗TfR抗体)与包含(G)n序列的分子载荷(参见，例如Proft T.Sortase-mediated protein ligation:an emerging biotechnologytool for protein modification and immobilization.Biotechnol Lett.2010,32(1):1-10.)。In some embodiments, a non-cleavable joint can be used. Generally speaking, a non-cleavable joint is not easily degraded in a cell or physiological environment. In some embodiments, a non-cleavable joint comprises an optionally substituted alkyl, wherein the substitution may include halogen, hydroxyl, oxygen species and other common substitutions. In some embodiments, the joint may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated polysaccharide, one or more sugars that cannot be degraded by an enzyme, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, a biphenyl, a polyethylene glycol or an equivalent compound repeating unit, an acid ester, an amide, a sulfonamide and/or (e.g., and) an alkoxy-amine joint. In some embodiments, sortase-mediated ligation will be used to covalently link a muscle targeting agent (e.g., an anti-TfR antibody) comprising a LPXT sequence to a molecular payload comprising a (G)n sequence (see, e.g., Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1): 1-10.).
在一些实施方案中，接头可包含经取代的亚烷基、任选经取代的亚烯基、任选经取代的亚炔基、任选经取代的亚环烷基、任选经取代的亚环烯基、任选经取代的亚芳基、还包含至少一个选自N、O和S的杂原子的任选经取代的杂亚芳基；还包含至少一个选自N、O和S的杂原子的任选经取代的亚杂环基；亚氨基、任选经取代的氮物质、任选经取代的氧物质O、任选经取代的硫物质或聚(环氧烷烃)，例如聚环氧乙烷或聚环氧丙烷。In some embodiments, the linker can comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino group, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide.
iii.接头缀合iii. Linker conjugation
在一些实施方案中，接头通过磷酸酯、硫醚、醚、碳-碳键、氨基甲酸酯或酰胺键与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接。在一些实施方案中，接头通过磷酸酯或硫代磷酸酯基团例如寡核苷酸主链的末端磷酸酯与寡核苷酸连接。在一些实施方案中，接头通过肌肉靶向剂(例如，抗TfR抗体)上存在的赖氨酸或半胱氨酸残基与该肌肉靶向剂(例如，抗TfR抗体)连接。In some embodiments, the linker is connected to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular payload via a phosphate, thioether, ether, carbon-carbon bond, carbamate, or amide bond. In some embodiments, the linker is connected to the oligonucleotide via a phosphate or phosphorothioate group, such as a terminal phosphate of the oligonucleotide backbone. In some embodiments, the linker is connected to the muscle targeting agent (e.g., anti-TfR antibody) via a lysine or cysteine residue present on the muscle targeting agent (e.g., anti-TfR antibody).
在一些实施方案中，接头通过叠氮化物和炔烃之间的环加成反应与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接以形成三唑，其中叠氮化物和炔烃可位于肌肉靶向剂(例如，抗TfR抗体)、分子载荷或接头上。在一些实施方案中，炔烃可以是环炔烃，例如环辛炔。在一些实施方案中，炔烃可以是双环壬炔(也称为双环[6.1.0]壬炔或BCN)或经取代的双环壬炔。在一些实施方案中，环辛烷如国际专利申请公开WO2011136645中所述，其于2011年11月3日公开，标题为“Fused Cyclooctyne Compounds And Their Use InMetal-free Click Reactions”。BCN的exo-和endo-形式二者均可用于连接缀合，并且本文中提供的式(C)、(D)、(E)、(F)、(G)和(H)中的BCN可以是endo-或exo-BCN。In some embodiments, the linker is connected to a muscle targeting agent (e.g., an anti-TfR antibody) and/or (e.g., and) a molecular payload to form a triazole by a cycloaddition reaction between an azide and an alkyne, wherein the azide and the alkyne may be located on the muscle targeting agent (e.g., an anti-TfR antibody), the molecular payload, or the linker. In some embodiments, the alkyne may be a cycloalkyne, such as a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, which was published on November 3, 2011, entitled "Fused Cyclooctyne Compounds And Their Use InMetal-free Click Reactions". Both the exo- and endo- forms of BCN can be used for connection conjugation, and the BCN in formulas (C), (D), (E), (F), (G), and (H) provided herein may be endo- or exo-BCN.
在一些实施方案中，叠氮化物可以是包含叠氮化物的糖或碳水化合物分子。在一些实施方案中，叠氮化物可以是6-叠氮基-6-脱氧半乳糖或6-叠氮基-N-乙酰半乳糖胺。在一些实施方案中，包含叠氮化物的糖或碳水化合物分子如国际专利申请公开WO2016170186中所述，其于2016年10月27日公开，标题为“Process For The Modification Of AGlycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”。在一些实施方案中，在叠氮化物和炔烃之间进行环加成反应以形成三唑，其中叠氮化物和炔烃可位于肌肉靶向剂(例如，抗TfR抗体)、分子载荷或接头上，如国际专利申请公开WO2014065661，其于2014年5月1日公开，标题为“Modified antibody,antibody-conjugate and process for the preparationthereof”；或国际专利申请公开WO2016170186，其于2016年10月27日公开，标题为“ProcessFor The Modification Of A Glycoprotein Using A Glycosyltransferase That Is OrIs Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”中所述。In some embodiments, the azide can be a sugar or carbohydrate molecule comprising azide. In some embodiments, the azide can be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the sugar or carbohydrate molecule comprising azide is as described in International Patent Application Publication WO2016170186, which was published on October 27, 2016, entitled "Process For The Modification Of AGlycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase". In some embodiments, a cycloaddition reaction is performed between an azide and an alkyne to form a triazole, wherein the azide and the alkyne can be located on a muscle targeting agent (e.g., an anti-TfR antibody), a molecular payload, or a linker, as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”; or International Patent Application Publication WO2016170186, published on October 27, 2016, entitled “Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”.
在一些实施方案中，接头还包含间隔区，例如聚乙二醇间隔区或酰基/胺甲酰基磺酰胺间隔区，例如HydraSpaceTM间隔区。在一些实施方案中，间隔区如Verkade,J.M.M.etal.,“A Polar Sulfamide Spacer Significantly Enhances the Manufacturability,Stability,and Therapeutic Index of Antibody-Drug Conjugates”,Antibodies,2018,7,12中所述。In some embodiments, the linker further comprises a spacer, such as a polyethylene glycol spacer or an acyl/carbamoyl sulfonamide spacer, such as a HydraSpaceTM spacer. In some embodiments, the spacer is as described in Verkade, JMMetal., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.
在一些实施方案中，接头通过亲二烯体与二烯/杂二烯之间的第尔斯-阿尔德反应(Diels-Alder reaction)与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接，其中亲二烯体和二烯/杂二烯可位于肌肉靶向剂(例如，抗TfR抗体)、分子载荷或接头上。在一些实施方案中，接头通过其他周环反应(pericyclic reaction)，例如烯反应与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接。在一些实施方案中，接头通过酰胺、硫代酰胺或磺酰胺键合反应与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接。在一些实施方案中，接头通过缩合反应与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接，以形成存在于接头与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷之间的肟、腙或氨基脲基团。In some embodiments, the linker is connected to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular payload via a Diels-Alder reaction between a dienophile and a diene/heterodiene, wherein the dienophile and the diene/heterodiene may be located on the muscle targeting agent (e.g., anti-TfR antibody), the molecular payload, or the linker. In some embodiments, the linker is connected to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular payload via other pericyclic reactions, such as ene reactions. In some embodiments, the linker is connected to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) molecular payload via amide, thioamide, or sulfonamide bonding reactions. In some embodiments, the linker is attached to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular payload via a condensation reaction to form an oxime, hydrazone, or semicarbazide group present between the linker and the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular payload.
在一些实施方案中，接头通过亲核体(例如胺或羟基)和亲电体(例如羧酸、碳酸盐或醛)之间的共轭加成反应与肌肉靶向剂(例如，抗TfR抗体)和/或(例如，和)分子载荷连接。在一些实施方案中，在进行接头与肌肉靶向剂(例如，抗TfR抗体)或分子载荷之间的反应之前，亲核体可存在于接头上并且亲电体可存在于肌肉靶向剂(例如，抗TfR抗体)或分子载荷上。在一些实施方案中，在进行接头与肌肉靶向剂(例如，抗TfR抗体)或分子载荷之间的反应之前，亲电体可存在于接头上并且亲核体可存在于肌肉靶向剂(例如，抗TfR抗体)或分子载荷上。在一些实施方案中，亲电体可以是叠氮化物、五氟苯基、对硝基苯酚酯、硅中心、羰基、羧酸、酸酐、异氰酸酯、硫代异氰酸酯、琥珀酰亚胺酯、磺基琥珀酰亚胺酯、马来酰亚胺、烷基卤化物、烷基假卤化物、环氧化物、环硫化物、氮丙啶、芳基、活化的磷中心和/或(例如，和)活化的硫中心。在一些实施方案中，亲核体可以是任选经取代的烯烃、任选经取代的炔烃、任选经取代的芳基、任选经取代的杂环基、羟基、氨基、烷基氨基、苯胺基或硫醇基。In some embodiments, the linker is connected to the muscle targeting agent (e.g., anti-TfR antibody) and/or (e.g., and) the molecular payload by a conjugate addition reaction between a nucleophile (e.g., amine or hydroxyl) and an electrophile (e.g., carboxylic acid, carbonate, or aldehyde). In some embodiments, prior to the reaction between the linker and the muscle targeting agent (e.g., anti-TfR antibody) or the molecular payload, the nucleophile may be present on the linker and the electrophile may be present on the muscle targeting agent (e.g., anti-TfR antibody) or the molecular payload. In some embodiments, prior to the reaction between the linker and the muscle targeting agent (e.g., anti-TfR antibody) or the molecular payload, the electrophile may be present on the linker and the nucleophile may be present on the muscle targeting agent (e.g., anti-TfR antibody) or the molecular payload. In some embodiments, the electrophile can be an azide, a pentafluorophenyl, a p-nitrophenol ester, a silicon center, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimide ester, a sulfosuccinimide ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and/or (e.g., and) an activated sulfur center. In some embodiments, the nucleophile can be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl, an amino, an alkylamino, an anilino, or a thiol group.
在一些实施方案中，与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接的val-cit接头通过以下结构与抗TfR抗体缀合：In some embodiments, a val-cit linker connected to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated to an anti-TfR antibody via the following structure:
其中m为0至15中的任意数字。在一些实施方案中，m为4。wherein m is any number from 0 to 15. In some embodiments, m is 4.
在一些实施方案中，与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接的val-cit接头与具有以下结构的抗TfR抗体缀合：In some embodiments, a val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated to an anti-TfR antibody having the following structure:
其中m为0至15中的任意数字。在一些实施方案中，m为4。wherein m is any number from 0 to 15. In some embodiments, m is 4.
在一些实施方案中，与反应性化学部分(例如，用于点击化学缀合的SPAAC)连接并且与抗TfR抗体缀合的val-cit接头具有以下结构：In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to an anti-TfR antibody has the following structure:
其中n为0至15中的任意数字，其中m为0至15中的任意数字。在一些实施方案中，n为3和/或(例如，和)m为4。在一些实施方案中，寡核苷酸与包含式(H)结构的化合物共价连接，从而形成包含式(E)结构的复合物。应理解的是，在式(H)中与抗TfR1抗体相邻显示的酰胺是由与抗TfR1抗体的胺(例如赖氨酸ε胺)的反应产生的。wherein n is any number from 0 to 15, and wherein m is any number from 0 to 15. In some embodiments, n is 3 and/or (for example, and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound comprising the structure of formula (H) to form a complex comprising the structure of formula (E). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (H) is produced by reaction with an amine of the anti-TfR1 antibody (e.g., lysine epsilon amine).
在一些实施方案中，用于共价连接抗TfR抗体和分子载荷(例如，寡核苷酸)的val-cit接头包含以下结构：In some embodiments, the val-cit linker for covalently linking an anti-TfR antibody and a molecular payload (e.g., an oligonucleotide) comprises the following structure:
其中n为0至15中的任意数字，其中m为0至15中的任意数字。在一些实施方案中，n为3和/或(例如，和)m为4。在一些实施方案中，n为3和/或(例如，和)m为4。在一些实施方案中，X是抗体的NH(例如，来自赖氨酸的胺基的NH)、S(例如，来自半胱氨酸的巯基的S)或O(例如，来自丝氨酸、苏氨酸或酪氨酸的羟基的O)。在一些实施方案中，所述处理复合物的方法包括产生复合物的步骤。wherein n is any number from 0 to 15, wherein m is any number from 0 to 15. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NH from the amine group of lysine), S (e.g., S from the sulfhydryl group of cysteine), or O (e.g., O from the hydroxyl group of serine, threonine, or tyrosine) of the antibody. In some embodiments, the method of treating the complex comprises the step of generating the complex.
在一些实施方案中，产生复合物的方法包括：In some embodiments, the method of producing a complex comprises:
(i)获得包含以下结构的寡核苷酸：(i) obtaining an oligonucleotide comprising the following structure:
其中n为0至15(例如，3)；wherein n is 0 to 15 (e.g., 3);
(ii)获得包含以下结构的抗体：(ii) obtaining an antibody comprising the following structure:
其中m为0至15(例如，4)；以及wherein m is 0 to 15 (e.g., 4); and
(iii)使步骤(i)中的寡核苷酸和步骤(ii)中获得的抗体反应以获得复合物。(iii) reacting the oligonucleotide obtained in step (i) with the antibody obtained in step (ii) to obtain a complex.
在一些实施方案中，该方法包括：In some embodiments, the method comprises:
(i)获得包含以下结构的寡核苷酸：(i) obtaining an oligonucleotide comprising the following structure:
其中n为0至15(例如，3)，并且其中m为0至15(例如，4)；wherein n is 0 to 15 (e.g., 3), and wherein m is 0 to 15 (e.g., 4);
(ii)获得抗体；以及(ii) obtaining antibodies; and
(iii)使步骤(i)中的寡核苷酸和步骤(ii)中获得的抗体反应以获得复合物。(iii) reacting the oligonucleotide obtained in step (i) with the antibody obtained in step (ii) to obtain a complex.
在一些实施方案中，使用本文中所述方法产生的复合物包含以下结构：In some embodiments, the complex produced using the methods described herein comprises the following structure:
其中n为0至15(例如，3)，并且其中m为0至15(例如，4)，并且其中抗体通过赖氨酸共价连接。wherein n is 0 to 15 (eg, 3), and wherein m is 0 to 15 (eg, 4), and wherein the antibody is covalently linked via a lysine.
在一些实施方案中，本文中所述的复合物具有以下结构：In some embodiments, the complexes described herein have the following structure:
其中n为0至10中的任意数字，其中m为0至10中的任意数字。在一些实施方案中，n为3并且m为4。wherein n is any number from 0 to 10, wherein m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.
在结构式(I)中，在一些实施方案中，L1为间隔区，其为经取代或未经取代的脂肪族、经取代或未经取代的杂脂肪族、经取代或未经取代的碳环亚基、经取代或未经取代的杂环亚基、经取代或未经取代的亚芳基、经取代或未经取代的杂亚芳基、-O-，-N(RA)-，-S，-C(＝O)-，-C(＝O)O-，-C(＝O)NRA-，-NRAC(＝O)-，-NRAC(＝O)RA-，-C(＝O)RA-，-NRAC(＝O)O-，-NRAC(＝O)N(RA)-，-OC(＝O)-，-OC(＝O)O-，-OC(＝O)N(RA)-，-S(O)2NRA-，-NRAS(O)2-，或其组合，其中每个RA独立地为氢或者经取代或未经取代的烷基。在一些实施方案中，L1是In structural formula (I), in some embodiments, L1 is a spacer which is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclic substituent, a substituted or unsubstituted heterocyclic substituent, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, -O-, -N(RA )-, -S, -C(＝O)-, -C(＝O)O-, -C(＝O)NRA-,-NR AC(＝O)-,-NRAC (＝O)RA-, -C(＝O)RA-, -NRAC (＝O)O-, -NRAC (＝O)N(RA )-, -OC(＝O)-, -OC(＝O)O-, -OC(＝O)N(RA )-, -S(O)2NRA- ,-NRAS (O)2- , or a combination thereof, wherein each RA is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L1 is
其中L2是Where L2 is
其中a标记与式(I)的氨基甲酸酯部分直接连接的位点；并且b标记与寡核苷酸共价连接(直接或通过另外的化学部分)的位点。wherein a marks the site of direct attachment to the carbamate moiety of formula (I); and b marks the site of covalent attachment to the oligonucleotide (directly or via an additional chemical moiety).
在一些实施方案中，L1是：In some embodiments, L1 is:
其中a标记与式(I)的氨基甲酸酯部分直接连接的位点；并且b标记与寡核苷酸共价连接(直接或通过另外的化学部分)的位点。wherein a marks the site of direct attachment to the carbamate moiety of formula (I); and b marks the site of covalent attachment to the oligonucleotide (directly or via an additional chemical moiety).
在一些实施方案中，L1是In some embodiments, L1 is
在一些实施方案中，L1与寡核苷酸的5’磷酸连接。In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide.
在一些实施方案中，L1是任选的(例如，不需要存在的)。In some embodiments, L1 is optional (eg, need not be present).
应理解，在式(E)、式(F)和式(I)中与抗TfR1抗体相邻显示的酰胺是由与抗TfR1抗体的胺(例如赖氨酸ε胺)的反应产生的。It is understood that the amide shown adjacent to the anti-TfRl antibody in Formula (E), Formula (F), and Formula (I) results from a reaction with an amine of the anti-TfRl antibody (eg, the lysine epsilon amine).
D.抗体-分子载荷复合物的一些实例D. Some Examples of Antibody-Molecular Loading Complexes
本文中还提供了复合物的一些非限制性实例，所述复合物包含与本文中所述的任何分子载荷(例如，寡核苷酸)共价连接的本文中所述的任一种肌肉靶向剂(例如，抗TfR抗体)。在一些实施方案中，抗TfR抗体(例如，表2中提供的任一种肌肉靶向剂(例如，抗TfR抗体))通过接头与分子载荷(例如，寡核苷酸)共价连接。可以使用本文中描述的任意接头。在一些实施方案中，如果分子载荷是寡核苷酸，则接头与寡核苷酸的5’端、3’端或内部共价连接。在一些实施方案中，接头通过巯基反应性键联(例如，通过抗TfR抗体中的半胱氨酸)与抗TfR抗体共价连接。在一些实施方案中，接头(例如，Val-cit接头)通过n胺基(例如，通过抗体中的赖氨酸)与抗体(例如，本文中所述的抗TfR抗体)共价连接。在一些实施方案中，分子载荷是寡核苷酸(例如，靶向表1中列出的萎缩基因(atrophy gene)的寡核苷酸)。Also provided herein are some non-limiting examples of complexes, the complexes comprising any muscle targeting agent described herein (e.g., anti-TfR antibody) covalently linked to any molecular payload (e.g., oligonucleotide) described herein. In some embodiments, an anti-TfR antibody (e.g., any muscle targeting agent (e.g., anti-TfR antibody) provided in Table 2) is covalently linked to a molecular payload (e.g., oligonucleotide) via a linker. Any linker described herein can be used. In some embodiments, if the molecular payload is an oligonucleotide, the linker is covalently linked to the 5' end, 3' end, or interior of the oligonucleotide. In some embodiments, the linker is covalently linked to the anti-TfR antibody via a sulfhydryl-reactive linkage (e.g., via cysteine in an anti-TfR antibody). In some embodiments, a linker (e.g., a Val-cit linker) is covalently linked to an antibody (e.g., an anti-TfR antibody described herein) via an n amine group (e.g., via lysine in an antibody). In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide targeting an atrophy gene listed in Table 1).
下面提供了包含通过Val-cit接头与分子载荷共价连接的抗TfR抗体的复合物的结构的一个实例：An example of the structure of a complex comprising an anti-TfR antibody covalently linked to a molecular cargo via a Val-cit linker is provided below:
其中接头通过巯基反应性键联(例如，通过抗体中的半胱氨酸)与抗体共价连接。在一些实施方案中，分子载荷是寡核苷酸(例如，靶向表1中列出的萎缩基因的寡核苷酸)。wherein the linker is covalently attached to the antibody via a thiol-reactive linkage (eg, via a cysteine in the antibody). In some embodiments, the molecular payload is an oligonucleotide (eg, an oligonucleotide targeting an atrophy gene listed in Table 1).
下面提供了包含通过Val-cit接头与分子载荷共价连接的抗TfR抗体的复合物的结构的另一个实例：Another example of the structure of a complex comprising an anti-TfR antibody covalently linked to a molecular cargo via a Val-cit linker is provided below:
其中n为0至15中的数字，其中m为0至15中的数字，其中接头通过胺基(例如，在赖氨酸残基上)与抗体共价连接，和/或(例如，和)其中接头与寡核苷酸(例如，在5’端、3’端或内部)共价连接。在一些实施方案中，接头通过赖氨酸与抗体共价连接，接头在5’端与寡核苷酸共价连接，n为3并且m为4。在一些实施方案中，分子载荷是寡核苷酸(例如，靶向表1中列出的萎缩基因的寡核苷酸)。wherein n is a number from 0 to 15, wherein m is a number from 0 to 15, wherein the linker is covalently linked to the antibody via an amine group (e.g., on a lysine residue), and/or (e.g., and) wherein the linker is covalently linked to the oligonucleotide (e.g., at the 5' end, the 3' end, or internally). In some embodiments, the linker is covalently linked to the antibody via a lysine, the linker is covalently linked to the oligonucleotide at the 5' end, n is 3 and m is 4. In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide targeting an atrophy gene listed in Table 1).
应理解，抗体可与具有不同化学计量的寡核苷酸共价连接，该特性可被称为药物抗体比(DAR)，其中“药物”是寡核苷酸。在一些实施方案中，一个寡核苷酸与一个抗体共价连接(DAR＝1)。在一些实施方案中，两个寡核苷酸与一个抗体共价连接(DAR＝2)。在一些实施方案中，三个寡核苷酸与一个抗体共价连接(DAR＝3)。在一些实施方案中，四个寡核苷酸与一个抗体共价连接(DAR＝4)。在一些实施方案中，提供了不同复合物的混合物，每种复合物具有不同的DAR。在一些实施方案中，这样的混合物中的复合物的平均DAR可在1至3、1至4、1至5或更大的范围内。可通过将寡核苷酸缀合至抗体上的不同位点和/或通过将多聚体缀合至抗体上的一个或更多个位点来提高DAR。例如，可通过将单个寡核苷酸缀合至抗体上的两个不同位点或通过将二聚体寡核苷酸缀合至抗体的单个位点来实现DAR为2。It should be understood that antibodies can be covalently linked to oligonucleotides with different stoichiometries, and this property can be referred to as drug-antibody ratio (DAR), where "drug" is an oligonucleotide. In some embodiments, one oligonucleotide is covalently linked to one antibody (DAR=1). In some embodiments, two oligonucleotides are covalently linked to one antibody (DAR=2). In some embodiments, three oligonucleotides are covalently linked to one antibody (DAR=3). In some embodiments, four oligonucleotides are covalently linked to one antibody (DAR=4). In some embodiments, a mixture of different complexes is provided, each complex having a different DAR. In some embodiments, the average DAR of the complex in such a mixture can be in the range of 1 to 3, 1 to 4, 1 to 5 or more. DAR can be increased by conjugating oligonucleotides to different sites on the antibody and/or by conjugating a polymer to one or more sites on the antibody. For example, DAR of 2 can be achieved by conjugating a single oligonucleotide to two different sites on the antibody or by conjugating a dimer oligonucleotide to a single site of the antibody.
在一些实施方案中，本文中所述的复合物包含与寡核苷酸共价连接的转铁蛋白受体抗体(例如，如本文中所述的抗体或其任意变体)。在一些实施方案中，本文中所述的复合物包含通过接头(例如，Val-cit接头)与寡核苷酸共价连接的转铁蛋白受体抗体(例如，如本文中所述的抗体或其任意变体)。在一些实施方案中，接头(例如，Val-cit接头)与寡核苷酸的5’端、3’端或内部共价连接。在一些实施方案中，接头(例如，Val-cit接头)通过硫醇反应性键联(例如，通过抗体中的半胱氨酸)与抗体(例如，如本文中所述的抗体或其任意变体)共价连接。In some embodiments, the complex described herein comprises a transferrin receptor antibody (e.g., an antibody as described herein or any variant thereof) covalently linked to an oligonucleotide. In some embodiments, the complex described herein comprises a transferrin receptor antibody (e.g., an antibody as described herein or any variant thereof) covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker). In some embodiments, a linker (e.g., a Val-cit linker) is covalently linked to the 5' end, 3' end, or interior of an oligonucleotide. In some embodiments, a linker (e.g., a Val-cit linker) is covalently linked to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine in an antibody).
在一些实施方案中，本文中所述的复合物包含与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含与表3所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2和CDR-H3；以及与表3所示的CDR-L1、CDR-L2和CDR-L3相同的CDR-L1、CDR-L2和CDR-L3。In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to CDR-H1, CDR-H2, and CDR-H3 shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to CDR-L1, CDR-L2, and CDR-L3 shown in Table 3.
在一些实施方案中，本文中所述的复合物包含与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:33的氨基酸序列的VH和具有SEQID NO:34的氨基酸序列的VL。在一些实施方案中，本文中所述的复合物包含与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:35的氨基酸序列的VH和具有SEQ ID NO:36的氨基酸序列的VL。In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36.
在一些实施方案中，本文中所述的复合物包含与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:39的氨基酸序列的重链和具有SEQ ID NO:40的氨基酸序列的轻链。在一些实施方案中，本文中所述的复合物包含与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:41的氨基酸序列的重链和具有SEQ ID NO:42的氨基酸序列的轻链。In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42.
在一些实施方案中，本文中所述的复合物包含通过接头(例如，Val-cit接头)与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含与表3中所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2和CDR-H3；以及与表3中所示的CDR-L1、CDR-L2和CDR-L3相同的CDR-L1、CDR-L2和CDR-L3。In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3.
在一些实施方案中，本文中所述的复合物包含通过接头(例如，Val-cit接头)与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:33的氨基酸序列的VH和具有SEQ ID NO:34的氨基酸序列的VL。在一些实施方案中，本文中所述的复合物包含通过接头(例如，Val-cit接头)与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:35的氨基酸序列的VH和具有SEQ ID NO:36的氨基酸序列的VL。In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36.
在一些实施方案中，本文中所述的复合物包含通过接头(例如，Val-cit接头)与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:39的氨基酸序列的重链和具有SEQ ID NO:40的氨基酸序列的轻链。在一些实施方案中，本文中所述的复合物包含通过接头(例如，Val-cit接头)与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:41的氨基酸序列的重链和具有SEQID NO:42的氨基酸序列的轻链。In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42.
在一些实施方案中，本文中所述的复合物包含通过Val-cit接头与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含与表3中所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2和CDR-H3；以及与表3中所示的CDR-L1、CDR-L2和CDR-L3相同的CDR-L1、CDR-L2和CDR-L3，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3, and wherein the complex comprises the following structure:
其中接头Val-cit接头与寡核苷酸的5’端、3’端或内部共价连接，并且其中Val-cit接头通过巯基反应性键联(例如，通过抗体中的半胱氨酸)与抗体(例如，本文中所述的抗体或其任意变体)共价连接。wherein the linker Val-cit linker is covalently linked to the 5' end, 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is covalently linked to the antibody (e.g., an antibody described herein or any variant thereof) via a sulfhydryl-reactive linkage (e.g., via a cysteine in the antibody).
在一些实施方案中，本文中所述的复合物包含通过Val-cit接头与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:33的氨基酸序列的VH和具有SEQ ID NO:34的氨基酸序列的VL，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34, and wherein the complex comprises the following structure:
其中接头Val-cit接头与寡核苷酸的5’端、3’端或内部共价连接，并且其中Val-cit接头通过巯基反应性键联(例如，通过抗体中的半胱氨酸)与抗体(例如，本文中所述的抗体或其任意变体)共价连接。wherein the linker Val-cit linker is covalently linked to the 5' end, 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is covalently linked to the antibody (e.g., an antibody described herein or any variant thereof) via a sulfhydryl-reactive linkage (e.g., via a cysteine in the antibody).
在一些实施方案中，本文中所述的复合物包含通过Val-cit接头与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:35的氨基酸序列的VH和具有SEQ ID NO:36的氨基酸序列的VL，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36, and wherein the complex comprises the following structure:
其中接头Val-cit接头与寡核苷酸的5’端、3’端或内部共价连接，并且其中Val-cit接头通过硫醇反应性键联(例如，通过抗体中的半胱氨酸)与抗体(例如，如本文中所述的抗体或其任意变体)共价连接。wherein the linker Val-cit linker is covalently linked to the 5' end, 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is covalently linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
在一些实施方案中，本文中所述的复合物包含通过Val-cit接头与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:39的氨基酸序列的重链和具有SEQ ID NO:40的氨基酸序列的轻链，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40, and wherein the complex comprises the following structure:
其中接头Val-cit接头与寡核苷酸的5’端、3’端或内部共价连接，并且其中Val-cit接头通过硫醇反应性键联(例如，通过抗体中的半胱氨酸)与抗体(例如，如本文中所述的抗体或其任意变体)共价连接。wherein the linker Val-cit linker is covalently linked to the 5' end, 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is covalently linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
在一些实施方案中，本文中所述的复合物包含通过Val-cit接头与寡核苷酸共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:41的氨基酸序列的重链和具有SEQ ID NO:42的氨基酸序列的轻链，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42, and wherein the complex comprises the following structure:
其中接头Val-cit接头与寡核苷酸的5’端、3’端或内部共价连接，并且其中Val-cit接头通过硫醇反应性键联(例如，通过抗体中的半胱氨酸)与抗体(例如，如本文中所述的抗体或其任意变体)共价连接。wherein the linker Val-cit linker is covalently linked to the 5' end, 3' end or internally of the oligonucleotide, and wherein the Val-cit linker is covalently linked to the antibody (e.g., an antibody as described herein or any variant thereof) through a thiol-reactive linkage (e.g., through a cysteine in the antibody).
在一些实施方案中，本文中所述的复合物包含通过赖氨酸与寡核苷酸的5’端共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含与表3中所示的CDR-H1、CDR-H2和CDR-H3相同的CDR-H1、CDR-H2和CDR-H3；以及与表3中所示的CDR-L1、CDR-L2和CDR-L3相同的CDR-L1、CDR-L2和CDR-L3，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide via a lysine, wherein the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 3; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 3, and wherein the complex comprises the following structure:
其中n为3并且m为4。在一些实施方案中，寡核苷酸是靶向表1中列出的萎缩基因的寡核苷酸。wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting an atrophy gene listed in Table 1.
在一些实施方案中，本文中所述的复合物包含通过赖氨酸与寡核苷酸的5’端共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:33的氨基酸序列的VH和具有SEQ ID NO:34的氨基酸序列的VL，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide via a lysine, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 34, and wherein the complex comprises the following structure:
其中n为3并且m为4。在一些实施方案中，寡核苷酸是靶向表1中列出的萎缩基因的寡核苷酸。在一些实施方案中，本文中所述的复合物包含通过赖氨酸与寡核苷酸5’端共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:35的氨基酸序列的VH和具有SEQ ID NO:36的氨基酸序列的VL，并且其中所述复合物包含以下结构：wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting an atrophy gene listed in Table 1. In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of the oligonucleotide via a lysine, wherein the transferrin receptor antibody comprises a VH having an amino acid sequence of SEQ ID NO: 35 and a VL having an amino acid sequence of SEQ ID NO: 36, and wherein the complex comprises the following structure:
其中n为3并且m为4。在一些实施方案中，寡核苷酸是靶向表1中列出的萎缩基因的寡核苷酸。wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting an atrophy gene listed in Table 1.
在一些实施方案中，本文中所述的复合物包含通过赖氨酸与寡核苷酸的5’端共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:39的氨基酸序列的重链和具有SEQ ID NO:40的氨基酸序列的轻链，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide via a lysine, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 39 and a light chain having an amino acid sequence of SEQ ID NO: 40, and wherein the complex comprises the following structure:
其中n为3并且m为4。在一些实施方案中，寡核苷酸是靶向表1中列出的萎缩基因的寡核苷酸。wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting an atrophy gene listed in Table 1.
在一些实施方案中，本文中所述的复合物包含通过赖氨酸与寡核苷酸的5’端共价连接的转铁蛋白受体抗体，其中所述转铁蛋白受体抗体包含具有SEQ ID NO:41的氨基酸序列的重链和具有SEQ ID NO:42的氨基酸序列的轻链，并且其中所述复合物包含以下结构：In some embodiments, the complex described herein comprises a transferrin receptor antibody covalently linked to the 5' end of an oligonucleotide via a lysine, wherein the transferrin receptor antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 41 and a light chain having an amino acid sequence of SEQ ID NO: 42, and wherein the complex comprises the following structure:
其中n为3并且m为4。在一些实施方案中，寡核苷酸是靶向表1中列出的萎缩基因的寡核苷酸。wherein n is 3 and m is 4. In some embodiments, the oligonucleotide is an oligonucleotide targeting an atrophy gene listed in Table 1.
应理解，在具有式(E)的复合物的一些实例中，与抗TfR1抗体相邻显示的酰胺是由与抗TfR1抗体的胺(例如赖氨酸ε胺)反应产生的。It will be appreciated that in some examples of complexes having formula (E), the amide displayed adjacent to the anti-TfRl antibody results from reaction with an amine (eg, lysine epsilon amine) of the anti-TfRl antibody.
III.制剂III. Preparation
本文中提供的复合物可以任何合适的方式配制。通常来说，本文中提供的复合物以适用于药物用途的方式配制。例如，可使用使降解最小化、促进递送和/或摄取或者为制剂中的复合物提供另一种有益特性的制剂将复合物递送至对象。在一些实施方案中，本文中提供了包含复合物和可药用载体的组合物。可适当地配制这样的组合物使得当施用于对象时，无论是施用至靶细胞的直接环境中或全身性施用，足够量的复合物都能进入靶肌细胞。在一些实施方案中，复合物被配制在缓冲溶液例如磷酸盐缓冲盐水溶液、脂质体、胶束结构和衣壳中。The complexes provided herein can be formulated in any suitable manner. Generally speaking, the complexes provided herein are formulated in a manner suitable for pharmaceutical use. For example, the complexes can be delivered to the subject using a formulation that minimizes degradation, promotes delivery and/or uptake, or provides another beneficial property for the complexes in the formulation. In some embodiments, compositions comprising complexes and pharmaceutically acceptable carriers are provided herein. Such compositions can be appropriately formulated so that when applied to the subject, whether it is applied to the direct environment of the target cell or systemically, a sufficient amount of the complex can enter the target muscle cell. In some embodiments, the complex is formulated in a buffer solution such as a phosphate buffered saline solution, a liposome, a micellar structure, and a capsid.
应理解，在一些实施方案中，组合物可分别包含本文中提供的复合物的一种或更多种组分(例如，肌肉靶向剂、接头、分子载荷或它们中任一者的前体分子)。It is understood that, in some embodiments, a composition can separately comprise one or more components of a complex provided herein (eg, a muscle targeting agent, a linker, a molecular cargo, or a precursor molecule of any of them).
在一些实施方案中，复合物被配制在水或水溶液(例如，用pH调节的水)中。在一些实施方案中，复合物被配制在碱性缓冲水溶液(例如，PBS)中。在一些实施方案中，本文中公开的制剂包含赋形剂。在一些实施方案中，赋形剂赋予组合物以改善的稳定性、改善的吸收、改善的溶解性和/或活性成分的治疗性增强。在一些实施方案中，赋形剂是缓冲剂(例如，柠檬酸钠、磷酸钠、tris碱或氢氧化钠)或载剂(例如，缓冲溶液、矿脂(petrolatum)、二甲基亚砜或矿物质油)。In some embodiments, the complex is formulated in water or an aqueous solution (e.g., water adjusted with pH). In some embodiments, the complex is formulated in an alkaline buffered aqueous solution (e.g., PBS). In some embodiments, the formulation disclosed herein comprises an excipient. In some embodiments, the excipient imparts the composition with improved stability, improved absorption, improved solubility, and/or therapeutic enhancement of the active ingredient. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris alkali, or sodium hydroxide) or a carrier (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).
在一些实施方案中，将复合物或其组分(例如，寡核苷酸或抗体)冻干用于延长其保质期，并随后在使用(例如，施用于对象)之前制成溶液。因此，包含本文中所述的复合物或其组分的组合物中的赋形剂可以是冻干保护剂(例如，甘露醇、乳糖、聚乙二醇或聚乙烯吡咯烷酮)或崩解温度调节剂(例如，右旋糖酐、ficoll或明胶)。In some embodiments, the complex or its components (e.g., oligonucleotides or antibodies) are lyophilized to extend their shelf life and then made into a solution before use (e.g., administration to a subject). Thus, the excipient in the composition comprising the complex or its components described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol or polyvinyl pyrrolidone) or a disintegration temperature regulator (e.g., dextran, ficoll or gelatin).
在一些实施方案中，药物组合物被配制成与其预期施用途径相容。施用途径的一些实例包括肠胃外施用，例如静脉内、皮内、皮下施用。通常来说，施用途径是静脉内或皮下的。在一些实施方案中，施用途径是肌外肠胃外施用。In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Some examples of route of administration include parenteral administration, such as intravenous, intradermal, subcutaneous administration. Generally speaking, the route of administration is intravenous or subcutaneous. In some embodiments, the route of administration is extramuscular parenteral administration.
适用于可注射使用的药物组合物包括无菌水溶液(在具有水溶性时)或分散体，以及用于即时制备无菌可注射溶液或分散体的无菌粉末。载体可以是溶剂或分散介质，其包含例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)，及其合适的混合物。在一些实施方案中，组合物中的制剂包含等张剂，例如糖、多元醇例如甘露醇、山梨醇以及氯化钠。无菌可注射溶液可通过将所需量的复合物与以上列举的成分之一或组合(根据需要)一起并入选定的溶剂中，然后过滤灭菌来制备。Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when water-soluble) or dispersions, and sterile powders for instant preparation of sterile injectable solutions or dispersions. Carriers can be solvents or dispersion media, which include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the preparation in the composition includes isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Sterile injectable solutions can be prepared by incorporating the desired amount of the complex into a selected solvent together with one of the ingredients listed above or in combination (as required), and then filter sterilizing.
在一些实施方案中，组合物可包含至少约0.1％的复合物或其组分，或者更多，尽管一种或更多种活性成分的百分比可为总组合物的重量或体积的约1％至约80％或更多。本领域技术人员将考虑制备这样的药物制剂的因素例如溶解性、生物利用度、生物学半衰期、施用途径、产品保质期以及其他药理学考虑因素，并且因此多种剂量和治疗方案可以是期望的。In some embodiments, the composition may contain at least about 0.1% of the complex or its components, or more, although the percentage of one or more active ingredients may be from about 1% to about 80% or more by weight or volume of the total composition. One skilled in the art will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations in preparing such pharmaceutical formulations, and therefore a variety of dosages and treatment regimens may be desired.
IV.使用/治疗方法IV. Use/treatment methods
如本文中所述的包含与分子载荷共价的肌肉靶向剂的复合物在治疗肌肉疾病(例如罕见肌肉疾病)中有效。在一些实施方案中，复合物在治疗表1中提供的肌肉疾病中有效。在一些实施方案中，肌肉疾病与疾病等位基因相关，例如，特定肌肉疾病的疾病等位基因可包含表1所列相应基因的遗传改变。The complexes comprising a muscle targeting agent covalently attached to a molecular payload as described herein are effective in treating muscle diseases (e.g., rare muscle diseases). In some embodiments, the complexes are effective in treating the muscle diseases provided in Table 1. In some embodiments, the muscle disease is associated with a disease allele, for example, a disease allele of a particular muscle disease may comprise a genetic alteration of a corresponding gene listed in Table 1.
在一些实施方案中，对象可以是人对象、非人灵长类对象、啮齿动物对象或任何合适的哺乳动物对象。在一些实施方案中，对象可患有表1中提供的肌肉疾病。In some embodiments, the subject can be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject can be suffering from a muscle disease provided in Table 1.
本公开内容的一个方面包括涉及向对象施用有效量的本文中所述的复合物的方法。在一些实施方案中，可向有治疗需要的对象施用有效量的包含含有与分子载荷共价的肌肉靶向剂的复合物的药物组合物。在一些实施方案中，可通过合适的途径施用包含如本文中所述的复合物的药物组合物，所述途径可包括静脉内施用，例如作为推注(bolus)或通过在一段时间内的连续输注。在一些实施方案中，可通过肌内、腹膜内、脑脊髓内、皮下、关节内、滑膜内或鞘内途径进行静脉内施用。在一些实施方案中，药物组合物可以是固体形式、水性形式或液体形式。在一些实施方案中，可将水性或液体形式雾化或冻干。在一些实施方案中，雾化或冻干形式可用水溶液或液体溶液重构。One aspect of the present disclosure includes methods involving administering an effective amount of a complex as described herein to a subject. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently attached to a molecular payload may be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, for example as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial or intrathecal routes. In some embodiments, the pharmaceutical composition may be in solid form, aqueous form or liquid form. In some embodiments, an aqueous or liquid form may be atomized or lyophilized. In some embodiments, an atomized or lyophilized form may be reconstituted with an aqueous solution or a liquid solution.
用于静脉内施用的组合物可包含多种载体，例如植物油、二甲基乳酰胺、二甲基甲酰胺、乳酸乙酯、碳酸乙酯、豆蔻酸异丙酯、乙醇以及多元醇(甘油、丙二醇、液体聚乙二醇等)。对于静脉内注射，水溶性抗体可通过滴注方法施用，通过所述方法输注包含抗体和生理学上可接受的赋形剂的药物制剂。生理上可接受的赋形剂可包括，例如5％右旋糖、0.9％盐水、林格液(Ringer’s solution)或其他合适的赋形剂。可将肌内制剂，例如抗体的合适可溶性盐形式的无菌制剂溶解在药用赋形剂例如注射用水、0.9％盐水或5％葡萄糖溶液中并施用。Compositions for intravenous administration may include a variety of carriers, such as vegetable oils, dimethyl lactamide, dimethyl formamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by a drip method, by which a pharmaceutical preparation comprising an antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile preparations in the form of suitable soluble salts of antibodies, may be dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution and administered.
在一些实施方案中，包含含有与分子载荷共价的肌肉靶向剂的复合物的药物组合物通过位点特异性或局部递送技术施用。这些技术的一些实例包括复合物的可植入储库源、局部递送导管、位点特异性载体、直接注射或直接应用。In some embodiments, the pharmaceutical composition comprising a complex containing a muscle targeting agent covalently attached to a molecular payload is administered by site-specific or local delivery techniques. Some examples of these techniques include implantable reservoir sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.
在一些实施方案中，包含含有与分子载荷共价的肌肉靶向剂的复合物的药物组合物以赋予对象治疗作用的有效浓度施用。如本领域技术人员所公认的，有效量根据疾病的严重程度、所治疗对象的独特特征(例如年龄、身体状况、健康或体重)、治疗的持续时间、任何同时治疗的性质、施用途径和相关因素而变化。这些相关因素是本领域技术人员已知的，并且仅通过常规实验即可解决。在一些实施方案中，有效浓度是被认为对患者安全的最大剂量。在一些实施方案中，有效浓度将是提供最大效力的最低的可能浓度。In some embodiments, the pharmaceutical composition comprising a complex containing a muscle targeting agent covalently attached to a molecular payload is administered at an effective concentration to confer a therapeutic effect on the subject. As recognized by those skilled in the art, the effective amount varies according to the severity of the disease, the unique characteristics of the treated subject (e.g., age, physical condition, health, or weight), the duration of treatment, the nature of any simultaneous treatment, the route of administration, and related factors. These related factors are known to those skilled in the art and can be resolved only by routine experimentation. In some embodiments, the effective concentration is the maximum dose that is considered safe for the patient. In some embodiments, the effective concentration will be the lowest possible concentration that provides maximum efficacy.
经验考虑因素(例如复合物在对象中的半衰期)通常将有助于确定用于治疗的药物组合物的浓度。施用频率可凭经验确定和调整以使治疗效力最大化。Empirical considerations, such as the half-life of the complex in the subject, will generally help determine the concentration of the pharmaceutical composition to use for treatment. The frequency of administration can be empirically determined and adjusted to maximize therapeutic efficacy.
通常来说，对于本文中所述的任何复合物的施用，根据上述因素，例如安全性或效力，初始候选剂量可为约1至100mg/kg或更高。在一些实施方案中，将施用一次治疗。在一些实施方案中，将每天、每两周、每周、每两个月、每月或以提供最大效力的同时使对对象的安全风险最小化的任何时间间隔施用治疗。通常来说，可在整个治疗过程中监测效力和治疗以及安全风险。Generally speaking, for the administration of any complex described herein, the initial candidate dose may be about 1 to 100 mg/kg or higher, depending on the above factors, such as safety or efficacy. In some embodiments, the treatment will be administered once. In some embodiments, the treatment will be administered daily, every two weeks, weekly, every two months, monthly, or at any time interval that provides maximum efficacy while minimizing safety risks to the subject. Generally speaking, the efficacy and treatment and safety risks can be monitored throughout the treatment process.
可使用任何合适的方法评估治疗的效力。在一些实施方案中，可通过对与肌肉疾病相关的症状的观察进行评价来评估治疗的效力。The effectiveness of treatment can be assessed using any suitable method.In some embodiments, the effectiveness of treatment can be assessed by evaluating the observation of symptoms associated with the muscle disease.
在一些实施方案中，将包含含有与分子载荷共价的肌肉靶向剂的本文中所述的复合物的药物组合物以相对于对照(例如治疗之前基因表达的基线水平)足以抑制至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少95％的靶基因活性或表达的有效浓度施用于对象。In some embodiments, a pharmaceutical composition comprising a complex described herein that comprises a muscle targeting agent covalently attached to a molecular payload is administered to a subject at an effective concentration sufficient to inhibit the activity or expression of a target gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% relative to a control (e.g., baseline level of gene expression prior to treatment).
在一些实施方案中，药物组合物可包含多于一种包含与分子载荷共价的肌肉靶向剂的复合物。在一些实施方案中，药物组合物还可包含用于治疗对象(例如，患有肌肉疾病(例如表1中提供的肌肉疾病)的人对象)的任何其他合适的治疗剂。在一些实施方案中，其他治疗剂可增强或补充本文中所述的复合物的效力。在一些实施方案中，其他治疗剂可发挥功能来治疗不同于本文中所述的复合物的症状或疾病。In some embodiments, the pharmaceutical composition may include more than one complex comprising a muscle targeting agent covalently attached to a molecular payload. In some embodiments, the pharmaceutical composition may also include any other suitable therapeutic agent for treating a subject, e.g., a human subject suffering from a muscle disease, e.g., a muscle disease provided in Table 1. In some embodiments, the other therapeutic agent may enhance or supplement the efficacy of the complexes described herein. In some embodiments, the other therapeutic agent may function to treat a symptom or disease other than the complexes described herein.
实施例Example
实施例1:包含与寡核苷酸连接的抗体的复合物的合成(缀合方法1-预反应缀合)Example 1: Synthesis of a complex comprising an antibody linked to an oligonucleotide (Conjugation method 1 - pre-reaction conjugation)
产生了肌肉靶向复合物，其包含通过组织蛋白酶可切割接头与抗转铁蛋白受体hIgG1-κFab抗体(抗TfR Fab)共价连接的靶向DMPK的反义寡核苷酸。A muscle targeting complex was generated comprising an antisense oligonucleotide targeting DMPK covalently linked to an anti-transferrin receptor hIgG1-κ Fab antibody (anti-TfR Fab) via a cathepsin cleavable linker.
使用1mL CaptureSelectTM CH1-XL柱(Thermo Fisher，Loughborough，UK)从CHO细胞培养上清液中纯化抗TfR Fab。使用1×DPBS来洗涤柱，并随后使用50mM乙酸钠pH 4.0来洗脱蛋白质。随后将蛋白质缓冲液交换到20mM柠檬酸钠，150mM NaCl，pH 6.0中。然后通过制备型SEC使用HiLoadTM 16/60SuperdexTM 75pg柱(GE Healthcare，Little Chalfont，UK)用20mM柠檬酸钠，150mM NaCl，pH 6.0作为流动相进一步纯化Fab。将包含单体蛋白质的峰级分合并，浓缩并过滤灭菌，然后根据A280nm使用基于预测的氨基酸序列的消光系数(Ec(0.1％))进行定量。然后通过还原和非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE)、分析尺寸排阻色谱-高效液相色谱(size-exclusion chromatography-high performance liquidchromatography，SEC-HPLC)和Biacore单循环动力学(single-cycle kinetic，SCK)来分析经纯化的Fab。在继续缀合反应之前，将抗TfR Fab缓冲液交换到50mM HEPES pH 7.5中。Anti-TfR Fab was purified from CHO cell culture supernatant using 1mL CaptureSelectTM CH1-XL column (Thermo Fisher, Loughborough, UK). 1 × DPBS was used to wash the column, and 50mM sodium acetate pH 4.0 was then used to elute the protein. The protein buffer was then exchanged into 20mM sodium citrate, 150mM NaCl, pH 6.0. Fab was then further purified by preparative SEC using HiLoadTM 16/60SuperdexTM 75pg column (GE Healthcare, Little Chalfont, UK) with 20mM sodium citrate, 150mM NaCl, pH 6.0 as mobile phase. The peak fractions containing monomeric protein were merged, concentrated and sterilized by filtration, and then quantified according to A280nm using an extinction coefficient (Ec (0.1%)) based on the predicted amino acid sequence. The purified Fab was then analyzed by reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), analytical size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC), and Biacore single-cycle kinetic (SCK). Before continuing the conjugation reaction, the anti-TfR Fab buffer was exchanged into 50 mM HEPES pH 7.5.
产生了包含寡核苷酸(例如，带电荷寡核苷酸)与叠氮化物-缬氨酸-瓜氨酸接头的接头/载荷化合物。将寡核苷酸(Na+加合物)以200mg/mL溶解在不含RNAse的水中。用无水二甲基甲酰胺(dimethylformamide，DMF)将该溶液稀释至10mg/mL。然后向溶液添加25倍摩尔过量的三丁胺。将接头分子(叠氮化物-PEG3-Val-Cit-PAB-PNP，以20mg/mL溶解在DMF中)在室温(约25℃)下以2倍摩尔过量添加至寡核苷酸溶液，持续120分钟。使用茚三酮(Kaiser测试)来测量反应完成，然后使用醇沉淀猝灭反应。通过添加0.1v/v 3M NaCl溶液，随后添加3体积的-80℃异丙醇来完成醇沉淀。然后将溶液充分混合，并随后允许在-20℃下沉淀1小时。将经沉淀的溶液离心(以4300×g；8℃)30分钟，并倾析溶剂。用不含RNase的水中的-80℃的80％乙醇(相当于起始反应的一体积)洗涤沉淀，并离心(以4300×g；8℃)20分钟。然后倾析乙醇，并将沉淀(含有包含寡核苷酸和叠氮化物-缬氨酸-瓜氨酸接头的化合物)在37℃下干燥10分钟。将包含寡核苷酸和叠氮化物-缬氨酸-瓜氨酸接头的接头/载荷化合物重悬于不含核酸酶的水的20％乙腈中，浓度为20mg/mL。A joint/load compound comprising an oligonucleotide (e.g., a charged oligonucleotide) and an azide-valine-citrulline joint is produced. Oligonucleotide (Na+ adduct) is dissolved in RNAse-free water at 200 mg/mL. The solution is diluted to 10 mg/mL with anhydrous dimethylformamide (DMF). Then 25 times of molar excess of tributylamine is added to the solution. The joint molecule (azide-PEG3-Val-Cit-PAB-PNP, dissolved in DMF at 20 mg/mL) is added to the oligonucleotide solution at room temperature (about 25°C) with 2 times of molar excess for 120 minutes. The reaction is measured using ninhydrin (Kaiser test) to complete, and then the reaction is quenched using alcohol precipitation. Alcohol precipitation is completed by adding 0.1v/v 3M NaCl solution, followed by the addition of 3 volumes of -80°C isopropanol. The solution is then fully mixed, and then allowed to precipitate at -20°C for 1 hour. The precipitated solution was centrifuged (at 4300×g; 8°C) for 30 minutes and the solvent was decanted. The precipitate was washed with 80% ethanol at -80°C in RNase-free water (equivalent to one volume of the starting reaction) and centrifuged (at 4300×g; 8°C) for 20 minutes. The ethanol was then decanted and the precipitate (containing the compound comprising the oligonucleotide and the azide-valine-citrulline linker) was dried at 37°C for 10 minutes. The linker/load compound comprising the oligonucleotide and the azide-valine-citrulline linker was resuspended in 20% acetonitrile in nuclease-free water at a concentration of 20 mg/mL.
随后在以下条件下进行预反应：在室温下，使5.87μM寡核苷酸-PAB-VC-PEG3-叠氮化物(结构在图1A中示出)和5.34mM endo-BCN-PEG4-PFP酯(1.1:1.0摩尔:摩尔当量；结构在图1B中示出)在60:40v/v％的DMA与25mM 2-(N-吗啉代)乙磺酸(MES)pH 5.5缓冲液中组合。A pre-reaction was then performed under the following conditions: 5.87 μM oligo-PAB-VC-PEG3-azide (structure shown in FIG. 1A ) and 5.34 mM endo-BCN-PEG4-PFP ester (1.1:1.0 mole:molar equivalents; structure shown in FIG. 1B ) were combined in 60:40 v/v % DMA and 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5 buffer at room temperature.
通过RP-C18UPLC每30分钟(在5分钟、35分钟和65分钟时间点时)重复注入，通过在220nm处观察endo-BCN-PEG4-PFP酯起始物质的消失来监测产生寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯(结构在图1C中示出)的预反应的完成。当相对于5分钟时间点剩余少于5％的endo-BCN-PEG4-PFP酯时，则确定反应完成。将粗制预反应混合物在没有任何纯化的情况下立即进行缀合反应。总的预反应时间为90分钟，定义为将endo-BCN-PEG4-PFP酯添加至预反应与将该反应混合物添加至Fab缀合反应之间的时间。Injection is repeated every 30 minutes (at 5 minutes, 35 minutes and 65 minutes time points) by RP-C18UPLC, and the completion of the pre-reaction producing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester (structure is shown in Figure 1C) is monitored by observing the disappearance of endo-BCN-PEG4-PFP ester starting material at 220nm. When less than 5% endo-BCN-PEG4-PFP ester is left relative to the 5 minute time point, the reaction is determined to be complete. The crude pre-reaction mixture is immediately subjected to conjugation reaction without any purification. The total pre-reaction time is 90 minutes, which is defined as the time between adding endo-BCN-PEG4-PFP ester to the pre-reaction and adding the reaction mixture to the Fab conjugation reaction.
寡核苷酸(例如，带电荷寡核苷酸)与抗TfR Fab的缀合涉及在Fab的溶剂可及赖氨酸残基与预反应步骤中产生的寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的活化PFP酯之间的酰胺键的形成。Conjugation of an oligonucleotide (e.g., a charged oligonucleotide) to an anti-TfR Fab involves the formation of an amide bond between a solvent accessible lysine residue of the Fab and the activated PFP ester of the oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester generated in a pre-reaction step.
用以下溶液反应物参数进行缀合反应：抗TfR Fab(45mg，6mg/mL，125μM)和以终浓度为750μM的寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯(1.0:6.0mol:mol当量的抗TfR Fab与寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯)。寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯浓度假设为预反应中BCN 100％转化成寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯。最终的反应混合物由15:85v/v％的DMA与25mM HEPES pH 7.5缓冲液组成。通过添加适量的反应物和储备溶液，在20mL玻璃闪烁小瓶中建立反应。将反应在室温(约25℃)下进行20小时。反应的开始被定义为将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的预反应混合物添加至抗TfR Fab溶液的时间。停止时间定义为在随后的纯化之前pH调节的开始。缀合反应的总持续时间为20小时。The conjugation reaction was carried out with the following solution reactant parameters: anti-TfR Fab (45 mg, 6 mg/mL, 125 μM) and oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester (1.0:6.0 mol:mol equivalents of anti-TfR Fab and oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester) at a final concentration of 750 μM. The concentration of oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester was assumed to be 100% conversion of BCN into oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester in the pre-reaction. The final reaction mixture consisted of 15:85 v/v% DMA and 25 mM HEPES pH 7.5 buffer. The reaction was set up in a 20 mL glass scintillation vial by adding an appropriate amount of reactants and stock solutions. The reaction was carried out at room temperature (about 25 ° C) for 20 hours. The start of the reaction was defined as the time when the pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester was added to the anti-TfR Fab solution. The stop time was defined as the start of pH adjustment before subsequent purification. The total duration of the conjugation reaction was 20 hours.
在反应20小时之后，通过SDS-PAGE来测试粗制缀合物混合物并通过密度测定法进行分析，以确定药物抗体比(DAR)和％未缀合Fab。After 20 hours of reaction, the crude conjugate mixture was tested by SDS-PAGE and analyzed by densitometry to determine the drug to antibody ratio (DAR) and % unconjugated Fab.
实施例2：包含与寡核苷酸连接的肌肉靶向抗体的复合物的纯化Example 2: Purification of complexes comprising muscle targeting antibodies linked to oligonucleotides
使用色谱成功地将来自实施例1的粗制反应产物纯化。将抗TfR Fab-寡核苷酸缀合物、未缀合的抗TfR Fab和未缀合的寡核苷酸的混合物在不含核酸酶的水中以1:3稀释，并用500mM MES缓冲液将混合物的pH调节至5.7。然后将该溶液以8mg/mL树脂的生物分子浓度装载到陶瓷羟基磷灰石(HA)柱(HiLoad-26mm×40cm柱，CHTTM 40μm树脂；来自Biorad，目录#732-4324)上[装载流量:线性113cm/小时，26mm ID-10.0mL/分钟体积流量，停留时间-21.4分钟]。用5CV的洗涤溶液(5mM Na2HPO4，25mM NaCl pH 7.0)洗涤HA柱以去除未连接寡核苷酸。在去除未连接寡核苷酸之后，在配制缓冲液(100mM Na2HPO4，100mM NaCl，pH 7.6)中使包含与寡核苷酸共价连接的抗TfR Fab的复合物从HA柱中洗脱。The crude reaction product from Example 1 was successfully purified using chromatography. A mixture of anti-TfR Fab-oligonucleotide conjugate, unconjugated anti-TfR Fab and unconjugated oligonucleotide was diluted 1:3 in nuclease-free water and the pH of the mixture was adjusted to 5.7 with 500 mM MES buffer. The solution was then loaded onto a ceramic hydroxyapatite (HA) column (HiLoad-26 mm×40 cm column, CHT™ 40 μm resin; from Biorad, catalog # 732-4324) at a biomolecule concentration of 8 mg/mL resin [loading flow: linear 113 cm/hour, 26 mm ID-10.0 mL/minute volume flow, residence time-21.4 minutes]. The HA column was washed with 5 CV of a wash solution (5 mM Na2 HPO4 , 25 mM NaCl pH 7.0) to remove unattached oligonucleotides. After removal of unligated oligonucleotides, the complex containing the anti-TfR Fab covalently linked to the oligonucleotides was eluted from the HA column in formulation buffer (100 mMNa2HPO4 , 100 mM NaCl,pH 7.6).
通过SDS-PAGE和分析型SEC来分析分离和经纯化的抗TfR Fab-寡核苷酸缀合物以表明未连接寡核苷酸的完全去除，以及通过ELISA分析人TfR1/cyno TfR1结合和内毒素水平。SEC色谱图显示来自HA树脂的洗脱级分提供了基本上纯化的包含与寡核苷酸共价连接的抗TfR Fab的复合物。此外，HA流通物(例如，洗涤级分)包含未连接寡核苷酸。这些结果表明，可使用羟基磷灰石树脂从未连接寡核苷酸中纯化复合物，这是以其他方式无法实现的出乎意料的纯化结果。The anti-TfR Fab-oligonucleotide conjugates separated and purified were analyzed by SDS-PAGE and analytical SEC to show the complete removal of unconnected oligonucleotides, and human TfR1/cyno TfR1 binding and endotoxin levels were analyzed by ELISA.SEC chromatograms show that the eluted fractions from HA resin provide substantially purified anti-TfR Fab complexes covalently attached to oligonucleotides. In addition, HA flowables (e.g., wash fractions) include unconnected oligonucleotides. These results show that hydroxyapatite resins can be used to purify complexes from unconnected oligonucleotides, which is an unexpected purification result that cannot be achieved otherwise.
检查了用于纯化来自实施例1的包含与寡核苷酸共价连接的抗TfR Fab的复合物、未连接寡核苷酸和未连接抗TfR Fab的粗制混合物的数种可替代策略，包括阳离子交换(cation exchange，CEX)和阴离子交换(anion exchange，AEX)树脂。发现没有一种可替代策略像此处所述方法(使用陶瓷羟基磷灰石树脂)一样有效。Several alternative strategies were examined for purification of the crude mixture of complexes containing anti-TfR Fab covalently linked to oligonucleotides, unlinked oligonucleotides, and unlinked anti-TfR Fabs from Example 1, including cation exchange (CEX) and anion exchange (AEX) resins. None of the alternative strategies were found to be as effective as the method described here (using ceramic hydroxyapatite resin).
实施例3：BCN与Fab的比率对Fab-寡核苷酸缀合反应的作用Example 3: Effect of the ratio of BCN to Fab on Fab-oligonucleotide conjugation reaction
为了研究来自预反应混合物的BCN与抗TfR的比率的作用，根据实施例1中描述的通用方案进行了一组反应。To investigate the effect of the ratio of BCN to anti-TfR from the pre-reaction mixture, a set of reactions was performed according to the general protocol described in Example 1.
使用以下最终反应条件进行寡核苷酸-PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使2.43mM寡核苷酸-PAB-VC-PEG3-叠氮化物与1.62mM endo-BCN-PEG4-PFP酯(1.5∶1mol∶mol比)在DMA和25mM MES pH 5.5缓冲液的1∶1混合物中反应。总预反应体积为0.17mL，并且使预反应步骤在室温下进行4小时。The following final reaction conditions were used for the pre-reaction between oligonucleotide-PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester: 2.43 mM oligonucleotide-PAB-VC-PEG3-azide was reacted with 1.62 mM endo-BCN-PEG4-PFP ester (1.5:1 mol:mol ratio) in a 1:1 mixture of DMA and 25 mM MES pH 5.5 buffer. The total pre-reaction volume was 0.17 mL, and the pre-reaction step was performed at room temperature for 4 hours.
在预反应完成后，将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化)的预反应混合物用于建立一系列抗TfR Fab缀合反应。使用相对于Fab的1.0、1.3、1.6、1.9、2.2和2.5摩尔当量的来自预反应产物混合物的BCN进行独立的抗TfR Fab缀合反应。所有其他反应条件在不同的缀合中保持不变。在缀合反应混合物中，Fab浓度为6mg/mL，其中在20mM HEPES pH 7.5缓冲液中有15v/v％DMA以及1mg总Fab/反应。将反应在室温下进行20小时。通过SDS-PAGE密度测定法来确定每种缀合物的最终平均DAR和％未缀合Fab(D0)，并且结果在表4中示出。SDS-PAGE凝胶在图2中示出。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester is used to set up a series of anti-TfR Fab conjugation reactions. Independent anti-TfR Fab conjugation reactions are carried out using BCN from pre-reaction product mixtures relative to 1.0, 1.3, 1.6, 1.9, 2.2 and 2.5 molar equivalents of Fab. All other reaction conditions remain unchanged in different conjugations. In the conjugation reaction mixture, the Fab concentration is 6 mg/mL, wherein there are 15 v/v% DMA and 1 mg total Fab/reaction in 20 mM HEPES pH 7.5 buffer. The reaction is carried out at room temperature for 20 hours. The final average DAR and % unconjugated Fab (D0) of each conjugate are determined by SDS-PAGE density determination, and the results are shown in Table 4. SDS-PAGE gel is shown in Figure 2.
表4。Table 4.
实施例4：BCN与Fab的比率和反应条件对Fab-寡核苷酸缀合反应的作用Example 4: Effect of BCN to Fab ratio and reaction conditions on Fab-oligonucleotide conjugation reaction
为了研究来自预反应混合物的BCN与抗TfR的比率的作用，以及在寡核苷酸-Fab缀合反应中寡核苷酸和Fab浓度的作用，根据实施例1中描述的通用方案进行了一组反应。To investigate the effect of the ratio of BCN to anti-TfR from the pre-reaction mixture, and the effect of oligonucleotide and Fab concentrations in the oligonucleotide-Fab conjugation reaction, a set of reactions were performed according to the general protocol described in Example 1.
使用以下反应条件进行寡核苷酸-PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使6.15mM寡核苷酸-PAB-VC-PEG3-叠氮化物与6.15mM endo-BCN-PEG4-PFP酯(1∶1mol∶mol比)在DMA和25mM MES pH 5.5缓冲液(60∶40DMA∶MES)的混合物中反应，总反应体积为0.11mL。使预反应在室温下进行110分钟。The following reaction conditions were used for the pre-reaction between oligonucleotide-PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester: 6.15 mM oligonucleotide-PAB-VC-PEG3-azide was reacted with 6.15 mM endo-BCN-PEG4-PFP ester (1:1 mol:mol ratio) in a mixture of DMA and 25 mM MES pH 5.5 buffer (60:40 DMA:MES) in a total reaction volume of 0.11 mL. The pre-reaction was allowed to proceed at room temperature for 110 minutes.
在预反应完成后，将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化的)预反应混合物用于建立一系列抗TfR Fab缀合反应。使用相对于Fab的2、3、4、5、6、8和10摩尔当量的来自预反应产物混合物的BCN进行独立的抗TfR Fab缀合反应。每次反应中的Fab浓度为10mg/mL，其中每次反应中总Fab为0.65mg。除DMA含量之外，用于缀合的所有其他反应条件在不同的缀合反应中均保持不变。使用2、3、4、5和6摩尔当量的BCN的反应在50mM HEPES pH 7.5缓冲液中的15v/v％DMA中于23至25℃下进行20小时。使用8和10摩尔当量的BCN的反应在50mM HEPES pH 7.5缓冲液中的20v/v％DMA中于23至25℃下进行19小时。通过SDS-PAGE密度测定法确定每种缀合物的最终平均DAR，其结果在表5中示出。SDS-PAGE凝胶在图3中示出。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester is used to establish a series of anti-TfR Fab conjugation reactions. Independent anti-TfR Fab conjugation reactions are carried out using 2, 3, 4, 5, 6, 8 and 10 molar equivalents of BCN from the pre-reaction product mixture relative to Fab. The Fab concentration in each reaction is 10 mg/mL, wherein the total Fab in each reaction is 0.65 mg. Except for DMA content, all other reaction conditions for conjugation remain unchanged in different conjugation reactions. The reaction using 2, 3, 4, 5 and 6 molar equivalents of BCN was carried out in 15v/v%DMA in 50mM HEPES pH 7.5 buffer at 23 to 25°C for 20 hours. The reaction using 8 and 10 molar equivalents of BCN was carried out in 20v/v%DMA in 50mM HEPES pH 7.5 buffer at 23 to 25°C for 19 hours. The final average DAR for each conjugate was determined by SDS-PAGE densitometry, the results of which are shown in Table 5. The SDS-PAGE gel is shown in FIG3.
表5。Table 5.
实施例5：Fab-寡核苷酸缀合和纯化Example 5: Fab-oligonucleotide conjugation and purification
使用以下反应条件进行寡核苷酸-PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使2.61mM寡核苷酸-PAB-VC-PEG3-叠氮化物与1.74mM endo-BCN-PEG4-PFP酯(1.5∶1.0mol∶mol当量)在DMA和25mM MES pH 5.5缓冲液的1∶1v/v％混合物中反应，总反应体积为0.44mL。使预反应在室温下进行4.75小时。通过RP C18 UPLC随时间测量endo-BCN-PEG4-PFP酯起始物质的峰面积的减少来监测预反应的完成(图4)。Use the following reaction conditions to carry out the pre-reaction between oligonucleotide-PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester: 2.61mM oligonucleotide-PAB-VC-PEG3-azide and 1.74mM endo-BCN-PEG4-PFP ester (1.5: 1.0mol: mol equivalent) are reacted in 1: 1v/v% mixture of DMA and 25mM MES pH 5.5 buffer, and the total reaction volume is 0.44mL. Make the pre-reaction carry out 4.75 hours at room temperature. The reduction of the peak area of endo-BCN-PEG4-PFP ester starting material is measured over time by RP C18 UPLC to monitor the completion of the pre-reaction (Fig. 4).
在预反应完成后，将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化的)预反应混合物用于建立一系列抗TfR Fab缀合反应。使用2.2∶1mol∶mol当量的相对于Fab的来自预反应产物混合物的寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯进行抗TfRFab缀合。缀合反应中的Fab浓度为6mg/mL，并且反应以10mg总Fab的规模进行。最终反应混合物的组成为20mM HEPES pH 7.5缓冲液中15v/v％DMA。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester is used to establish a series of anti-TfR Fab conjugation reactions. Anti-TfRFab conjugation is performed using 2.2: 1 mol: mol equivalents of oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester from the pre-reaction product mixture relative to Fab. The Fab concentration in the conjugation reaction is 6 mg/mL, and the reaction is carried out on a scale of 10 mg total Fab. The composition of the final reaction mixture is 15 v/v% DMA in 20 mM HEPES pH 7.5 buffer.
在室温下反应19小时之后，通过色谱来纯化粗制缀合物产物混合物以去除游离寡核苷酸物质。首先，在装载到1mL CHT I型羟基磷灰石柱上之后，用5倍柱体积(columnvolume，CV)的100％缓冲液A(10mM磷酸钠和10mM氯化钠，pH 6.5)洗涤缀合物。然后用10CV的缓冲液A与缓冲液B的95:5混合物(100mM磷酸钠和100mM氯化钠，pH 7.5)以1mL/分钟的流量进行第二次洗涤。最后，用10CV的缓冲液A与缓冲液B的90:10混合物以1ml/分钟的流量进行第三次洗涤。在洗涤步骤之后，用100％缓冲液B以1ml/分钟的流量洗脱缀合物。该方法得到7.4mg(74％产率)的抗TfR Fab-寡核苷酸缀合物，平均DAR为1.30。经纯化的抗TfR Fab-寡核苷酸缀合物的SEC色谱图和SDS-PAGE凝胶分别在图5和图6中示出。After reacting for 19 hours at room temperature, the crude conjugate product mixture is purified by chromatography to remove free oligonucleotide material.First, after being loaded on 1mL CHT I type hydroxyapatite column, the conjugate is washed with 100% buffer A (10mM sodium phosphate and 10mM sodium chloride, pH 6.5) of 5 times of column volume (column volume, CV).Then the 95:5 mixture (100mM sodium phosphate and 100mM sodium chloride, pH 7.5) of the buffer A of 10CV and buffer B is washed for the second time with a flow rate of 1mL/ minute.Finally, the 90:10 mixture of the buffer A of 10CV and buffer B is washed for the third time with a flow rate of 1ml/ minute.After the washing step, the conjugate is eluted with 100% buffer B with a flow rate of 1ml/ minute.The method obtains 7.4mg (74% yield) of anti-TfR Fab-oligonucleotide conjugate, and average DAR is 1.30. The SEC chromatogram and SDS-PAGE gel of the purified anti-TfR Fab-oligonucleotide conjugate are shown in Figure 5 and Figure 6, respectively.
实施例6：包含与寡核苷酸连接的抗体的复合物的合成(缀合方法2-两步缀合)Example 6: Synthesis of complexes comprising antibodies linked to oligonucleotides (Conjugation method 2 - two-step conjugation)
产生了肌肉靶向复合物，其包含通过组织蛋白酶可切割接头与抗转铁蛋白受体hIgG1-κ抗体(抗TfR抗体)共价连接的反义寡核苷酸。A muscle targeting complex was generated comprising an antisense oligonucleotide covalently linked to an anti-transferrin receptor hIgG1-κ antibody (anti-TfR antibody) via a cathepsin cleavable linker.
在15L分批补料培养中，使抗TfR抗体在CHO-K1SP细胞(Genscript)中稳定表达。经由MabSelectSure蛋白A亲和树脂纯化上清液，用20mM磷酸盐洗涤，并用pH＝3.5的50mM柠檬酸钠洗脱。用1.0M NaOH中和洗脱物，并通过尺寸排阻色谱(SEC)进一步精制至DPBS pH＝7.4，并通过切向流过滤(TFF)浓缩至10.0mg/mL。Anti-TfR antibodies were stably expressed in CHO-K1SP cells (Genscript) in a 15L fed-batch culture. The supernatant was purified via MabSelectSure protein A affinity resin, washed with 20 mM phosphate, and eluted with 50 mM sodium citrate at pH = 3.5. The eluate was neutralized with 1.0 M NaOH and further refined to DPBS pH = 7.4 by size exclusion chromatography (SEC) and concentrated to 10.0 mg/mL by tangential flow filtration (TFF).
使用FragIt固相支持的IdeS酶(1.0gAb/10mL树脂，在室温下在摇瓶中以125rpm进行120分钟)，将经纯化的抗体位点特异性切割(在CPAPELLG-GPSVF(SEQ ID NO:45)的序列处)成F(ab’)2片段。使用CaptureSelect FcXL(ThermoScientific)以25mg/mL的结合容量在HiLoad柱(26mm×40cm，[装载流量：线性113cm/小时，26mm ID-10.0ml/分钟体积流量，停留时间-21.2分钟])上去除FC结构域和任何未切割IgG。收集包含经纯化的F(ab’)2的流通物，并通过SDS-PAGE(4％至12％NuPAGE，MES pH＝7.0，160v，40分钟)和分析型HPLC-SEC(Zorbax GF-250，9.4mm×250mm，PBS pH＝7.2，25℃)来验证是否有任何突破。将F(ab’)2片段用每铰链硫醇80摩尔过量的半胱胺-HCl(ChemImpex#02839)在37℃下还原成Fab’，持续90分钟。然后立即用蛋白L色谱(GE#17547855，10×系列)，使用标准pH梯度(50mMNa3C6H5O7，pH＝2.6，经过20个柱体积从60％到100％梯度)将Fab’纯化使其远离未还原的F(ab’)2和游离半胱胺。也可以重组产生抗TfR Fab。The purified antibody was site-specifically cleaved (at the sequence of CPAPELLG-GPSVF (SEQ ID NO: 45)) into F(ab')2 fragments using Fragit solid phase supported IdeS enzyme (1.0 gAb/10 mL resin, 120 min at room temperature in a shake flask at 125 rpm). The FC domain and any uncleaved IgG were removed on a HiLoad column (26 mm x 40 cm, [loading flow: linear 113 cm/hour, 26 mm ID-10.0 ml/min volume flow, residence time-21.2 min]) using CaptureSelect FcXL (ThermoScientific) with a binding capacity of 25 mg/mL. The flow-through containing the purified F(ab')2 was collected and verified for any breakthrough by SDS-PAGE (4% to 12% NuPAGE, MES pH = 7.0, 160v, 40 minutes) and analytical HPLC-SEC (Zorbax GF-250, 9.4mm x 250mm, PBS pH = 7.2, 25°C). The F(ab')2 fragment was reduced to Fab' with 80 molar excess of cysteamine-HCl (ChemImpex #02839) per hinge thiol at 37°C for 90 minutes. The Fab' was then immediately purified away from unreduced F(ab')2 and free cysteamine using protein L chromatography (GE #17547855, 10x series) using a standard pH gradient (50mM Na3C6H5O7, pH = 2.6, gradient from 60% to 100% over 20 column volumes). Anti-TfR Fabs can also be produced recombinantly.
用乙腈(HPLC级)以1:10v/v稀释抗TfR Fab，并使其与5摩尔过量的endo-BCN-PEG3-PFP((endo)双环壬炔-PEG3-五氟苯基)(式(C))；以20mg/mL溶解在DMSO中)在室温(约22.5℃)下反应2小时。在反应之后，将抗TfR Fab BCN溶液进行无菌过滤或深度过滤以去除沉淀的BCN。然后使用LCMS和叠氮化物-A488(20摩尔过量)对过滤的溶液的平均反应性BCN部分进行测定，持续90分钟。以5滤液体积用10kDa-TFF(1.2巴，Sartorius VivaFlow 200)来纯化并入BCN的Fab(>1.3摩尔的BCN/摩尔的Fab)以去除游离BCN。通过分析型HPLC-SEC(Tosoh，TSKgel SuperSW mAb HR，7.8mm×300mm)来验证游离BCN的完全去除。经BCN修饰的抗TfR Fab的回收>90％的起始物质。将并入BCN的经纯化Fab进行下一步骤。Anti-TfR Fab was diluted with acetonitrile (HPLC grade) at 1:10 v/v and reacted with 5 molar excess of endo-BCN-PEG3-PFP ((endo) bicyclononyne-PEG3-pentafluorophenyl) (Formula (C)); dissolved in DMSO at 20 mg/mL) at room temperature (about 22.5°C) for 2 hours. After the reaction, the anti-TfR Fab BCN solution was sterile filtered or deep filtered to remove precipitated BCN. The average reactive BCN portion of the filtered solution was then determined using LCMS and azide-A488 (20 molar excess) for 90 minutes. Fab incorporated with BCN (>1.3 moles of BCN/mole of Fab) was purified with 10kDa-TFF (1.2 bar, Sartorius VivaFlow 200) at 5 filtrate volumes to remove free BCN. Complete removal of free BCN was verified by analytical HPLC-SEC (Tosoh, TSKgel SuperSW mAb HR, 7.8 mm x 300 mm). Recovery of BCN-modified anti-TfR Fab was >90% of the starting material. The purified Fab incorporating BCN was carried forward to the next step.
产生了包含寡核苷酸(例如，带电荷寡核苷酸)和叠氮化物-缬氨酸-瓜氨酸接头的接头/载荷化合物。将寡核苷酸(Na+加合物)以200mg/mL溶解在不含RNAse的水中。用无水二甲基甲酰胺(DMF)将该溶液稀释至10mg/mL。然后向溶液添加25倍摩尔过量的三丁胺。将接头分子(叠氮化物-PEG3-Val-Cit-PAB-PNP(式(A))，以20mg/mL溶解在DMF中)在约25℃下以2倍摩尔过量添加至寡核苷酸溶液，持续120分钟。使用茚三酮(Kaiser测试)来测量反应完成，然后使用醇沉淀猝灭反应。通过添加0.1v/v 3M NaCl溶液，随后添加3体积的-80℃的异丙醇来完成醇沉淀。然后将溶液充分混合，并随后允许在-20℃下沉淀1小时。将沉淀的溶液离心(在4300×g；8℃)30分钟，并倾析溶剂。用不含RNase的水中的-80℃的80％乙醇(相当于开始反应的一体积)洗涤沉淀，并离心(4300×g；8℃)20分钟。然后倾析乙醇，并将沉淀(含有包含寡核苷酸和叠氮化物-缬氨酸-瓜氨酸接头的化合物)在37℃下干燥10分钟。将包含寡核苷酸和叠氮化物-缬氨酸-瓜氨酸接头的接头/载荷化合物重悬于不含核酸酶的水中的20％乙腈中，浓度为20mg/mL。A joint/load compound comprising an oligonucleotide (e.g., a charged oligonucleotide) and an azide-valine-citrulline joint is produced. The oligonucleotide (Na+ adduct) is dissolved in RNAse-free water at 200 mg/mL. The solution is diluted to 10 mg/mL with anhydrous dimethylformamide (DMF). Then 25 times of molar excess of tributylamine is added to the solution. The joint molecule (azide-PEG3-Val-Cit-PAB-PNP (formula (A)), dissolved in DMF at 20 mg/mL) is added to the oligonucleotide solution at about 25 ° C with 2 times of molar excess for 120 minutes. The reaction is measured using ninhydrin (Kaiser test) to complete, and then the reaction is quenched using alcohol precipitation. By adding 0.1v/v 3M NaCl solution, 3 volumes of -80 ° C of isopropanol are subsequently added to complete the alcohol precipitation. The solution is then fully mixed, and then allowed to precipitate at -20 ° C for 1 hour. The precipitated solution was centrifuged (at 4300×g; 8°C) for 30 minutes and the solvent was decanted. The precipitate was washed with 80% ethanol at -80°C in RNase-free water (equivalent to one volume of the starting reaction) and centrifuged (4300×g; 8°C) for 20 minutes. The ethanol was then decanted and the precipitate (containing the compound comprising the oligonucleotide and the azide-valine-citrulline linker) was dried at 37°C for 10 minutes. The linker/load compound comprising the oligonucleotide and the azide-valine-citrulline linker was resuspended in 20% acetonitrile in nuclease-free water at a concentration of 20 mg/mL.
使经BCN修饰的抗TfR Fab与2.5摩尔当量/BCN的接头/包含寡核苷酸与叠氮缬氨酸瓜氨酸接头的载荷化合物在室温(约25℃)下反应2小时。The BCN-modified anti-TfR Fab was reacted with 2.5 molar equivalents/BCN of linker/load compound comprising an oligonucleotide and an azidovaline-citrulline linker at room temperature (about 25° C.) for 2 hours.
通过SDS-PAGE和分析型SEC来评价偶联反应的完成，其通过密度测定法表明偶联效率为78％。The completion of the coupling reaction was assessed by SDS-PAGE and analytical SEC, which indicated a coupling efficiency of 78% by densitometry.
测试了通过实施例1中所述的缀合方法产生的缀合物和通过实施例6中所述的缀合方法产生的缀合物的生物活性。两种缀合物均包含DMPK靶向寡核苷酸(ASO1)，并有效地敲低了RD细胞中的DMPK mRNA水平(图7)。The biological activities of the conjugate produced by the conjugation method described in Example 1 and the conjugate produced by the conjugation method described in Example 6 were tested. Both conjugates contained a DMPK targeting oligonucleotide (ASO1) and effectively knocked down DMPK mRNA levels in RD cells ( FIG. 7 ).
实施例7：包含与电荷中性寡核苷酸连接的抗体的复合物的合成(缀合方法2-两步缀合)Example 7: Synthesis of a complex comprising an antibody linked to a charge-neutral oligonucleotide (Conjugation method 2 - two-step conjugation)
产生了肌肉靶向复合物，其包含通过组织蛋白酶可切割接头与抗转铁蛋白(抗TfR)受体Fab抗体共价连接的寡核苷酸(例如，电荷中性寡核苷酸)。可重组产生抗TfR Fab(例如，在CHO细胞中)并进行纯化。所使用的寡核苷酸是长度为30个核苷酸的磷酸二酰胺吗啉代寡聚物(PMO)。A muscle targeting complex was generated comprising an oligonucleotide (e.g., a charge neutral oligonucleotide) covalently linked to an anti-transferrin (anti-TfR) receptor Fab antibody via a cathepsin cleavable linker. Anti-TfR Fabs can be recombinantly produced (e.g., in CHO cells) and purified. The oligonucleotide used was a 30 nucleotide phosphorodiamidate morpholino oligomer (PMO).
用丙二醇将抗TfR Fab稀释至最终浓度为40％v/v丙二醇，并与5倍摩尔过量的endo-BCN-PEG3-PFP(endo-双环壬炔-PEG3-五氟苯基，以20mg/mL浓度溶解在DMSO中)在室温(约22.5℃)下孵育2小时。预期标记应产生2.0至2.5摩尔的BCN/摩尔的Fab。在标记后，将反应产物进行无菌过滤或深度过滤以去除沉淀的BCN。然后使用LCMS(ThermoFisherMAbPac RP 4um 2.1×100mm，#088647；流动相A：100％UPLC级水中的0.1％甲酸；流动相B：100％UPLC级乙腈中的0.1％甲酸；流量0.3mL/分钟；柱温70℃；源内CID 20eV；正极性；喷雾电压3.5千伏；扫描范围1000至3000m/z)来测定过滤的溶液的平均反应性BCN部分。Anti-TfR Fab was diluted with propylene glycol to a final concentration of 40% v/v propylene glycol and incubated with a 5-fold molar excess of endo-BCN-PEG3-PFP (endo-bicyclononyne-PEG3-pentafluorophenyl, dissolved in DMSO at a concentration of 20 mg/mL) at room temperature (about 22.5°C) for 2 hours. It is expected that labeling should produce 2.0 to 2.5 moles of BCN/mole of Fab. After labeling, the reaction product was sterile filtered or deep filtered to remove precipitated BCN. The average reactive BCN fraction of the filtered solution was then determined using LCMS (ThermoFisher MAbPac RP 4um 2.1×100mm, #088647; mobile phase A: 0.1% formic acid in 100% UPLC grade water; mobile phase B: 0.1% formic acid in 100% UPLC grade acetonitrile; flow rate 0.3 mL/min; column temperature 70°C; in-source CID 20 eV; positive polarity; spray voltage 3.5 kV; scan range 1000 to 3000 m/z).
将标记程度(degree of labeling，DOL)>2.3的抗TfR用于缀合的下一步骤，并将其使用10kDa分子量截止值(1.2巴)，5滤液体积，通过切向流过滤纯化到pH 7.2的PBS中的10％异丙醇中以去除游离BCN和丙二醇。通过分析型HPLC-SEC(Waters Xbridge蛋白BEHSEC 3.5um，7.8×300mm，0.3mL/分钟，100mM PO4，100mM NaCl，15％v/v乙腈pH 7.0)来验证BCN和丙二醇的完全去除。粗制产物和经纯化产物的SEC迹线在图8中示出。经BCN标记的抗TfR的回收>90％的起始物质。将经纯化的溶液浓缩至3.5mg/mL，用于进一步的缀合步骤。Anti-TfR with a degree of labeling (DOL)>2.3 was used for the next step of conjugation and purified by tangential flow filtration into 10% isopropanol in PBS at pH 7.2 using a 10 kDa molecular weight cutoff (1.2 bar), 5 filtrate volumes to remove free BCN and propylene glycol. Complete removal of BCN and propylene glycol was verified by analytical HPLC-SEC (Waters Xbridge protein BEHSEC 3.5um, 7.8×300mm, 0.3mL/min, 100mM PO4 , 100mM NaCl, 15% v/v acetonitrile pH 7.0). The SEC traces of the crude product and the purified product are shown in Figure 8. Recovery of anti-TfR labeled with BCN>90% of the starting material. The purified solution was concentrated to 3.5mg/mL for further conjugation steps.
在单独的反应中，寡核苷酸(例如，电荷中性寡核苷酸)与接头分子缀合。将寡核苷酸以35mg/mL在37℃下溶解在无水DMSO中。将接头分子(叠氮化物-PEG3-Val-Cit-PAB-PNP)以40mg/mL溶解在无水DMF中，并将其以2.7倍摩尔过量用3倍摩尔过量的N,N-二异丙基乙胺(DIPEA)添加至寡核苷酸。使该接头缀合反应在室温(约22.5℃)下进行2小时。使用茚三酮测定(Kaiser测试)来测量反应的进程和完成，然后通过丙酮沉淀猝灭反应。In a separate reaction, an oligonucleotide (e.g., a charge neutral oligonucleotide) is conjugated to a linker molecule. The oligonucleotide is dissolved in anhydrous DMSO at 37°C at 35mg/mL. The linker molecule (azide-PEG3-Val-Cit-PAB-PNP) is dissolved in anhydrous DMF at 40mg/mL, and is added to the oligonucleotide with 2.7 times molar excess of 3 times molar excess of N,N-diisopropylethylamine (DIPEA). The linker conjugation reaction is carried out at room temperature (about 22.5°C) for 2 hours. The progress and completion of the reaction are measured using ninhydrin determination (Kaiser test), and the reaction is then quenched by acetone precipitation.
通过将8体积的冷丙酮添加至产物溶液来进行沉淀，并通过在8℃下以3500×g离心20分钟使沉淀物沉淀。然后用3体积的丙酮来洗涤沉淀以去除残留的游离接头，并再次在8℃下以3500×g离心20分钟。然后将经纯化寡核苷酸-接头以30mg/mL的浓度溶解在不含核酸酶的水中的20％v/v乙腈中。通过在0.1N HCl中的光密度(optical density，OD)来测量浓度和产率，表明产率大于90％。对粗制接头/寡核苷酸缀合反应产物(图9)和经纯化寡核苷酸-接头(图10)进行分析型RP-HPLC(Waters BEH-C18，4.6mm×150mm，0.5mL/分钟，在水中的5％至90％v/v乙腈，30分钟运行时间)以确定通过沉淀和洗涤步骤去除了游离接头。还进行了对经纯化寡核苷酸-接头的验证性LCMS(图11)。Precipitation is carried out by adding 8 volumes of cold acetone to the product solution, and the precipitate is precipitated by centrifugation at 3500 × g for 20 minutes at 8 ° C. The precipitate is then washed with 3 volumes of acetone to remove the remaining free joints, and again at 8 ° C with 3500 × g for 20 minutes. The purified oligonucleotide-joint is then dissolved in 20% v/v acetonitrile in water without nuclease at a concentration of 30 mg/mL. Concentration and yield are measured by optical density (optical density, OD) in 0.1N HCl, indicating that the yield is greater than 90%. Analytical RP-HPLC (Waters BEH-C18, 4.6 mm × 150 mm, 0.5 mL/ minute, 5% to 90% v/v acetonitrile in water, 30 minutes running time) is performed to determine that the free joint is removed by precipitation and washing steps. A confirmatory LCMS of the purified oligonucleotide-linker was also performed (Figure 11).
为了缀合抗TfR和寡核苷酸，将经BCN标记的抗体与5倍摩尔过量的寡核苷酸-PAB-VC-PEG3-叠氮化物(图1A)在玻璃瓶中在室温(约22.5℃)下混合过夜。To conjugate anti-TfR and oligonucleotide, BCN-labeled antibody was mixed with 5-fold molar excess of oligonucleotide-PAB-VC-PEG3-azide ( FIG. 1A ) in a glass vial at room temperature (about 22.5° C.) overnight.
通过SDS-PAGE(图12)和分析型SEC分析(图13)来评价反应的完成，其通过密度测定法表明少于10％的未连接抗TfR抗体(DAR0)和90％的缀合效率。The completion of the reaction was assessed by SDS-PAGE ( FIG. 12 ) and analytical SEC analysis ( FIG. 13 ), which showed less than 10% unattached anti-TfR antibody (DAR0) and 90% conjugation efficiency by densitometry.
实施例8.用于制备Fab-寡核苷酸(电荷中性寡核苷酸)缀合物的缀合方法Example 8. Conjugation method for preparing Fab-oligonucleotide (charge-neutral oligonucleotide) conjugates
(缀合方法1–预反应缀合)(Conjugation Method 1 – Pre-reaction conjugation)
该实施例描述了由通过val-cit组织蛋白酶可切割肽接头与抗转铁蛋白受体(抗TfR)Fab抗体共价连接的寡核苷酸构成的缀合物的制备。抗可重组产生TfR Fab(例如，在CHO细胞中)并进行纯化。所使用的寡核苷酸是长度为30个核苷酸的磷酸二酰胺吗啉代寡聚物(PMO)。在与Fab缀合之前，通过寡核苷酸-PAB-VC-PEG3-叠氮化物分子(图1A)的叠氮化物基团与endo-BCN-PEG4-PFP酯(图1B)异双官能交联剂上的应变双环壬炔部分之间的无铜3+2点击反应产生包含寡核苷酸和接头的中间体(“预反应”)。This embodiment describes the preparation of a conjugate consisting of an oligonucleotide covalently linked to an anti-transferrin receptor (anti-TfR) Fab antibody by a cleavable peptide linker of a val-cit cathepsin. Anti-TfR Fab can be recombinantly produced (e.g., in CHO cells) and purified. The oligonucleotide used is a phosphorodiamidate morpholino oligomer (PMO) of 30 nucleotides in length. Prior to conjugation with Fab, an intermediate ("pre-reaction") comprising an oligonucleotide and a linker is produced by a copper-free 3+2 click reaction between the azide group of an oligonucleotide-PAB-VC-PEG3-azide molecule (FIG. 1A) and the strained bicyclic nonyne moiety on an endo-BCN-PEG4-PFP ester (FIG. 1B) heterobifunctional crosslinker.
将冻干的寡核苷酸-PAB-VC-PEG3-叠氮化物(98.1mg)在4mL玻璃Wheaton小瓶中溶解于0.32mL MilliQ水中。在溶解之后，添加0.32mL的N,N-二甲基乙酰胺(DMA)并将混合物轻轻搅拌5至10分钟。在继续之前，仔细检查小瓶以确保寡核苷酸-PAB-VC-PEG3-叠氮化物完全溶解并且没有残留物保留在玻璃小瓶的壁上。用Nanodrop UV/vis仪器，通过使用包含最终浓度为0.1M HCl的1:1DMA:水中稀释25倍、50倍和100倍的等分试样，在265nm下使用318,050M-1cm-1的消光系数来确定在1:1DMA:水中的寡核苷酸-PAB-VC-PEG3-叠氮化物储备溶液的浓度。添加HCl以确保浓度测量的准确性。对每种稀释度的计算浓度求平均以确定10.1mM的溶液浓度。The lyophilized oligonucleotide-PAB-VC-PEG3-azide (98.1mg) is dissolved in 0.32mL MilliQ water in 4mL glass Wheaton vials. After dissolving, 0.32mL of N, N-dimethylacetamide (DMA) is added and the mixture is gently stirred for 5 to 10 minutes. Before continuing, the vial is carefully checked to ensure that the oligonucleotide-PAB-VC-PEG3-azide is completely dissolved and no residue is retained on the wall of the glass vial. With Nanodrop UV/vis instrument, by using a 1:1DMA: aliquots diluted 25 times, 50 times and 100 times in water containing a final concentration of 0.1M HCl, the extinction coefficient of 318,050M-1 cm-1 is used at 265nm to determine the concentration of the oligonucleotide-PAB-VC-PEG3-azide stock solution in 1:1DMA: water. HCl is added to ensure the accuracy of concentration measurement. The calculated concentrations for each dilution were averaged to determine a solution concentration of 10.1 mM.
通过将约25mg的endo-BCN-PEG4-PFP酯油称重到4mL玻璃Wheaton小瓶中来制备endo-BCN-PEG4-PFP酯的32.5mg/mL(53.5mM)储备溶液。然后添加适当体积的DMA以得到32.5mg/mL的储备溶液。A 32.5 mg/mL (53.5 mM) stock solution of endo-BCN-PEG4-PFP ester was prepared by weighing approximately 25 mg of endo-BCN-PEG4-PFP ester oil into a 4 mL glass Wheaton vial. An appropriate volume of DMA was then added to yield a 32.5 mg/mL stock solution.
在以下最终溶液反应条件下进行预反应：在室温下，5.87μM(6.5μmol)寡核苷酸-PAB-VC-PEG3-叠氮化物、5.34mM endo-BCN-PEG4-PFP酯(1.1:1.0mol:mol当量)在60:40v/v％DMA与25mM 2-(N-吗啉代)乙磺酸(MES)pH 5.5缓冲液中。如在表6中所示，通过添加适量的反应物和储备溶液，在4mL玻璃Wheaton小瓶中进行反应。预反应的最终总体积为1.11mL。Pre-reaction was performed under the following final solution reaction conditions: at room temperature, 5.87 μM (6.5 μmol) oligonucleotide-PAB-VC-PEG3-azide, 5.34 mM endo-BCN-PEG4-PFP ester (1.1: 1.0 mol: mol equivalent) in 60: 40 v/v% DMA and 25 mM 2-(N-morpholino) ethanesulfonic acid (MES) pH 5.5 buffer. As shown in Table 6, by adding the appropriate amount of reactants and stock solutions, the reaction was performed in a 4 mL glass Wheaton vial. The final total volume of the pre-reaction was 1.11 mL.
表6.Table 6.
通过RP-C18UPLC每30分钟(在5分钟、35分钟和65分钟时间点)重复注入，通过在220nm处观察endo-BCN-PEG4-PFP酯起始物质的消失来监测产生寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯(图1C)的预反应的完成。该IPC表明反应在65分钟时完成(图14)。当相对于5分钟时间点剩余少于5％的endo-BCN-PEG4-PFP酯时，则确定反应完成(表7)。将粗制预反应混合物在没有任何纯化的情况下立即进行缀合反应。总的预反应时间为90分钟，定义为将endo-BCN-PEG4-PFP酯添加至预反应与将该反应混合物添加至Fab缀合反应之间的时间。Injection was repeated every 30 minutes (at 5 minutes, 35 minutes and 65 minutes time points) by RP-C18UPLC, and the completion of the pre-reaction producing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester (Fig. 1C) was monitored by observing the disappearance of the endo-BCN-PEG4-PFP ester starting material at 220nm. The IPC showed that the reaction was completed (Fig. 14) at 65 minutes. When the endo-BCN-PEG4-PFP ester was less than 5% relative to the 5 minutes time point, the reaction was determined to be complete (Table 7). The crude pre-reaction mixture was immediately subjected to conjugation reaction without any purification. The total pre-reaction time was 90 minutes, which was defined as the time between adding endo-BCN-PEG4-PFP ester to the pre-reaction and adding the reaction mixture to the Fab conjugation reaction.
表7.基于RP-C18 UPLC测量对endo-BCN-PEG4-PFP酯起始物质进行的定量。Table 7. Quantification of endo-BCN-PEG4-PFP ester starting material based on RP-C18 UPLC measurement.
寡核苷酸与抗TfR Fab的缀合涉及在Fab的溶剂可及赖氨酸残基与预反应步骤中产生的寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的活化PFP酯(图1C)之间的酰胺键的形成。Conjugation of the oligonucleotide to the anti-TfR Fab involves the formation of an amide bond between a solvent accessible lysine residue of the Fab and the activated PFP ester of the oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester generated in the pre-reaction step ( FIG. 1C ).
在设置缀合反应之前，将在20mM柠檬酸钠、100mM氯化钠中配制的抗TfR Fab经缓冲液交换到50mM HEPES pH 7.5中。将抗TfR Fab(10mL，10.15mg/mL)装载到50mM HEPES pH7.5平衡的NAP-25脱盐柱(4×2.5mL)上，并用50mM HEPES pH 7.5(4×3.5mL)洗脱。将洗脱物合并并用Amicon Ultra-15 10kDa离心过滤单元以4000rcf旋转进行浓缩，以使体积减少至2.86mL。通过Nanodrop UV/vis测量缓冲液所得抗TfR Fab的浓度为31.75mg/mL。Before setting up the conjugation reaction, the anti-TfR Fab prepared in 20mM sodium citrate, 100mM sodium chloride was buffer exchanged into 50mM HEPES pH 7.5. Anti-TfR Fab (10mL, 10.15mg/mL) was loaded onto a NAP-25 desalting column (4×2.5mL) balanced with 50mM HEPES pH7.5 and eluted with 50mM HEPES pH 7.5 (4×3.5mL). The eluates were combined and concentrated with an Amicon Ultra-15 10kDa centrifugal filter unit at 4000rcf to reduce the volume to 2.86mL. The concentration of the anti-TfR Fab obtained by the Nanodrop UV/vis measurement buffer was 31.75mg/mL.
用以下最终溶液反应物量进行缀合反应：抗TfR Fab(45mg，6mg/mL，125μM)和最终理论浓度为750μM的寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯(6.0:1.0mol:mol当量的寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯vs抗TfR Fab)。寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯浓度假设为预反应中BCN 100％转化成寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯。最终的反应混合物由15:85v/v％DMA与25mM HEPES pH 7.5缓冲液组成。如在表8中所示，通过添加适量的反应物和储备溶液，在20mL玻璃闪烁小瓶中建立反应。将反应在室温(约25℃)下进行20小时。反应的开始定义为将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的预反应混合物添加至抗TfR Fab溶液的时间。缀合反应的总持续时间为20小时。The conjugation reaction was carried out with the following final solution reactant amount: anti-TfR Fab (45 mg, 6 mg/mL, 125 μM) and oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester (6.0:1.0 mol:mol equivalents of oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester vs anti-TfR Fab) with a final theoretical concentration of 750 μM. The concentration of oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester was assumed to be 100% conversion of BCN into oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester in the pre-reaction. The final reaction mixture consisted of 15:85 v/v% DMA and 25 mM HEPES pH 7.5 buffer. As shown in Table 8, the reaction was set up in a 20 mL glass scintillation vial by adding an appropriate amount of reactant and stock solution. The reaction was carried out for 20 hours at room temperature (about 25 ° C). The start of the reaction was defined as the time when the pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester was added to the anti-TfR Fab solution. The total duration of the conjugation reaction was 20 hours.
表8.Table 8.
在反应20小时之后，通过SDS-PAGE测试粗制缀合混合物并通过密度测定法进行分析以确定药物抗体比(DAR)和％未缀合Fab。结果见于图15和表9。After 20 hours of reaction, the crude conjugation mixture was tested by SDS-PAGE and analyzed by densitometry to determine the drug antibody ratio (DAR) and % unconjugated Fab. The results are shown in Figure 15 and Table 9.
表9.Table 9.
实施例9.抗TfR Fab-寡核苷酸缀合物的纯化Example 9. Purification of anti-TfR Fab-oligonucleotide conjugates
在实施例8中描述的抗TfR Fab-寡核苷酸缀合物的合成之后，进行了两部分纯化过程。首先，通过羟基磷灰石(HA)色谱去除游离载荷。然后将HA洗脱物经缓冲液交换到最终制剂中。在45mg规模的Fab下，用30kDa离心过滤装置进行最终的缓冲液交换。在装载到HA柱上之前，将来自实施例8的粗制反应产物(抗TfR Fab-寡核苷酸缀合物)稀释，并将pH从7.5调节至5.7。首先，通过添加16.5mL的在水中的15v/v％DMA来稀释7.5mL粗制缀合物，并彻底混合溶液。向该混合物添加0.75mL的500mM MES(pH 3.3)以将pH下调至5.7。After the synthesis of the anti-TfR Fab-oligonucleotide conjugate described in Example 8, a two-part purification process was carried out. First, free load was removed by hydroxyapatite (HA) chromatography. The HA eluate was then buffer exchanged into the final formulation. Under 45mg of Fab, final buffer exchange was performed with a 30kDa centrifugal filter device. Before being loaded onto the HA column, the crude reaction product (anti-TfR Fab-oligonucleotide conjugate) from Example 8 was diluted, and the pH was adjusted to 5.7 from 7.5. First, 7.5mL of crude conjugate was diluted by adding 16.5mL of 15v/v%DMA in water, and the solution was thoroughly mixed. 0.75mL of 500mM MES (pH 3.3) was added to the mixture to adjust the pH down to 5.7.
使用5mL Bio Rad CHT I型(陶瓷羟基磷灰石)盒在AKTA纯色谱系统上进行色谱纯化以去除未反应的寡核苷酸物质。在装载来自反应混合物制备步骤的稀释的缀合物合并物之前，使用15：85v/v％的DMA与10mM磷酸钠pH 5.8缓冲液，根据制造商说明来制备CHT盒并使其平衡。在平衡之后，以5mL/分钟的流量装载缀合物合并物。在装载缀合物之后，用10mM磷酸钠缓冲液(pH 5.8)中的15：85v/v％的DMA将柱洗涤最少7CV。在洗涤完成之后，用包含15：85v/v％的DMA的100mM磷酸钠pH 7.6缓冲液以5mL/分钟的流量，通过阶梯梯度开始洗脱。收集通过在260nm和280nm处监测而鉴定的整个洗脱峰并将其合并。Use 5mL Bio Rad CHT I type (ceramic hydroxyapatite) box to carry out chromatographic purification on AKTA pure chromatography system to remove unreacted oligonucleotide material.Before loading the conjugate merger of dilution from reaction mixture preparation step, use 15: 85v/v% DMA and 10mM sodium phosphate pH 5.8 buffer, prepare CHT box according to manufacturer's instructions and make it balance.After balance, load conjugate merger with the flow rate of 5mL/ minute.After loading conjugate, use 15: 85v/v% DMA in 10mM sodium phosphate buffer (pH 5.8) by post washing minimum 7CV.After washing is complete, use 100mM sodium phosphate pH 7.6 buffer containing 15: 85v/v% DMA with the flow rate of 5mL/ minute, start elution by step gradient.Collect the whole elution peak identified by monitoring at 260nm and 280nm and merge it.
通过SEC进行的HA柱装载步骤期间对流通物的分析(图16)表明在流通物中存在很少或不存在Fab-寡核苷酸缀合物。仅在约10.5分钟和约11.3分钟时观察到由于寡核苷酸载荷物质引起的峰。相反地，对合并的洗脱峰的SEC分析仅显示出缀合物物质(图17)，其中由于不同寡核苷酸(例如，PMO)载荷装载下缀合物的尺寸差异，而具有多个峰和肩峰(shoulder)。在10.5或11.3分钟时没有观察到载荷物质的峰。通过SEC色谱来估计HA纯化的相对于Fab的缀合物质量平衡。这是通过注入24μg的来自粗制缀合反应产物的Fab(4μL注入，浓度为6μg/mL)，以及注入理论上24μg的假设100％回收的来自HA洗脱物合并物的Fab来实现的。HA洗脱物合并物为13.9mL，理论上包含45mg的Fab，理论浓度为3.24μg/μL。为了实现注入24μg的Fab，使用了7.4μL注入。这两个SEC色谱图的重叠(图18)表明Fab作为Fab-寡核苷酸缀合物几乎完全回收。9.1分钟时的峰高用于估计在HA色谱纯化之后反应产物的97％回收。The analysis of the flowable material during the HA column loading step by SEC (Figure 16) shows that there is little or no Fab-oligonucleotide conjugate in the flowable material. Only at about 10.5 minutes and about 11.3 minutes, the peak caused by the oligonucleotide load material is observed. On the contrary, the SEC analysis of the merged elution peak only shows the conjugate material (Figure 17), wherein due to the size difference of the conjugate under different oligonucleotide (e.g., PMO) load loading, there are multiple peaks and shoulders. At 10.5 or 11.3 minutes, no peak of load material is observed. The conjugate mass balance relative to Fab of HA purification is estimated by SEC chromatography. This is achieved by injecting 24 μg of Fab from the crude conjugation reaction product (4 μL injection, concentration of 6 μg/mL), and injecting 24 μg of theoretically assumed 100% recovery of the Fab from the HA eluate merger. The HA eluate merger is 13.9mL, theoretically containing 45mg of Fab, and the theoretical concentration is 3.24 μg/μL. To achieve an injection of 24 μg of Fab, a 7.4 μL injection was used. Overlay of these two SEC chromatograms (Figure 18) shows almost complete recovery of the Fab as Fab-oligonucleotide conjugate. The peak height at 9.1 minutes was used to estimate 97% recovery of the reaction product after HA chromatography purification.
在45mg反应规模下，使用Amicon Ultra-15 30kDa离心过滤装置进行将HA洗脱物经缓冲液交换到50mM His(pH 6.0)中。首先，通过以4000rcf旋转该柱，将HA洗脱物合并物浓缩至约1.5mL。随后通过添加3mL的50mM His(pH 6.0)进行缓冲液交换，并通过以4000rcf离心进行浓缩，直至体积达到约1.5mL。使用相当于15体积的缓冲液重复该步骤总共5轮，以产生最终纯化的抗TfR Fab-寡核苷酸缀合物。然后用另外的50mM His(pH 6.0)将所得约1.5mL经纯化缀合物(在50mM His中，pH 6.0)稀释至最终体积为3.0mL。Under 45mg reaction scale, Amicon Ultra-15 30kDa centrifugal filter device is used to carry out HA eluate through buffer exchange into 50mM His (pH 6.0).First, by rotating the column at 4000rcf, the HA eluate merger is concentrated to about 1.5mL.Subsequently, buffer exchange is carried out by adding 3mL of 50mM His (pH 6.0), and by centrifugation at 4000rcf, it is concentrated until the volume reaches about 1.5mL. This step is repeated for a total of 5 rounds using a buffer equivalent to 15 volumes, to produce the final purified anti-TfR Fab-oligonucleotide conjugate.Then, with another 50mM His (pH 6.0), the obtained approximately 1.5mL purified conjugate (in 50mM His, pH 6.0) is diluted to a final volume of 3.0mL.
通过SEC、SDS-PAGE密度测定法和BCA来分析最终经纯化的抗TfR Fab-寡核苷酸缀合物。最终缀合物的SEC(在图19A和19B中示出)与HA纯化之后缀合物合并物的相应SEC数据几乎相同，表明纯化过程没有诱导高分子量物质的形成。通过SDS-PAGE密度测定法(在图20中示出的SDS-PAGE凝胶)来计算平均DAR、DAR物质分布和未缀合Fab的百分比，并且用来自Li-Cor Biosciences的Image Studio Lite软件包进行数据分析(计算结果在表10中示出)。经纯化的抗TfR Fab-寡核苷酸缀合物的最终平均DAR为1.96，包括8.1％未缀合抗TfRFab。通过BCA测定测量的蛋白质浓度为10.5mg/mL，表明终产物中总共有31.4mg的缀合物，总工艺产率为70％。The final purified anti-TfR Fab-oligonucleotide conjugate is analyzed by SEC, SDS-PAGE densitometry and BCA.The SEC of the final conjugate (shown in Figures 19 A and 19B) is almost the same as the corresponding SEC data of the conjugate merger after HA purification, indicating that the purification process does not induce the formation of high molecular weight substances. The average DAR, DAR material distribution and the percentage of unconjugated Fab are calculated by SDS-PAGE densitometry (SDS-PAGE gel shown in Figure 20), and data analysis (calculation result is shown in Table 10) is performed with the Image Studio Lite software package from Li-Cor Biosciences. The final average DAR of the purified anti-TfR Fab-oligonucleotide conjugate is 1.96, including 8.1% unconjugated anti-TfRFab. The protein concentration measured by BCA determination is 10.5mg/mL, indicating that there are a total of 31.4mg of conjugates in the final product, and the total process yield is 70%.
表10.经纯化的Fab-寡核苷酸缀合物产物的平均DAR和DAR分布。Table 10. Average DAR and DAR distribution of purified Fab-oligonucleotide conjugate products.
实施例10.BCN与Fab的比率和反应条件对Fab-寡核苷酸缀合反应的作用Example 10. Effect of BCN to Fab ratio and reaction conditions on Fab-oligonucleotide conjugation reaction
为了研究来自预反应混合物的BCN与抗TfR的比率的作用，以及寡核苷酸-Fab缀合反应中寡核苷酸和Fab浓度的作用，根据实施例8中描述的通用方案进行了一组反应。所使用的寡核苷酸是长度为30个核苷酸的PMO。To investigate the effect of the ratio of BCN to anti-TfR from the pre-reaction mixture, and the effect of oligonucleotide and Fab concentrations in the oligonucleotide-Fab conjugation reaction, a set of reactions was performed according to the general protocol described in Example 8. The oligonucleotide used was a PMO of 30 nucleotides in length.
在第一组反应中，使用以下条件进行寡核苷酸PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使2.44mM寡核苷酸PAB-VC-PEG3-叠氮化物与1.63mM endo-BCN-PEG4-PFP酯(1.5∶1mol∶mol比)在DMA和25mM MES pH 5.5缓冲液的1∶1混合物中反应。总的预反应体积为0.37mL，并且使预反应步骤在室温(约25℃)下进行18小时。In the first set of reactions, the pre-reaction between oligonucleotide PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 2.44 mM oligonucleotide PAB-VC-PEG3-azide was reacted with 1.63 mM endo-BCN-PEG4-PFP ester (1.5:1 mol:mol ratio) in a 1:1 mixture of DMA and 25 mM MES pH 5.5 buffer. The total pre-reaction volume was 0.37 mL, and the pre-reaction step was performed at room temperature (about 25° C.) for 18 hours.
在预反应完成后，将包含寡核苷酸PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化)的预反应混合物用于建立一系列抗TfR Fab缀合反应。使用相对于Fab的2、4、6和10摩尔当量的来自预反应混合物的BCN进行抗TfR Fab缀合反应。所有其他反应条件在不同的缀合中保持不变。在缀合反应混合物中，Fab浓度为3mg/mL，其中在50mM HEPES pH 7.5缓冲液中的15v/v％DMA中每次反应总Fab为1mg。将缀合反应在23至25℃下进行18小时。通过SDS-PAGE密度测定法来确定每种缀合物的最终DAR和DAR物质分布，并且结果在表11中示出。SDS-PAGE凝胶的图像在图21中示出。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide PAB-VC-PEG3-triazole-PEG4-PFP ester is used to establish a series of anti-TfR Fab conjugation reactions. Anti-TfR Fab conjugation reactions are carried out using BCN from the pre-reaction mixture relative to 2, 4, 6 and 10 molar equivalents of Fab. All other reaction conditions remain unchanged in different conjugations. In the conjugation reaction mixture, the Fab concentration is 3 mg/mL, wherein the total Fab per reaction is 1 mg in 15v/v% DMA in 50mM HEPES pH 7.5 buffer. The conjugation reaction is carried out at 23 to 25°C for 18 hours. The final DAR and DAR material distribution of each conjugate are determined by SDS-PAGE density determination, and the results are shown in Table 11. The image of SDS-PAGE gel is shown in Figure 21.
表11.Table 11.
在第二组反应中，使用以下条件进行寡核苷酸-PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使6.77mM寡核苷酸-PAB-VC-PEG3-叠氮化物与4.84mMendo-BCN-PEG4-PFP酯(1.4:1mol:mol比)在DMA和25mM MES pH 5.5缓冲液的1:1混合物中反应。总的预反应体积为0.160mL，并且使预反应步骤在室温(约25℃)下进行90分钟。In the second set of reactions, the pre-reaction between oligonucleotide-PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 6.77 mM oligonucleotide-PAB-VC-PEG3-azide was reacted with 4.84 mM endo-BCN-PEG4-PFP ester (1.4:1 mol:mol ratio) in a 1:1 mixture of DMA and 25 mM MES pH 5.5 buffer. The total pre-reaction volume was 0.160 mL, and the pre-reaction step was performed at room temperature (about 25° C.) for 90 minutes.
在预反应完成后，将包含寡核苷酸PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化的)预反应混合物用于建立一系列抗TfR Fab缀合反应。使用相对于Fab的2、4、5、6和8摩尔当量的来自预反应混合物的BCN进行抗TfR Fab缀合反应。所有其他反应条件在不同的缀合中保持不变。在缀合反应混合物中，Fab浓度为6mg/mL，其中在50mM HEPES pH 7.5缓冲液中的15v/v％DMA中每次反应的总Fab为1mg。将缀合反应在23至25℃下进行19小时。通过SDS-PAGE密度测定法来确定每种缀合物的最终平均DAR，并且结果在表12中示出。SDS-PAGE凝胶的图像在图22中示出。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide PAB-VC-PEG3-triazole-PEG4-PFP ester is used to set up a series of anti-TfR Fab conjugation reactions. Anti-TfR Fab conjugation reactions are carried out using BCN from the pre-reaction mixture relative to 2, 4, 5, 6 and 8 molar equivalents of Fab. All other reaction conditions remain unchanged in different conjugations. In the conjugation reaction mixture, the Fab concentration is 6 mg/mL, and the total Fab of each reaction in 15v/v%DMA in 50mM HEPES pH 7.5 buffer is 1 mg. The conjugation reaction is carried out at 23 to 25°C for 19 hours. The final average DAR of each conjugate is determined by SDS-PAGE density determination, and the results are shown in Table 12. The image of SDS-PAGE gel is shown in Figure 22.
表12.Table 12.
在第三组反应中，使用以下条件进行寡核苷酸-PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使6.77mM寡核苷酸-PAB-VC-PEG3-叠氮化物与4.84mMendo-BCN-PEG4-PFP酯(1.4:1mol:mol比)在DMA和25mM MES pH 5.5缓冲液的60:40混合物中反应。总的预反应体积为0.160mL，并且使预反应步骤在室温(约25℃)下进行90分钟。In the third set of reactions, the following conditions were used for the pre-reaction between oligonucleotide-PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester: 6.77 mM oligonucleotide-PAB-VC-PEG3-azide was reacted with 4.84 mM endo-BCN-PEG4-PFP ester (1.4:1 mol:mol ratio) in a 60:40 mixture of DMA and 25 mM MES pH 5.5 buffer. The total pre-reaction volume was 0.160 mL, and the pre-reaction step was carried out at room temperature (about 25° C.) for 90 minutes.
在预反应完成后，将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化的)预反应混合物用于建立一系列抗TfR Fab缀合反应。使用相对于Fab的2、4、5、6、8和10摩尔当量的来自预反应混合物的BCN进行抗TfR Fab缀合反应。所有其他反应条件在不同的缀合中保持不变。在缀合反应混合物中，Fab浓度为6mg/mL，其中在50mM HEPES pH 7.5缓冲液中的15v/v％DMA中每次反应总Fab为1mg。将缀合反应在室温(约25℃)下进行19小时。通过SDS-PAGE密度测定法来确定每种缀合物的最终平均DAR和DAR0百分比，并且结果在表13中示出(反应J、K、L、M、N、O)。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester is used to set up a series of anti-TfR Fab conjugation reactions. Anti-TfR Fab conjugation reactions are carried out using BCN from the pre-reaction mixture relative to 2, 4, 5, 6, 8 and 10 molar equivalents of Fab. All other reaction conditions remain unchanged in different conjugations. In the conjugation reaction mixture, the Fab concentration is 6 mg/mL, wherein the total Fab of each reaction is 1 mg in 15v/v%DMA in 50mM HEPES pH 7.5 buffer. The conjugation reaction is carried out at room temperature (about 25°C) for 19 hours. The final average DAR and DAR0 percentages of each conjugate are determined by SDS-PAGE density determination, and the results are shown in Table 13 (reactions J, K, L, M, N, O).
在第四组反应中，使用以下条件进行寡核苷酸-PAB-VC-PEG3-叠氮化物与endo-BCN-PEG4-PFP酯之间的预反应：使6.77mM寡核苷酸-PAB-VC-PEG3-叠氮化物与6.45mMendo-BCN-PEG4-PFP酯(1.05:1mol:mol比)在DMA和25mM MES pH 5.5缓冲液的60:40混合物中反应。总的预反应体积为0.080mL，并且使预反应步骤在室温(约25℃)下进行160分钟。In the fourth set of reactions, the pre-reaction between oligonucleotide-PAB-VC-PEG3-azide and endo-BCN-PEG4-PFP ester was performed using the following conditions: 6.77 mM oligonucleotide-PAB-VC-PEG3-azide was reacted with 6.45 mM endo-BCN-PEG4-PFP ester (1.05:1 mol:mol ratio) in a 60:40 mixture of DMA and 25 mM MES pH 5.5 buffer. The total pre-reaction volume was 0.080 mL, and the pre-reaction step was performed at room temperature (about 25° C.) for 160 minutes.
在预反应完成后，将包含寡核苷酸-PAB-VC-PEG3-三唑-PEG4-PFP酯的粗制(即，未经纯化的)预反应混合物用于建立一系列抗TfR Fab缀合反应。基于预反应中BCN的浓度，使用相对于Fab的5、6和7摩尔当量的来自预反应混合物的BCN进行抗TfR Fab缀合反应。所有其他反应条件在不同的缀合中保持不变。在缀合反应混合物中，Fab浓度为6mg/mL或12mg/mL，其中每次反应中总Fab分别为0.6mg和1.2mg。所有反应均在25mM HEPES pH 7.5缓冲液中的15v/v％DMA中进行。将缀合反应在室温(约25℃)下进行约18小时。通过SDS-PAGE密度测定法来确定每种缀合物的最终平均DAR和DAR0百分比，并且结果在表13中示出(反应P、Q、R、S)。After the pre-reaction is completed, the crude (i.e., unpurified) pre-reaction mixture containing oligonucleotide-PAB-VC-PEG3-triazole-PEG4-PFP ester is used to set up a series of anti-TfR Fab conjugation reactions. Based on the concentration of BCN in the pre-reaction, the anti-TfR Fab conjugation reaction is carried out using BCN from the pre-reaction mixture relative to 5, 6 and 7 molar equivalents of Fab. All other reaction conditions remain unchanged in different conjugations. In the conjugation reaction mixture, the Fab concentration is 6mg/mL or 12mg/mL, and the total Fab is 0.6mg and 1.2mg respectively in each reaction. All reactions are carried out in 15v/v%DMA in 25mM HEPES pH 7.5 buffer. The conjugation reaction is carried out for about 18 hours at room temperature (about 25°C). The final average DAR and DAR0 percentages of each conjugate are determined by SDS-PAGE density determination, and the results are shown in Table 13 (reaction P, Q, R, S).
表13.Table 13.
实施例11.预反应endo-BCN-PEG4-PFP酯水解Example 11. Pre-reacted endo-BCN-PEG4-PFP ester hydrolysis
模拟预反应测试用于通过RP-UPLC监测PFP酯水解的速率。使用以下最终反应条件进行预反应：1.63mM endo-BCN-PEG4-PFP酯，在1:1DMA与25mM MES pH 5.5缓冲液中，在20至25℃下持续20小时。UPLC色谱条件在第5.4.3节中提供。每小时将样品注入在UPLC上以监测作为时间函数的endo-BCN-PEG4-PFP酯峰面积的减少，从而监测起始试剂的PFP酯水解的速率。另外，还评估了endo-BCN-PEG4-PFP酯与寡核苷酸-PAB-VC-PEG3-叠氮化物(寡核苷酸是PMO)之间的点击反应的速率。使用以下条件进行预反应：1.63mM endo-BCN-PEG4-PFP酯、2.47mM寡核苷酸-PAB-VC-PEG3-叠氮化物接头-载荷(1:1.5摩尔比)，在1:1DMA与25mM MESpH 5.5缓冲液中，在20至25℃下持续20小时。对于该测试，通过RP-UPLC每30分钟、1小时或5小时监测反应中endo-BCN-PEG4-PFP酯峰面积的减少，总反应持续时间为20小时。结果在图3中示出。The simulated pre-reaction test was used to monitor the rate of PFP ester hydrolysis by RP-UPLC. The pre-reaction was performed using the following final reaction conditions: 1.63mM endo-BCN-PEG4-PFP ester in 1:1 DMA with 25mM MES pH 5.5 buffer for 20 hours at 20 to 25°C. The UPLC chromatographic conditions are provided in Section 5.4.3. Samples were injected onto the UPLC every hour to monitor the decrease in the endo-BCN-PEG4-PFP ester peak area as a function of time, thereby monitoring the rate of PFP ester hydrolysis of the starting reagent. In addition, the rate of the click reaction between endo-BCN-PEG4-PFP ester and oligonucleotide-PAB-VC-PEG3-azide (oligonucleotide is PMO) was also evaluated. The following conditions were used for the pre-reaction: 1.63 mM endo-BCN-PEG4-PFP ester, 2.47 mM oligonucleotide-PAB-VC-PEG3-azide linker-load (1: 1.5 molar ratio), in 1: 1 DMA with 25 mM MES pH 5.5 buffer, at 20 to 25 ° C for 20 hours. For this test, the reduction of the endo-BCN-PEG4-PFP ester peak area in the reaction was monitored by RP-UPLC every 30 minutes, 1 hour or 5 hours, and the total reaction duration was 20 hours. The results are shown in Figure 3.
实施例12.初始抗体-寡核苷酸分析Example 12. Initial Antibody-Oligonucleotide Analysis
评价反应混合物在产生高DAR产物中的效率。最初，通过SEC-UPLC分析来评价粗制反应混合物、样品流通物和合并的HA洗脱物(图24A至24C)。粗制反应混合物包含抗体-寡核苷酸缀合物以及未缀合寡核苷酸(图24A)。样品流通物显示出在存在未缀合寡核苷酸的情况下存在高DAR物质(图24B)。未缀合残留寡核苷酸存在于合并的HA洗脱物中(图24C)。这些结果在缓冲液交换的最终缀合物中是一致的(图25)，其导致总产率为62％。通过SEC-UPLC对合并的HA洗脱物和样品流通物的进一步分析表明，在pH 7.3下，在包含100mM磷酸钠和10％MeCN的流动相缓冲液中损失缀合物(图26A至26B)。确定在相同反应条件下的最终抗体片段-药物缀合物(FDC)混合物包含未缀合寡核苷酸(潜在的寡核苷酸二聚体)和47.2％回收的缀合寡核苷酸(图27)。HA纯化色谱图显示出低缀合物纯化和未缀合寡核苷酸的存在(图28A至28B)。总之，这些结果表明需要改善的反应条件以产生更高浓度的抗体-寡核苷酸缀合物。The efficiency of reaction mixture in producing high DAR product is evaluated. Initially, crude reaction mixture, sample flow-through and HA eluate (Figure 24 A to 24C) merged are evaluated by SEC-UPLC analysis. Crude reaction mixture comprises antibody-oligonucleotide conjugate and unconjugated oligonucleotide (Figure 24 A). Sample flow-through shows that there is high DAR material (Figure 24 B) in the presence of unconjugated oligonucleotide. Unconjugated residual oligonucleotide is present in the HA eluate merged (Figure 24 C). These results are consistent (Figure 25) in the final conjugate of buffer exchange, and it causes that total yield is 62%. Further analysis of the HA eluate merged and sample flow-through by SEC-UPLC shows that at pH 7.3, conjugate (Figure 26 A to 26B) is lost in the mobile phase buffer comprising 100mM sodium phosphate and 10%MeCN. It was determined that the final antibody fragment-drug conjugate (FDC) mixture under the same reaction conditions contained unconjugated oligonucleotides (potential oligonucleotide dimers) and 47.2% recovered conjugated oligonucleotides (Figure 27). The HA purification chromatogram showed low conjugate purification and the presence of unconjugated oligonucleotides (Figures 28A to 28B). In summary, these results indicate that improved reaction conditions are needed to produce higher concentrations of antibody-oligonucleotide conjugates.
实施例13.反应条件Example 13. Reaction conditions
为了确定产生高浓度抗体-寡核苷酸缀合物的条件，改变了反应条件。然而，提高IPA浓度和降低磷酸钠浓度显示出改善了在样品施加期间未缀合寡核苷酸的流通物和FDC的保留(图30A至30C)。最后，提高DMA浓度改善了反应效率，导致更高数目的抗体-寡核苷酸缀合物和更低数目的未缀合寡核苷酸(表14)。In order to determine the conditions for producing high concentration antibody-oligonucleotide conjugates, reaction conditions were changed. However, improving IPA concentration and reducing sodium phosphate concentration demonstrated the retention (Figure 30 A to 30 C) of the flowable material and FDC that improved the unconjugated oligonucleotide during sample application. Finally, improving DMA concentration improved reaction efficiency, resulting in higher number of antibody-oligonucleotide conjugates and lower number of unconjugated oligonucleotide (Table 14).
表14.Table 14.
另一些实施方案Other implementation plans
1.混合物，其包含复合物和未连接寡核苷酸，所述复合物各自包含与一个或更多个寡核苷酸共价连接的抗体，其中所述混合物通过包括以下的方法来产生：1. A mixture comprising complexes and unattached oligonucleotides, each of the complexes comprising an antibody covalently attached to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i)将寡核苷酸与val-cit接头连接以获得第一中间体；(i) connecting the oligonucleotide to a val-cit linker to obtain a first intermediate;
(ii)将步骤(i)中所获得的第一中间体与包含双环壬炔的化合物连接以获得第二中间体；以及(ii) connecting the first intermediate obtained in step (i) with a compound containing a bicyclononyne to obtain a second intermediate; and
(iii)将步骤(ii)中所获得的第二中间体与抗体连接以获得所述复合物；(iii) connecting the second intermediate obtained in step (ii) to an antibody to obtain the complex;
其中所述包含双环壬炔的化合物以小于步骤(ii)中所述化合物的起始量的5％的量存在于步骤(iii)的反应中，任选地其中所述寡核苷酸在5’端与所述val-cit接头连接和/或所述抗体通过赖氨酸连接。wherein the bicyclononyne-containing compound is present in the reaction of step (iii) in an amount less than 5% of the starting amount of the compound in step (ii), optionally wherein the oligonucleotide is linked to the val-cit linker at the 5' end and/or the antibody is linked via a lysine.
2.混合物，其包含复合物和未连接寡核苷酸，所述复合物各自包含与一个或更多个寡核苷酸共价连接的抗体，其中所述混合物通过包括以下的方法来产生：2. A mixture comprising complexes and unattached oligonucleotides, each of the complexes comprising an antibody covalently attached to one or more oligonucleotides, wherein the mixture is produced by a method comprising:
(i)将所述寡核苷酸与式(A)接头连接：(i) connecting the oligonucleotide to a linker of formula (A):
其中n为3；以提供式(B)寡核苷酸：wherein n is 3; to provide an oligonucleotide of formula (B):
其中n为3；Where n is 3;
(ii)使式(B)寡核苷酸与式(C)化合物接触：(ii) contacting an oligonucleotide of formula (B) with a compound of formula (C):
其中m为4；以提供式(D)寡核苷酸：wherein m is 4; to provide an oligonucleotide of formula (D):
其中n为3并且m为4；以及wherein n is 3 and m is 4; and
(iii)使式(D)寡核苷酸与抗体接触以提供式(E)复合物：(iii) contacting the oligonucleotide of formula (D) with an antibody to provide a complex of formula (E):
其中n为3并且m为4；Where n is 3 and m is 4;
其中式(C)化合物以小于步骤(ii)的反应中式(C)化合物起始量的5％的量存在于步骤(iii)的反应混合物中。The compound of formula (C) is present in the reaction mixture of step (iii) in an amount less than 5% of the starting amount of the compound of formula (C) in the reaction of step (ii).
3.处理复合物的方法，所述复合物各自包含与一个或更多个寡核苷酸共价连接的抗体，所述方法包括：3. A method of treating complexes, each of which comprises an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i)使实施方案1或实施方案2所述的混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂在使所述复合物吸附至所述混合模式树脂的条件下接触，其中所述混合物包含的含有炔基的未连接抗体为痕量；以及(i) contacting the mixture of embodiment 1 or embodiment 2 with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions such that the complex is adsorbed to the mixed mode resin, wherein the mixture comprises a trace amount of unlinked antibody containing an alkynyl group; and
(ii)在所述复合物从所述混合模式树脂上解离的条件下从所述混合模式树脂洗脱所述复合物。(ii) eluting the complex from the mixed mode resin under conditions whereby the complex dissociates from the mixed mode resin.
4.实施方案3所述的方法，其中步骤(i)中的所述混合物的pH为5.0至6.0。4. The method of embodiment 3, wherein the pH of the mixture in step (i) is 5.0 to 6.0.
5.实施方案3或实施方案4所述的方法，其中步骤(i)中的所述混合物未经过预先纯化。5. The method of embodiment 3 or embodiment 4, wherein the mixture in step (i) is not pre-purified.
6.实施方案3至5中任一项所述的方法，其中步骤(i)中的所述混合物包含的磷酸根离子和/或氯离子为痕量。6. The method of any one of embodiments 3 to 5, wherein the mixture in step (i) comprises phosphate ions and/or chloride ions in trace amounts.
7.实施方案3至5中任一项所述的方法，其中步骤(i)中的所述混合物不包含磷酸根离子和/或氯离子。7. The method of any one of embodiments 3 to 5, wherein the mixture in step (i) does not contain phosphate ions and/or chloride ions.
8.实施方案3至5中任一项所述的方法，其中所述混合模式树脂是磷灰石树脂。8. The method of any one of embodiments 3 to 5, wherein the mixed mode resin is an apatite resin.
9.实施方案8所述的方法，其中所述磷灰石树脂是羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂或氯磷灰石树脂。9. The method of embodiment 8, wherein the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.
10.实施方案3至9中任一项所述的方法，其中在步骤(i)中，所述未连接寡核苷酸不吸附至所述混合模式树脂。10. The method of any one of embodiments 3 to 9, wherein in step (i), the unligated oligonucleotide is not adsorbed to the mixed mode resin.
11.实施方案3至9中任一项所述的方法，其中在步骤(i)中，一些或所有的所述未连接寡核苷酸吸附至所述混合模式树脂。11. The method of any one of embodiments 3 to 9, wherein in step (i), some or all of the unligated oligonucleotides are adsorbed to the mixed mode resin.
12.实施方案11所述的方法，其还包括在步骤(i)与步骤(ii)之间用包含最高为20mM的磷酸根离子和/或最高为30mM的氯离子的洗涤溶液来洗涤所述混合模式树脂，任选地其中所述溶液包含最高为10mM的磷酸根离子和/或最高为25mM的氯离子。12. The method of embodiment 11 further comprises washing the mixed mode resin between step (i) and step (ii) with a washing solution comprising up to 20 mM phosphate ions and/or up to 30 mM chloride ions, optionally wherein the solution comprises up to 10 mM phosphate ions and/or up to 25 mM chloride ions.
13.实施方案12所述的方法，其中所述洗涤溶液的pH为5.0至7.6。13. The method of embodiment 12, wherein the pH of the washing solution is 5.0 to 7.6.
14.实施方案12或实施方案13所述的方法，其中在所述洗涤步骤中，大多数或所有所述未连接寡核苷酸从所述混合模式树脂中去除。14. The method of embodiment 12 or embodiment 13, wherein during the washing step, most or all of the unligated oligonucleotides are removed from the mixed mode resin.
15.实施方案3至14中任一项所述的方法，其中步骤(ii)包括向所述混合模式树脂施加包含至少30mM的磷酸根离子和/或至少50mM的氯离子的洗脱溶液以洗脱所述复合物，任选地其中所述洗脱溶液包含至少100mM的磷酸根离子和/或至少100mM的氯离子。15. The method of any one of embodiments 3 to 14, wherein step (ii) comprises applying an elution solution comprising at least 30 mM phosphate ions and/or at least 50 mM chloride ions to the mixed mode resin to elute the complex, optionally wherein the elution solution comprises at least 100 mM phosphate ions and/or at least 100 mM chloride ions.
16.实施方案15所述的方法，其中所述洗脱溶液的pH为7.5至8.5。16. The method of embodiment 15, wherein the pH of the elution solution is 7.5 to 8.5.
17.实施方案3至16中任一项所述的方法，其中所述抗体是全长IgG、Fab片段、Fab’片段、F(ab’)2片段、scFv或Fv片段。17. The method of any one of embodiments 3 to 16, wherein the antibody is a full-length IgG, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv or a Fv fragment.
18.实施方案3至17中任一项所述的方法，其中所述抗体是抗转铁蛋白受体抗体。18. The method of any one of embodiments 3 to 17, wherein the antibody is an anti-transferrin receptor antibody.
19.实施方案3至18中任一项所述的方法，其中所述寡核苷酸是单链的。19. The method of any one of embodiments 3 to 18, wherein the oligonucleotide is single-stranded.
20.实施方案19所述的方法，其中所述寡核苷酸是反义寡核苷酸，任选地是间隔聚体。20. The method of embodiment 19, wherein the oligonucleotide is an antisense oligonucleotide, optionally a gapmer.
21.实施方案20所述的方法，其中所述寡核苷酸是双链寡核苷酸的一条链，任选地其中所述双链寡核苷酸是siRNA，并且任选地其中所述一条链是siRNA的有义链。21. The method of embodiment 20, wherein the oligonucleotide is one strand of a double-stranded oligonucleotide, optionally wherein the double-stranded oligonucleotide is a siRNA, and optionally wherein the one strand is the sense strand of the siRNA.
22.实施方案3至21中任一项所述的方法，其中所述寡核苷酸包含至少一个经修饰核苷酸间键联，任选地其中所述至少一个经修饰核苷酸间键联是硫代磷酸酯键联。22. The method of any one of embodiments 3 to 21, wherein the oligonucleotide comprises at least one modified internucleotide linkage, optionally wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
23.实施方案3至22中任一项所述的方法，其中所述寡核苷酸包含一个或更多个经修饰核苷酸，任选地其中所述经修饰核苷酸包含2’-O-甲氧基乙基核糖(MOE)、锁核酸(LNA)、2’-氟修饰或吗啉代修饰。23. The method of any one of embodiments 3 to 22, wherein the oligonucleotide comprises one or more modified nucleotides, optionally wherein the modified nucleotide comprises 2'-O-methoxyethyl ribose (MOE), locked nucleic acid (LNA), 2'-fluoro modification or morpholino modification.
24.实施方案3至23中任一项所述的方法，其中所述寡核苷酸的长度为10至50个核苷酸，任选地长度为15至25个核苷酸。24. The method of any one of embodiments 3 to 23, wherein the oligonucleotide is 10 to 50 nucleotides in length, optionally 15 to 25 nucleotides in length.
25.实施方案3至24中任一项所述的方法，其中所述抗体与所述寡核苷酸的5’共价连接。25. The method of any one of embodiments 3 to 24, wherein the antibody is covalently linked to the 5' of the oligonucleotide.
26.实施方案3至25中任一项所述的方法，其中所述抗体与所述寡核苷酸的3’共价连接。26. The method of any one of embodiments 3 to 25, wherein the antibody is covalently linked to the 3' of the oligonucleotide.
27.实施方案3至26中任一项所述的方法，其中所述抗体通过赖氨酸连接。27. The method of any one of embodiments 3 to 26, wherein the antibody is linked via lysine.
28.实施方案2至27中任一项所述的方法，其中从步骤(ii)中获得的洗脱物包含的未连接寡核苷酸的水平不可检测。28. The method of any one of embodiments 2 to 27, wherein the eluate obtained from step (ii) comprises undetectable levels of unligated oligonucleotides.
29.处理复合物的方法，所述复合物各自包含与一个或更多个电荷中性寡核苷酸共价连接的抗体，所述方法包括：29. A method of treating complexes, each of which comprises an antibody covalently linked to one or more charge-neutral oligonucleotides, the method comprising:
(i)使包含有机溶剂、所述复合物和未连接电荷中性寡核苷酸的混合物与包含带正电荷的金属位点和带负电荷的离子位点的混合模式树脂在使所述复合物吸附至所述混合模式树脂的条件下接触，以及(i) contacting a mixture comprising an organic solvent, the complex and unattached neutrally charged oligonucleotides with a mixed mode resin comprising positively charged metal sites and negatively charged ionic sites under conditions such that the complex is adsorbed to the mixed mode resin, and
(ii)在所述复合物从所述混合模式树脂上解离的条件下从所述混合模式树脂洗脱所述复合物。(ii) eluting the complex from the mixed mode resin under conditions whereby the complex dissociates from the mixed mode resin.
30.实施方案29所述的方法，其中所述混合模式树脂是磷灰石树脂。30. The method of embodiment 29, wherein the mixed mode resin is an apatite resin.
31.实施方案2所述的方法，其中所述磷灰石树脂是羟基磷灰石树脂、陶瓷羟基磷灰石树脂、羟基氟磷灰石树脂、氟磷灰石树脂或氯磷灰石树脂。31. The method of embodiment 2, wherein the apatite resin is a hydroxyapatite resin, a ceramic hydroxyapatite resin, a hydroxyfluoroapatite resin, a fluoroapatite resin, or a chloroapatite resin.
32.实施方案29至31中任一项所述的方法，其中所述有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。32. The method of any one of embodiments 29 to 31, wherein the organic solvent is dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or propylene glycol (PG).
33.实施方案29至32中任一项所述的方法，其中所述有机溶剂在步骤(i)中的所述混合物中为5％至20％(v/v)，任选地其中所述有机溶剂在步骤(i)中的所述混合物中为15％(v/v)。33. The method of any one of embodiments 29 to 32, wherein the organic solvent is 5% to 20% (v/v) in the mixture in step (i), optionally wherein the organic solvent is 15% (v/v) in the mixture in step (i).
34.实施方案29至33中任一项所述的方法，其中步骤(i)中的所述混合物不包含磷酸根离子或氯离子。34. The method of any one of embodiments 29 to 33, wherein the mixture in step (i) does not contain phosphate ions or chloride ions.
35.实施方案29至34中任一项所述的方法，其中步骤(i)中的所述混合物还包含最高为10mM的磷酸根离子和/或最高为20mM的氯离子。35. The method of any one of embodiments 29 to 34, wherein the mixture in step (i) further comprises up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
36.实施方案29至35中任一项所述的方法，其中步骤(i)中的所述混合物的pH为5.0至6.0。36. The method of any one of embodiments 29 to 35, wherein the pH of the mixture in step (i) is 5.0 to 6.0.
37.实施方案29至36中任一项所述的方法，其中在步骤(i)中，所述未连接电荷中性寡核苷酸不吸附至所述混合模式树脂。37. The method of any one of embodiments 29 to 36, wherein in step (i), the unligated charge-neutral oligonucleotides are not adsorbed to the mixed mode resin.
38.实施方案29至37中任一项所述的方法，其还包括在步骤(i)与步骤(ii)之间用包含有机溶剂的洗涤溶液洗涤所述混合模式树脂，任选地其中所述有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。38. The method of any one of embodiments 29 to 37, further comprising washing the mixed mode resin with a washing solution comprising an organic solvent between step (i) and step (ii), optionally wherein the organic solvent is dimethylacetamide (DMA), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG).
39.实施方案38所述的方法，其中所述有机溶剂在所述洗涤溶液中为5％至20％(v/v)，任选地其中所述有机溶剂在所述洗涤溶液中为15％(v/v)。39. The method of embodiment 38, wherein the organic solvent is 5% to 20% (v/v) in the washing solution, optionally wherein the organic solvent is 15% (v/v) in the washing solution.
40.实施方案38或实施方案39所述的方法，其中所述洗涤溶液还包含最高为10mM的磷酸根离子和/或最高为20mM的氯离子。40. The method of embodiment 38 or embodiment 39, wherein the wash solution further comprises up to 10 mM phosphate ions and/or up to 20 mM chloride ions.
41.实施方案29至40中任一项所述的方法，其中步骤(ii)包括向所述混合模式树脂施加洗脱溶液以洗脱所述复合物，其中所述洗脱溶液包含有机溶剂，任选地其中所述有机溶剂是二甲基乙酰胺(DMA)、异丙醇(IPA)、二甲基亚砜(DMSO)、乙腈(ACN)或丙二醇(PG)。41. The method of any one of embodiments 29 to 40, wherein step (ii) comprises applying an elution solution to the mixed mode resin to elute the complex, wherein the elution solution comprises an organic solvent, optionally wherein the organic solvent is dimethylacetamide (DMA), isopropanol (IPA), dimethyl sulfoxide (DMSO), acetonitrile (ACN) or propylene glycol (PG).
42.实施方案41所述的方法，其中所述有机溶剂在所述洗脱溶液中为10％至20％(v/v)，任选地其中所述有机溶剂在所述洗脱溶液中为10％(v/v)。42. The method of embodiment 41, wherein the organic solvent is 10% to 20% (v/v) in the elution solution, optionally wherein the organic solvent is 10% (v/v) in the elution solution.
43.实施方案41或实施方案42所述的方法，其中所述洗脱溶液包含至少30mM的磷酸根离子，任选地其中所述洗脱溶液包含至少100mM的磷酸根离子。43. The method of embodiment 41 or embodiment 42, wherein the elution solution comprises at least 30 mM phosphate ions, optionally wherein the elution solution comprises at least 100 mM phosphate ions.
44.实施方案43所述的方法，其中所述洗脱溶液不包含氯离子。44. The method of embodiment 43, wherein the elution solution does not contain chloride ions.
45.实施方案41或实施方案42所述的方法，其中所述洗脱溶液包含逐渐提高浓度的磷酸根离子，任选地其中所述磷酸根离子的浓度从至少10mM提高至至少100mM。45. The method of embodiment 41 or embodiment 42, wherein the elution solution comprises a gradually increasing concentration of phosphate ions, optionally wherein the concentration of phosphate ions increases from at least 10 mM to at least 100 mM.
46.实施方案41至45中任一项所述的方法，其中所述洗脱溶液的pH为7.6至8.5。46. The method of any one of embodiments 41 to 45, wherein the pH of the elution solution is 7.6 to 8.5.
47.实施方案29至46中任一项所述的方法，其中所述抗体是全长IgG、Fab片段、Fab’片段、F(ab’)2片段、scFv或Fv片段。47. The method of any one of embodiments 29 to 46, wherein the antibody is a full-length IgG, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv or a Fv fragment.
48.实施方案29至47中任一项所述的方法，其中所述抗体是抗转铁蛋白受体抗体。48. The method of any one of embodiments 29 to 47, wherein the antibody is an anti-transferrin receptor antibody.
49.实施方案29至48中任一项所述的方法，其中所述电荷中性寡核苷酸是单链的。49. The method of any one of embodiments 29 to 48, wherein the charge-neutral oligonucleotide is single-stranded.
50.实施方案49所述的方法，其中所述电荷中性寡核苷酸是反义寡核苷酸。50. The method of embodiment 49, wherein the charge-neutral oligonucleotide is an antisense oligonucleotide.
51.实施方案29至50中任一项所述的方法，其中所述电荷中性寡核苷酸是磷酸二酰胺吗啉代寡聚物(PMO)。51. The method of any one of embodiments 29 to 50, wherein the charge-neutral oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
52.实施方案29至51中任一项所述的方法，其中所述电荷中性寡核苷酸的长度为10至50个核苷酸，任选地长度为20至30个核苷酸。52. The method of any one of embodiments 29 to 51, wherein the charge-neutral oligonucleotide is 10 to 50 nucleotides in length, optionally 20 to 30 nucleotides in length.
53.实施方案29至52中任一项所述的方法，其中所述抗体与所述电荷中性寡核苷酸的5’共价连接。53. The method of any one of embodiments 29 to 52, wherein the antibody is covalently linked to the 5' of the charge-neutral oligonucleotide.
54.实施方案29至53中任一项所述的方法，其中所述抗体与所述电荷中性寡核苷酸的3’共价连接。54. The method of any one of embodiments 29 to 53, wherein the antibody is covalently linked to the 3' of the charge-neutral oligonucleotide.
55.实施方案29至54中任一项所述的方法，其中所述抗体通过接头任选Val-cit接头与所述电荷中性寡核苷酸共价连接。55. The method of any one of embodiments 29 to 54, wherein the antibody is covalently linked to the charge-neutral oligonucleotide via a linker, optionally a Val-cit linker.
56.实施方案55所述的方法，其中所述接头包含以下结构：56. The method of embodiment 55, wherein the linker comprises the following structure:
其中n为3并且m为4。Where n is 3 and m is 4.
57.实施方案56所述的方法，其中所述复合物包含以下结构：57. The method of embodiment 56, wherein the complex comprises the following structure:
其中n为3并且m为4，并且其中所述抗体通过赖氨酸连接。wherein n is 3 and m is 4, and wherein the antibody is linked via a lysine.
58.实施方案29至57中任一项所述的方法，其中步骤(i)中的所述混合物中的复合物的平均药物抗体比(DAR)为至少约1.8。58. The method of any one of embodiments 29 to 57, wherein the average drug-antibody ratio (DAR) of the complexes in the mixture in step (i) is at least about 1.8.
59.实施方案29至58中任一项所述的方法，其中从步骤(ii)中获得的洗脱物包含的未连接电荷中性寡核苷酸的水平不可检测。59. The method of any one of embodiments 29 to 58, wherein the eluate obtained from step (ii) contains undetectable levels of unligated neutrally charged oligonucleotides.
60.实施方案29至59中任一项所述的方法，其中所述复合物通过将电荷中性寡核苷酸与抗体连接来产生，其中所述电荷中性寡核苷酸包含以下结构：60. The method of any one of embodiments 29 to 59, wherein the complex is produced by linking a charge-neutral oligonucleotide to an antibody, wherein the charge-neutral oligonucleotide comprises the following structure:
并且其中所述抗体包含以下结构：And wherein the antibody comprises the following structure:
其中n为3并且m为4。Where n is 3 and m is 4.
61.实施方案29至59中任一项所述的方法，其中所述复合物通过将电荷中性寡核苷酸与抗体连接来产生，其中所述电荷中性寡核苷酸包含以下结构：61. The method of any one of embodiments 29 to 59, wherein the complex is produced by linking a charge-neutral oligonucleotide to an antibody, wherein the charge-neutral oligonucleotide comprises the following structure:
其中n为3并且m为4。Where n is 3 and m is 4.
62.产生包含与一个或更多个寡核苷酸共价连接的抗体的复合物的方法，所述方法包括：62. A method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i)获得包含以下结构的寡核苷酸：(i) obtaining an oligonucleotide comprising the following structure:
其中n为3；Where n is 3;
(ii)获得包含以下结构的抗体：(ii) obtaining an antibody comprising the following structure:
其中m为4；以及where m is 4; and
(iii)使步骤(i)中的所述寡核苷酸与步骤(ii)中所获得的抗体反应以获得所述复合物。(iii) reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
63.产生包含与一个或更多个寡核苷酸共价连接的抗体的复合物的方法，所述方法包括：63. A method of producing a complex comprising an antibody covalently linked to one or more oligonucleotides, the method comprising:
(i)获得包含以下结构的寡核苷酸：(i) obtaining an oligonucleotide comprising the following structure:
其中n为3并且m为4；Where n is 3 and m is 4;
(ii)获得抗体；以及(ii) obtaining antibodies; and
(iii)使步骤(i)中的所述寡核苷酸和步骤(ii)中所获得的抗体反应以获得所述复合物。(iii) reacting the oligonucleotide in step (i) with the antibody obtained in step (ii) to obtain the complex.
64.实施方案62或实施方案63所述的方法，其中所述复合物包含以下结构：64. The method of embodiment 62 or embodiment 63, wherein the complex comprises the following structure:
其中n为3并且m为4，并且其中所述抗体通过赖氨酸连接。wherein n is 3 and m is 4, and wherein the antibody is linked via a lysine.
等同方案和术语Equivalent schemes and terminology
本文中举例说明性地描述的公开内容可在不存在本文中未具体公开的任何一个或更多个要素、一个或更多个限制的情况下适当地实践。因此，例如，在本文中的每种情况下，术语“包含/包括”、“基本上由......组成”和“由……组成”中的任一个可用其他两个术语中的任一个替换。已采用的术语和表达作为描述而非限制的术语使用，并且使用这样的术语和表达不旨在排除所示出和所描述的特征的任何等同形式或其一部分，而是应认识到，在所公开内容的范围内可进行多种修改。因此，应理解，尽管已通过一些优选的实施方案、任选的特征具体公开了本公开内容，但是本领域技术人员可获取本文中所公开概念的修改和变化，并且这样的修改和变化被认为是在本公开内容的范围内。The disclosure described illustratively herein can be appropriately practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, in each case herein, any one of the terms "comprising/including", "consisting essentially of" and "consisting of" can be replaced with any one of the other two terms. The terms and expressions adopted are used as descriptive rather than restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalent form or part thereof of the features shown and described, but it should be recognized that various modifications can be made within the scope of the disclosure. Therefore, it should be understood that although the disclosure has been specifically disclosed through some preferred embodiments, optional features, modifications and variations of the concepts disclosed herein can be obtained by those skilled in the art, and such modifications and variations are considered to be within the scope of the disclosure.
另外，在根据马库什组(Markush group)或其他替代组描述本公开内容的特征或方面的情况下，本领域技术人员将认识到，本公开内容也因此以马库什组或其他组的任何个体成员或成员亚组的方式描述。In addition, where features or aspects of the disclosure are described in terms of Markush groups or other alternative groupings, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other grouping.
应理解，在一些实施方案中，在描述寡核苷酸或其他核酸的结构时可参考序列表中所示的序列。在这样的实施方案中，实际的寡核苷酸或其他核酸与指定序列相比可具有一个或更多个替代核苷酸(例如，DNA核苷酸的RNA对应物或RNA核苷酸的DNA对应物)和/或者一个或更多个经修饰核苷酸和/或者一个或更多个经修饰核苷酸间键联和/或者一个或更多个其他修饰，同时保留与指定序列基本相同或相似的互补特性。It will be appreciated that in some embodiments, reference may be made to the sequences shown in the sequence listing when describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more substituted nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modifications compared to the specified sequence while retaining complementary properties that are substantially identical or similar to the specified sequence.
除非在本文中另外指明或与上下文明显矛盾，否则在描述本发明的上下文中(尤其是在所附权利要求书的上下文中)使用没有数量词修饰的名词将被解释为一个/种或更多个/种。除非另有说明，否则术语“包含”、“具有”、“包括”和“含有”将被解释为开放式术语(即，意指“包括但不限于”)。除非在本文中另有说明，否则本文中数值范围的记载仅旨在用作单独引用落入该范围内的每个单独值的速记方法，并且每个单独的值被并入说明书中，就好像它在本文中单独记载一样。除非本文中另外指出或者明显与上下文相矛盾，否则本文中所述的所有方法均可以以任何合适的顺序进行。除非另有说明，否则本文中提供的任何和所有实例或示例性语言(如“例如”)的使用仅仅旨在更好地举例说明本发明，并且不对本发明的范围构成限制。说明书中的语言均不应被解释为表示对本发明的实践必要的任何未要求保护的要素。Unless otherwise specified herein or obviously contradictory with the context, otherwise in the context of describing the present invention (especially in the context of the appended claims), the use of nouns without quantifier modification will be interpreted as one/kind or more/kind. Unless otherwise specified, the terms "comprising", "having", "including" and "containing" will be interpreted as open terms (that is, meaning "including but not limited to"). Unless otherwise specified herein, the recording of numerical ranges herein is only intended to be used as a shorthand method for quoting each individual value falling within the range individually, and each individual value is incorporated into the specification as if it is recorded separately in this article. Unless otherwise specified herein or obviously contradictory with the context, all methods described herein can be carried out in any suitable order. Unless otherwise specified, the use of any and all examples or exemplary languages (such as "for example") provided herein is only intended to better illustrate the present invention, and is not intended to limit the scope of the present invention. The language in the specification should not be interpreted as representing any unclaimed element necessary for the practice of the present invention.
本文中描述了本发明的一些实施方案。在阅读前述说明之后，那些实施实施方案的变化方案对于本领域普通技术人员可变得明显。Several embodiments of the invention are described herein. Variations of those implementing embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.
本发明人预期技术人员在适当时采用这样的变化方案，并且本发明人希望以除本文中具体描述的之外的方式实践本发明。因此，如适用法律所允许的，本发明包括在此所附权利要求书中所记载主题的所有修改方案和等同方案。此外，除非在本文中另外指明或在其他情况下与上下文明显矛盾，否则本发明涵盖其所有可能变化方案中的上述要素的任何组合。本领域技术人员将认识到或仅使用常规实验就能够确定本文中所述的本发明具体实施方案的许多等同方案。The inventors expect that the skilled person will adopt such variations when appropriate, and the inventors wish to practice the present invention in a manner other than that specifically described herein. Therefore, as permitted by applicable law, the present invention includes all modifications and equivalents of the subject matter described in the appended claims herein. In addition, unless otherwise specified herein or clearly contradictory with the context in other cases, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein using only routine experiments.
序列表Sequence Listing
<110> Dyne Therapeutics, Inc.<110> Dyne Therapeutics, Inc.
<120> 制备蛋白质-寡核苷酸复合物的方法<120> Method for preparing protein-oligonucleotide complex
<130> D0824.70043WO00<130> D0824.70043WO00
<140> 尚未分配<140> Not yet assigned
<141> 与此同时<141> Meanwhile
<150> US 63/074,439<150> US 63/074,439
<151> 2020-09-03<151> 2020-09-03
<150> US 63/074,436<150> US 63/074,436
<151> 2020-09-03<151> 2020-09-03
<160> 45<160> 45
<170> PatentIn version 3.5<170> PatentIn version 3.5
<210> 1<210> 1
<211> 760<211> 760
<212> PRT<212> PRT
<213> 智人（Homo sapiens）<213> Homo sapiens
<400> 1<400> 1
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly GluMet Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 151 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly AspPro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 3020 25 30
Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn AlaAsn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala
35 40 4535 40 45
Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser GlyAsp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly
50 55 6050 55 60
Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile GlySer Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly
65 70 75 8065 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys ThrPhe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 9585 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu ProGlu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro
100 105 110100 105 110
Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu LysGly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr IleArg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile
130 135 140130 135 140
Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser GlnLys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu PheLys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe
165 170 175165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln ValLys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly ArgLys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg
195 200 205195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser LysLeu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr LysAla Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240225 230 235 240
Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val IleLys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
245 250 255245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala GluVal Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys PheSer Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285275 280 285
Pro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu GlyPro Ile Val Asn Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr GlnThr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320305 310 315 320
Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln ThrPhe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly AspIle Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr SerCys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser
355 360 365355 360 365
Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu IleGlu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
370 375 380370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro AspLys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly AlaHis Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415405 410 415
Ala Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln MetAla Lys Ser Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser IlePhe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala ThrIle Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe ThrGlu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys ValTyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln AsnSer Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn
500 505 510500 505 510
Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn TrpVal Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp
515 520 525515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro PheAla Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu AspLeu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu LeuThr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575565 570 575
Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala GluIle Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu AsnVal Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn
595 600 605595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg AspLeu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
610 615 620610 615 620
Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu GlnLeu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg LeuTrp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655645 650 655
Thr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met LysThr Thr Asp Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys
660 665 670660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser ProLys Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro
675 680 685675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly SerTyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700690 695 700
Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg LysGly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys
705 710 715 720705 710 715 720
Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu AlaGln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly AspLeu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750740 745 750
Val Trp Asp Ile Asp Asn Glu PheVal Trp Asp Ile Asp Asn Glu Phe
755 760755 760
<210> 2<210> 2
<211> 760<211> 760
<212> PRT<212> PRT
<213> 恒河猴（Macaca mulatta）<213> Rhesus monkey (Macaca mulatta)
<400> 2<400> 2
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly GluMet Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 151 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly AspPro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 3020 25 30
Asn Ser His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn ThrAsn Ser His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn Thr
35 40 4535 40 45
Asp Asn Asn Thr Lys Pro Asn Gly Thr Lys Pro Lys Arg Cys Gly GlyAsp Asn Asn Thr Lys Pro Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly
50 55 6050 55 60
Asn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile GlyAsn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly
65 70 75 8065 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys ThrPhe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 9585 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu ProGlu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro
100 105 110100 105 110
Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu LysGlu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr IleArg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile
130 135 140130 135 140
Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser GlnLys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu PheLys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu Phe
165 170 175165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln ValLys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly GlyLys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly
195 200 205195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser LysLeu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr LysAla Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240225 230 235 240
Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val IleLys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
245 250 255245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala GluVal Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys PheSer Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285275 280 285
Pro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu GlyPro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr GlnThr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320305 310 315 320
Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln ThrPhe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly AspIle Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr SerCys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser
355 360 365355 360 365
Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu ThrGlu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Thr
370 375 380370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro AspLys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly AlaHis Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415405 410 415
Ala Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln MetAla Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser IlePhe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala ThrIle Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe ThrGlu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys ValTyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln AspSer Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asp
500 505 510500 505 510
Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn TrpVal Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn Trp
515 520 525515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro PheAla Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu AspLeu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu LeuThr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575565 570 575
Val Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala GluVal Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu AsnVal Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn
595 600 605595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg AspLeu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
610 615 620610 615 620
Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu GlnLeu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg LeuTrp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655645 650 655
Thr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met LysThr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met Lys
660 665 670660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser ProLys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro
675 680 685675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly SerTyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700690 695 700
Gly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg ArgGly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg
705 710 715 720705 710 715 720
Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu AlaGln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly AspLeu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750740 745 750
Val Trp Asp Ile Asp Asn Glu PheVal Trp Asp Ile Asp Asn Glu Phe
755 760755 760
<210> 3<210> 3
<211> 760<211> 760
<212> PRT<212> PRT
<213> 食蟹猴（Macaca fascicularis）<213> Crab-eating macaque (Macaca fascicularis)
<400> 3<400> 3
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly GluMet Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 151 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly AspPro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 3020 25 30
Asn Ser His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn ThrAsn Ser His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn Thr
35 40 4535 40 45
Asp Asn Asn Thr Lys Ala Asn Gly Thr Lys Pro Lys Arg Cys Gly GlyAsp Asn Asn Thr Lys Ala Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly
50 55 6050 55 60
Asn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile GlyAsn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly
65 70 75 8065 70 75 80
Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys ThrPhe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr
85 90 9585 90 95
Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu ProGlu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro
100 105 110100 105 110
Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu LysGlu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125115 120 125
Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr IleArg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile
130 135 140130 135 140
Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser GlnLys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser Gln
145 150 155 160145 150 155 160
Lys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu PheLys Asp Glu Asn Leu Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu Phe
165 170 175165 170 175
Lys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln ValLys Leu Ser Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val
180 185 190180 185 190
Lys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly GlyLys Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly
195 200 205195 200 205
Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser LysLeu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220210 215 220
Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr LysAla Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys
225 230 235 240225 230 235 240
Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val IleLys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
245 250 255245 250 255
Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala GluVal Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn Ala Glu
260 265 270260 265 270
Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys PheSer Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe
275 280 285275 280 285
Pro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu GlyPro Ile Val Lys Ala Asp Leu Ser Phe Phe Gly His Ala His Leu Gly
290 295 300290 295 300
Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr GlnThr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
305 310 315 320305 310 315 320
Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln ThrPhe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335325 330 335
Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly AspIle Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp
340 345 350340 345 350
Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr SerCys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser
355 360 365355 360 365
Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu ThrGlu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Thr
370 375 380370 375 380
Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro AspLys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp
385 390 395 400385 390 395 400
His Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly AlaHis Tyr Val Val Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala
405 410 415405 410 415
Ala Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln MetAla Lys Ser Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met
420 425 430420 425 430
Phe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser IlePhe Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile
435 440 445435 440 445
Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala ThrIle Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460450 455 460
Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe ThrGlu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr
465 470 475 480465 470 475 480
Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys ValTyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495485 490 495
Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln AspSer Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met Gln Asp
500 505 510500 505 510
Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn TrpVal Lys His Pro Val Thr Gly Arg Ser Leu Tyr Gln Asp Ser Asn Trp
515 520 525515 520 525
Ala Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro PheAla Ser Lys Val Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe
530 535 540530 535 540
Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu AspLeu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
545 550 555 560545 550 555 560
Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu LeuThr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575565 570 575
Val Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala GluVal Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu
580 585 590580 585 590
Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu AsnVal Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn
595 600 605595 600 605
Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg AspLeu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
610 615 620610 615 620
Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu GlnLeu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly Leu Ser Leu Gln
625 630 635 640625 630 635 640
Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg LeuTrp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu
645 650 655645 650 655
Thr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met LysThr Thr Asp Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met Lys
660 665 670660 665 670
Lys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser ProLys Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro
675 680 685675 680 685
Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly SerTyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700690 695 700
Gly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg ArgGly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg
705 710 715 720705 710 715 720
Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu AlaGln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735725 730 735
Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly AspLeu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp
740 745 750740 745 750
Val Trp Asp Ile Asp Asn Glu PheVal Trp Asp Ile Asp Asn Glu Phe
755 760755 760
<210> 4<210> 4
<211> 763<211> 763
<212> PRT<212> PRT
<213> 小家鼠（Mus musculus）<213> House mouse (Mus musculus)
<400> 4<400> 4
Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly GluMet Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu
1 5 10 151 5 10 15
Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly AspPro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp
20 25 3020 25 30
Asn Ser His Val Glu Met Lys Leu Ala Ala Asp Glu Glu Glu Asn AlaAsn Ser His Val Glu Met Lys Leu Ala Ala Asp Glu Glu Glu Asn Ala
35 40 4535 40 45
Asp Asn Asn Met Lys Ala Ser Val Arg Lys Pro Lys Arg Phe Asn GlyAsp Asn Asn Met Lys Ala Ser Val Arg Lys Pro Lys Arg Phe Asn Gly
50 55 6050 55 60
Arg Leu Cys Phe Ala Ala Ile Ala Leu Val Ile Phe Phe Leu Ile GlyArg Leu Cys Phe Ala Ala Ile Ala Leu Val Ile Phe Phe Leu Ile Gly
65 70 75 8065 70 75 80
Phe Met Ser Gly Tyr Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys GluPhe Met Ser Gly Tyr Leu Gly Tyr Cys Lys Arg Val Glu Gln Lys Glu
85 90 9585 90 95
Glu Cys Val Lys Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu ThrGlu Cys Val Lys Leu Ala Glu Thr Glu Glu Thr Asp Lys Ser Glu Thr
100 105 110100 105 110
Met Glu Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala AspMet Glu Thr Glu Asp Val Pro Thr Ser Ser Arg Leu Tyr Trp Ala Asp
115 120 125115 120 125
Leu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala AspLeu Lys Thr Leu Leu Ser Glu Lys Leu Asn Ser Ile Glu Phe Ala Asp
130 135 140130 135 140
Thr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu Ala GlyThr Ile Lys Gln Leu Ser Gln Asn Thr Tyr Thr Pro Arg Glu Ala Gly
145 150 155 160145 150 155 160
Ser Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile Glu Asn Gln Phe HisSer Gln Lys Asp Glu Ser Leu Ala Tyr Tyr Ile Glu Asn Gln Phe His
165 170 175165 170 175
Glu Phe Lys Phe Ser Lys Val Trp Arg Asp Glu His Tyr Val Lys IleGlu Phe Lys Phe Ser Lys Val Trp Arg Asp Glu His Tyr Val Lys Ile
180 185 190180 185 190
Gln Val Lys Ser Ser Ile Gly Gln Asn Met Val Thr Ile Val Gln SerGln Val Lys Ser Ser Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser
195 200 205195 200 205
Asn Gly Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr Val Ala PheAsn Gly Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr Val Ala Phe
210 215 220210 215 220
Ser Lys Pro Thr Glu Val Ser Gly Lys Leu Val His Ala Asn Phe GlySer Lys Pro Thr Glu Val Ser Gly Lys Leu Val His Ala Asn Phe Gly
225 230 235 240225 230 235 240
Thr Lys Lys Asp Phe Glu Glu Leu Ser Tyr Ser Val Asn Gly Ser LeuThr Lys Lys Asp Phe Glu Glu Leu Ser Tyr Ser Val Asn Gly Ser Leu
245 250 255245 250 255
Val Ile Val Arg Ala Gly Glu Ile Thr Phe Ala Glu Lys Val Ala AsnVal Ile Val Arg Ala Gly Glu Ile Thr Phe Ala Glu Lys Val Ala Asn
260 265 270260 265 270
Ala Gln Ser Phe Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Lys AsnAla Gln Ser Phe Asn Ala Ile Gly Val Leu Ile Tyr Met Asp Lys Asn
275 280 285275 280 285
Lys Phe Pro Val Val Glu Ala Asp Leu Ala Leu Phe Gly His Ala HisLys Phe Pro Val Val Glu Ala Asp Leu Ala Leu Phe Gly His Ala His
290 295 300290 295 300
Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn HisLeu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His
305 310 315 320305 310 315 320
Thr Gln Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro ValThr Gln Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val
325 330 335325 330 335
Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Lys Met GluGln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Lys Met Glu
340 345 350340 345 350
Gly Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Lys Leu GluGly Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Lys Leu Glu
355 360 365355 360 365
Leu Ser Gln Asn Gln Asn Val Lys Leu Ile Val Lys Asn Val Leu LysLeu Ser Gln Asn Gln Asn Val Lys Leu Ile Val Lys Asn Val Leu Lys
370 375 380370 375 380
Glu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr Glu GluGlu Arg Arg Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Tyr Glu Glu
385 390 395 400385 390 395 400
Pro Asp Arg Tyr Val Val Val Gly Ala Gln Arg Asp Ala Leu Gly AlaPro Asp Arg Tyr Val Val Val Gly Ala Gln Arg Asp Ala Leu Gly Ala
405 410 415405 410 415
Gly Val Ala Ala Lys Ser Ser Val Gly Thr Gly Leu Leu Leu Lys LeuGly Val Ala Ala Lys Ser Ser Val Gly Thr Gly Leu Leu Leu Lys Leu
420 425 430420 425 430
Ala Gln Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro SerAla Gln Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro Ser
435 440 445435 440 445
Arg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala ValArg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Val
450 455 460450 455 460
Gly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu LysGly Ala Thr Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys
465 470 475 480465 470 475 480
Ala Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser AsnAla Phe Thr Tyr Ile Asn Leu Asp Lys Val Val Leu Gly Thr Ser Asn
485 490 495485 490 495
Phe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly Lys IlePhe Lys Val Ser Ala Ser Pro Leu Leu Tyr Thr Leu Met Gly Lys Ile
500 505 510500 505 510
Met Gln Asp Val Lys His Pro Val Asp Gly Lys Ser Leu Tyr Arg AspMet Gln Asp Val Lys His Pro Val Asp Gly Lys Ser Leu Tyr Arg Asp
515 520 525515 520 525
Ser Asn Trp Ile Ser Lys Val Glu Lys Leu Ser Phe Asp Asn Ala AlaSer Asn Trp Ile Ser Lys Val Glu Lys Leu Ser Phe Asp Asn Ala Ala
530 535 540530 535 540
Tyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys PheTyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Val Ser Phe Cys Phe
545 550 555 560545 550 555 560
Cys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr TyrCys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Leu Asp Thr Tyr
565 570 575565 570 575
Glu Ala Leu Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg ThrGlu Ala Leu Thr Gln Lys Val Pro Gln Leu Asn Gln Met Val Arg Thr
580 585 590580 585 590
Ala Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp ValAla Ala Glu Val Ala Gly Gln Leu Ile Ile Lys Leu Thr His Asp Val
595 600 605595 600 605
Glu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser PheGlu Leu Asn Leu Asp Tyr Glu Met Tyr Asn Ser Lys Leu Leu Ser Phe
610 615 620610 615 620
Met Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met Gly LeuMet Lys Asp Leu Asn Gln Phe Lys Thr Asp Ile Arg Asp Met Gly Leu
625 630 635 640625 630 635 640
Ser Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Tyr Phe Arg Ala ThrSer Leu Gln Trp Leu Tyr Ser Ala Arg Gly Asp Tyr Phe Arg Ala Thr
645 650 655645 650 655
Ser Arg Leu Thr Thr Asp Phe His Asn Ala Glu Lys Thr Asn Arg PheSer Arg Leu Thr Thr Asp Phe His Asn Ala Glu Lys Thr Asn Arg Phe
660 665 670660 665 670
Val Met Arg Glu Ile Asn Asp Arg Ile Met Lys Val Glu Tyr His PheVal Met Arg Glu Ile Asn Asp Arg Ile Met Lys Val Glu Tyr His Phe
675 680 685675 680 685
Leu Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile PheLeu Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe
690 695 700690 695 700
Trp Gly Ser Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu LysTrp Gly Ser Gly Ser His Thr Leu Ser Ala Leu Val Glu Asn Leu Lys
705 710 715 720705 710 715 720
Leu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg AsnLeu Arg Gln Lys Asn Ile Thr Ala Phe Asn Glu Thr Leu Phe Arg Asn
725 730 735725 730 735
Gln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn Ala LeuGln Leu Ala Leu Ala Thr Trp Thr Ile Gln Gly Val Ala Asn Ala Leu
740 745 750740 745 750
Ser Gly Asp Ile Trp Asn Ile Asp Asn Glu PheSer Gly Asp Ile Trp Asn Ile Asp Asn Glu Phe
755 760755 760
<210> 5<210> 5
<211> 197<211> 197
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 5<400> 5
Phe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val Ile IlePhe Val Lys Ile Gln Val Lys Asp Ser Ala Gln Asn Ser Val Ile Ile
1 5 10 151 5 10 15
Val Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro Gly GlyVal Asp Lys Asn Gly Arg Leu Val Tyr Leu Val Glu Asn Pro Gly Gly
20 25 3020 25 30
Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu Val HisTyr Val Ala Tyr Ser Lys Ala Ala Thr Val Thr Gly Lys Leu Val His
35 40 4535 40 45
Ala Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr Pro ValAla Asn Phe Gly Thr Lys Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val
50 55 6050 55 60
Asn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe Ala GluAsn Gly Ser Ile Val Ile Val Arg Ala Gly Lys Ile Thr Phe Ala Glu
65 70 75 8065 70 75 80
Lys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu Ile TyrLys Val Ala Asn Ala Glu Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr
85 90 9585 90 95
Met Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser Phe PheMet Asp Gln Thr Lys Phe Pro Ile Val Asn Ala Glu Leu Ser Phe Phe
100 105 110100 105 110
Gly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe ProGly His Ala His Leu Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro
115 120 125115 120 125
Ser Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly Leu ProSer Phe Asn His Thr Gln Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro
130 135 140130 135 140
Asn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu PheAsn Ile Pro Val Gln Thr Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe
145 150 155 160145 150 155 160
Gly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp Ser ThrGly Asn Met Glu Gly Asp Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr
165 170 175165 170 175
Cys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr Val SerCys Arg Met Val Thr Ser Glu Ser Lys Asn Val Lys Leu Thr Val Ser
180 185 190180 185 190
Asn Val Leu Lys GluAsn Val Leu Lys Glu
195195
<210> 6<210> 6
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 6<400> 6
Ala Ser Ser Leu Asn Ile AlaAlas, Ser Ser, Leu, Asn, Ile Alas
1 51 5
<210> 7<210> 7
<211> 12<211> 12
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 7<400> 7
Ser Lys Thr Phe Asn Thr His Pro Gln Ser Thr ProSer Lys Thr Phe Asn Thr His Pro Gln Ser Thr Pro
1 5 101 5 10
<210> 8<210> 8
<211> 12<211> 12
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 8<400> 8
Thr Ala Arg Gly Glu His Lys Glu Glu Glu Leu IleThr Ala Arg Gly Glu His Lys Glu Glu Glu Leu Ile
1 5 101 5 10
<210> 9<210> 9
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 9<400> 9
Cys Gln Ala Gln Gly Gln Leu Val CysCys Gln Ala Gln Gly Gln Leu Val Cys
1 51 5
<210> 10<210> 10
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 10<400> 10
Cys Ser Glu Arg Ser Met Asn Phe CysCys Ser Glu Arg Ser Met Asn Phe Cys
1 51 5
<210> 11<210> 11
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 11<400> 11
Cys Pro Lys Thr Arg Arg Val Pro CysCys Pro Lys Thr Arg Arg Val Pro Cys
1 51 5
<210> 12<210> 12
<211> 20<211> 20
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 12<400> 12
Trp Leu Ser Glu Ala Gly Pro Val Val Thr Val Arg Ala Leu Arg GlyTrp Leu Ser Glu Ala Gly Pro Val Val Thr Val Arg Ala Leu Arg Gly
1 5 10 151 5 10 15
Thr Gly Ser TrpThr Gly Ser Trp
2020
<210> 13<210> 13
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 13<400> 13
Cys Met Gln His Ser Met Arg Val CysCys Met Gln His Ser Met Arg Val Cys
1 51 5
<210> 14<210> 14
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 14<400> 14
Asp Asp Thr Arg His Trp GlyAsp Asp Thr Arg His Trp Gly
1 51 5
<210> 15<210> 15
<400> 15<400> 15
000000
<210> 16<210> 16
<400> 16<400> 16
000000
<210> 17<210> 17
<211> 5<211> 5
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 17<400> 17
Ser Tyr Trp Met HisSer Tyr Trp Met His
1 51 5
<210> 18<210> 18
<211> 17<211> 17
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 18<400> 18
Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe LysGlu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe Lys
1 5 10 151 5 10 15
SerSer
<210> 19<210> 19
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 19<400> 19
Gly Thr Arg Ala Tyr His TyrGly Thr Arg Ala Tyr His Tyr
1 51 5
<210> 20<210> 20
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 20<400> 20
Arg Ala Ser Asp Asn Leu Tyr Ser Asn Leu AlaArg Ala Ser Asp Asn Leu Tyr Ser Asn Leu Ala
1 5 101 5 10
<210> 21<210> 21
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 21<400> 21
Asp Ala Thr Asn Leu Ala AspAsp Ala Thr Asn Leu Ala Asp
1 51 5
<210> 22<210> 22
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 22<400> 22
Gln His Phe Trp Gly Thr Pro Leu ThrGln His Phe Trp Gly Thr Pro Leu Thr
1 51 5
<210> 23<210> 23
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 23<400> 23
Gly Tyr Thr Phe Thr Ser TyrGly Tyr Thr Phe Thr Ser Tyr
1 51 5
<210> 24<210> 24
<211> 6<211> 6
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 24<400> 24
Asn Pro Thr Asn Gly ArgAsn Pro Thr Asn Gly Arg
1 51 5
<210> 25<210> 25
<211> 6<211> 6
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 25<400> 25
Thr Ser Tyr Trp Met HisThr Ser Tyr Trp Met His
1 51 5
<210> 26<210> 26
<211> 13<211> 13
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 26<400> 26
Trp Ile Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr AsnTrp Ile Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn
1 5 101 5 10
<210> 27<210> 27
<211> 6<211> 6
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 27<400> 27
Ala Arg Gly Thr Arg AlaAla Arg Gly Thr Arg Ala
1 51 5
<210> 28<210> 28
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 28<400> 28
Tyr Ser Asn Leu Ala Trp TyrTyr Ser Asn Leu Ala Trp Tyr
1 51 5
<210> 29<210> 29
<211> 10<211> 10
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 29<400> 29
Leu Leu Val Tyr Asp Ala Thr Asn Leu AlaLeu Leu Val Tyr Asp Ala Thr Asn Leu Ala
1 5 101 5 10
<210> 30<210> 30
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 30<400> 30
Gln His Phe Trp Gly Thr Pro LeuGln His Phe Trp Gly Thr Pro Leu
1 51 5
<210> 31<210> 31
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 31<400> 31
Gln His Phe Ala Gly Thr Pro Leu ThrGln His Phe Ala Gly Thr Pro Leu Thr
1 51 5
<210> 32<210> 32
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 32<400> 32
Gln His Phe Ala Gly Thr Pro LeuGln His Phe Ala Gly Thr Pro Leu
1 51 5
<210> 33<210> 33
<211> 116<211> 116
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 33<400> 33
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly AlaGln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 3020 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp IleTrp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 4535 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys PheGly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 6050 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala TyrLys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr CysMet Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser ValAla Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110100 105 110
Thr Val Ser SerThr Val Ser Ser
115115
<210> 34<210> 34
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 34<400> 34
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 151 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser AsnGlu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 3020 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu ValLeu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 4535 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln SerSer Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 8065 70 75 80
Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro LeuGlu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 9585 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu LysThr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105100 105
<210> 35<210> 35
<211> 116<211> 116
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 35<400> 35
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGlu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 3020 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp IleTrp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 4535 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys PheGly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 6050 55 60
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala TyrLys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met ValAla Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val
100 105 110100 105 110
Thr Val Ser SerThr Val Ser Ser
115115
<210> 36<210> 36
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 36<400> 36
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser AsnAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 3020 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu ValLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
35 40 4535 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro LeuGlu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 9585 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile LysThr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105100 105
<210> 37<210> 37
<211> 330<211> 330
<212> PRT<212> PRT
<213> 智人<213> Homo sapiens
<400> 37<400> 37
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser LysAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 151 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp TyrSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 3020 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr SerPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 4535 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr SerGly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 6050 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln ThrLeu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 8065 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp LysTyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 9585 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro CysLys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro ProPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr CysLys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn TrpVal Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg GluTyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuGlu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser AsnHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyLys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp GluGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe TyrLeu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu AsnPro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe PheAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly AsnLeu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr ThrVal Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly LysGln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330325 330
<210> 38<210> 38
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 38<400> 38
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp GluArg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 151 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn PheGln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 3020 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu GlnTyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 4535 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerSer Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 6050 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr GluThr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 8065 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser SerLys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 9585 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu CysPro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105100 105
<210> 39<210> 39
<211> 446<211> 446
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 39<400> 39
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly AlaGln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 3020 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp IleTrp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 4535 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys PheGly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 6050 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala TyrLys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr CysMet Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser ValAla Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu HisGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445435 440 445
<210> 40<210> 40
<211> 214<211> 214
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 40<400> 40
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly
1 5 10 151 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser AsnGlu Thr Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 3020 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu ValLeu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 4535 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln SerSer Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
65 70 75 8065 70 75 80
Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro LeuGlu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 9585 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala AlaThr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser GlyPro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaThr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser GlnLys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu SerGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val TyrSer Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys SerAla Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205195 200 205
Phe Asn Arg Gly Glu CysPhe Asn Arg Gly Glu Cys
210210
<210> 41<210> 41
<211> 446<211> 446
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 41<400> 41
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGlu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 3020 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp IleTrp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 4535 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys PheGly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 6050 55 60
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala TyrLys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met ValAla Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val
100 105 110100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
225 230 235 240225 230 235 240
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255245 250 255
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270260 265 270
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285275 280 285
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser ValLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
290 295 300290 295 300
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320305 310 315 320
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile SerLys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335325 330 335
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350340 345 350
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
355 360 365355 360 365
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380370 375 380
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
385 390 395 400385 390 395 400
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg TrpGly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
405 410 415405 410 415
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu HisGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430420 425 430
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysAsn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445435 440 445
<210> 42<210> 42
<211> 214<211> 214
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 42<400> 42
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser AsnAsp Arg Val Thr Ile Thr Cys Arg Ala Ser Asp Asn Leu Tyr Ser Asn
20 25 3020 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu ValLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val
35 40 4535 40 45
Tyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 6050 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro LeuGlu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Leu
85 90 9585 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala AlaThr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser GlyPro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaThr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser GlnLys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu SerGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val TyrSer Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys SerAla Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205195 200 205
Phe Asn Arg Gly Glu CysPhe Asn Arg Gly Glu Cys
210210
<210> 43<210> 43
<211> 226<211> 226
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 43<400> 43
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly AlaGln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 3020 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp IleTrp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 4535 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys PheGly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 6050 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala TyrLys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr CysMet Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser ValAla Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220210 215 220
Cys ProCys Pro
225225
<210> 44<210> 44
<211> 226<211> 226
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 44<400> 44
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGlu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 3020 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp IleTrp Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 4535 40 45
Gly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys PheGly Glu Ile Asn Pro Thr Asn Gly Arg Thr Asn Tyr Ile Glu Lys Phe
50 55 6050 55 60
Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala TyrLys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 9585 90 95
Ala Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met ValAla Arg Gly Thr Arg Ala Tyr His Tyr Trp Gly Gln Gly Thr Met Val
100 105 110100 105 110
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaThr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
115 120 125115 120 125
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys LeuPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140130 135 140
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyVal Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
145 150 155 160145 150 155 160
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
165 170 175165 170 175
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190180 185 190
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
195 200 205195 200 205
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrLys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220210 215 220
Cys ProCys Pro
225225
<210> 45<210> 45
<211> 13<211> 13
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 合成的<223> Synthetic
<400> 45<400> 45
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val PheCys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
1 5 101 5 10
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202063074436P | 2020-09-03 | 2020-09-03 | |
| US63/074,439 | 2020-09-03 | ||
| US63/074,436 | 2020-09-03 | ||
| PCT/US2021/049141WO2022051665A1 (en) | 2020-09-03 | 2021-09-03 | Methods of preparing protein-oligonucleotide complexes |
| Publication Number | Publication Date |
|---|---|
| CN116457015Atrue CN116457015A (en) | 2023-07-18 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202180074252.7APendingCN116457015A (en) | 2020-09-03 | 2021-09-03 | Method for preparing protein-oligonucleotide complex |
| Country | Link |
|---|---|
| CN (1) | CN116457015A (en) |
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| US7999085B2 (en)* | 2007-01-09 | 2011-08-16 | Bio-Rad Laboratories, Inc. | Enhanced capacity and purification of protein by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers |
| US20190092833A1 (en)* | 2014-11-21 | 2019-03-28 | Merck Sharp & Dohme Corp. | Insulin receptor partial agonists |
| WO2020028831A1 (en)* | 2018-08-02 | 2020-02-06 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating fibrodysplasia ossificans progressiva |
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| US7999085B2 (en)* | 2007-01-09 | 2011-08-16 | Bio-Rad Laboratories, Inc. | Enhanced capacity and purification of protein by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers |
| US20190092833A1 (en)* | 2014-11-21 | 2019-03-28 | Merck Sharp & Dohme Corp. | Insulin receptor partial agonists |
| WO2020028831A1 (en)* | 2018-08-02 | 2020-02-06 | Dyne Therapeutics, Inc. | Muscle targeting complexes and uses thereof for treating fibrodysplasia ossificans progressiva |
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