是埃博灵克斯股份有限公司(Ablynx N.V.)的注册商标]对于纳米抗体的概括描述参考以下的进一步描述，以及参考在本文中引用的现有技术，如，例如，在WO 08/020079(第16页)中描述的。In particular, immunoglobulin single variable domains may be

(as defined herein) or suitable fragments thereof. [Note:

is a registered trademark of Ablynx NV] For a general description of Nanobodies reference is made to the further description below, as well as to the prior art cited herein, such as, for example, in WO 08/020079( described on page 16).

对于VHH和纳米抗体的进一步描述，参考Muyldermans 2001(分子生物技术中的综述(Reviews in Molecular Biotechnology)74:277-302)的综述文章，以及参考作为一般背景技术提及的以下专利申请：VrijeUniversiteit Brussel的WO 94/04678、WO 95/04079和WO 96/34103；Unilever的WO 94/25591、WO 99/37681、WO 00/40968、WO 00/43507、WO00/65057、WO 01/40310、WO 01/44301、EP 1134231和WO 02/48193；Vlaams Instituutvoor Biotechnologie(VIB)的WO 97/49805、WO 01/21817、WO 03/035694、WO 03/054016和WO 03/055527；Algonomics N.V.和埃博灵克斯股份有限公司的WO 03/050531；加拿大国立研究委员会(National Research Council of Canada)的WO 01/90190；抗体研究所(Institute of Antibodies)的WO 03/025020；以及埃博灵克斯股份有限公司的WO 04/041867、WO 04/041862、WO 04/041865、WO 04/041863、WO 04/062551、WO 05/044858、WO06/40153、WO 06/079372、WO 06/122786、WO 06/122787和WO 06/122825和埃博灵克斯股份有限公司的另外的公布的专利申请。还参考在这些申请中提及的另外的现有技术，并且尤其参考在国际申请WO 06/040153的第41-43页提及的参考文献清单，该清单和参考文献通过引用结合在本文中。如在这些参考文献中描述的，纳米抗体(尤其是VHH序列和部分人源化的纳米抗体)的特征可以尤其在于在一个或多个构架序列中存在一个或多个“标志残基”。可以例如在WO 08/101985和WO 08/142164中找到纳米抗体的进一步描述，包括纳米抗体的人源化和/或骆驼源化，以及其他修饰、部分或片段、衍生物或“纳米抗体融合体”、多价构建体(包括接头序列的一些非限制性实例)和增加纳米抗体半衰期的不同修饰和它们的制备。For a further description of VHHs and Nanobodies, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302), and to the following patent application mentioned as general background art: VrijeUniversiteit Brussel WO 94/04678, WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/ 44301, EP 1134231 and WO 02/48193; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of Vlaams Instituutvoor Biotechnologie (VIB); Algonomics N.V. and Ebolinx WO 03/050531, Inc.; WO 01/90190, National Research Council of Canada; WO 03/025020, Institute of Antibodies; WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825 and additional published patent applications of Ebolinx Incorporated. Reference is also made to the additional prior art mentioned in these applications and in particular to the list of references mentioned on pages 41-43 of International Application WO 06/040153, which list and references are incorporated herein by reference. As described in these references, Nanobodies (especially VHH sequences and partially humanized Nanobodies) may be characterized in particular by the presence of one or more "Hallmark residues" in one or more framework sequences. A further description of Nanobodies, including humanization and/or camelization of Nanobodies, and other modifications, parts or fragments, derivatives or "Nanobody fusions" can be found, for example, in WO 08/101985 and WO 08/142164. ", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase the half-life of Nanobodies and their preparation.

因此，在本发明的意义中，术语“免疫球蛋白单可变结构域”或“单可变结构域”包括来源于非人来源、优选骆驼科动物、优选骆驼科动物重链抗体的多肽。如之前描述的，它们可以是人源化的。此外，该术语包括来源于非骆驼科动物来源例如小鼠或人的已经“骆驼源化”的多肽，例如在Davies和Riechmann 1994(FEBS 339:285-290)、1995(Biotechnol.13:475-479)、1996(Prot.Eng.9:531-537)和Riechmann和Muyldermans 1999(J.Immunol.Methods 231:25-38)中描述的。Thus, within the meaning of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from non-human sources, preferably camelids, preferably camelid heavy chain antibodies. They may be humanized as previously described. Furthermore, the term includes polypeptides derived from non-camelid sources such as mice or humans that have been "camelized", for example in Davies and Riechmann 1994 (FEBS 339:285-290), 1995 (Biotechnol. 13:475- 479), 1996 (Prot. Eng. 9:531-537) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231:25-38).

术语“免疫球蛋白单可变结构域”包括不同来源的免疫球蛋白序列，包括小鼠、大鼠、兔、驴、人和骆驼科动物免疫球蛋白序列。其还包括完全人的、人源化的或嵌合的免疫球蛋白序列。例如，它包括骆驼科动物免疫球蛋白序列和人源化骆驼科动物免疫球蛋白序列，或骆驼源化免疫球蛋白单可变结构域，例如由Ward等人，1989描述的骆驼源化dAb(参见例如WO 94/04678以及Davies和Riechmann 1994、1995和1996)和骆驼源化VH。The term "immunoglobulin single variable domain" includes immunoglobulin sequences of various origins, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. It also includes fully human, humanized or chimeric immunoglobulin sequences. For example, it includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized immunoglobulin single variable domains, such as the camelized dAb described by Ward et al., 1989 ( See eg WO 94/04678 and Davies and Riechmann 1994, 1995 and 1996) and camelized VH.

同样地，这样的免疫球蛋白单可变结构域可以以任何适合的方式来源于任何适合的来源，并且例如可以是自然存在的VHH序列(即，来自适合的骆驼科动物物种)或合成或半合成氨基酸序列，包括但不限于部分或完全“人源化”的VHH、“骆驼源化”的免疫球蛋白序列(并且尤其是骆驼源化VH)，以及通过以下技术获得的纳米抗体和/或VHH：如亲和力成熟(例如，从合成的、随机的或自然存在的免疫球蛋白序列如VHH序列开始)、CDR移植、镶盖术(veneering)、组合来源于不同免疫球蛋白序列的片段、使用重叠引物的PCR装配、和技术人员公知的用于改造免疫球蛋白序列的类似技术；或前述中任一项的任何适合的组合。Likewise, such immunoglobulin single variable domains may be derived in any suitable manner from any suitable source and, for example, may be naturally occurring VHH sequences (i.e. from a suitable camelid species) or synthetic or semi- Synthetic amino acid sequences, including but not limited to partially or fully "humanized" VHHs, "camelized" immunoglobulin sequences (and especially camelized VHs), and Nanobodies obtained by techniques and/or VHH: such as affinity maturation (e.g. starting from a synthetic, random or naturally occurring immunoglobulin sequence such as a VHH sequence), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, using PCR assembly of overlapping primers, and similar techniques known to the skilled artisan for engineering immunoglobulin sequences; or any suitable combination of any of the foregoing.

免疫球蛋白单可变结构域的氨基酸序列和结构可以被认为——然而不限于——由四个构架区或“FR”组成，其在本领域中和在本文中分别被称为“构架区1”或“FR1”；“构架区2”或“FR2”；“构架区3”或“FR3”；以及“构架区4”或“FR4”；所述构架区被三个互补决定区或“CDR”隔开，其在本领域中分别被称为“互补决定区1”或“CDR1”；“互补决定区2”或“CDR2”；以及“互补决定区3”或“CDR3”。The amino acid sequence and structure of an immunoglobulin single variable domain can be considered - however not limited to - to consist of four framework regions, or "FRs", which are referred to in the art and herein as "framework regions" respectively. 1" or "FR1"; "Framework Region 2" or "FR2"; "Framework Region 3" or "FR3"; and "Framework Region 4" or "FR4"; CDR", which are referred to in the art as "complementarity determining region 1" or "CDR1"; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3", respectively.

在免疫球蛋白单可变结构域中的氨基酸残基的总数量可以在110-120的范围内，优选为112-115，并且最优选113。The total number of amino acid residues in an immunoglobulin single variable domain may be in the range of 110-120, preferably 112-115, and most preferably 113.

如在WO 08/020079(通过引用结合在本文中)的第58和59页的q)段中进一步描述的，根据由Kabat等人给出的针对VH结构域的通用编号(“Kabat编号”)(“免疫学目标蛋白序列(Sequence of proteins ofimmunological interest)”,美国公众健康服务中心(USPublic HealthServices),NIH Bethesda,MD,公布号91)对免疫球蛋白单可变结构域的氨基酸残基进行编号，如在Riechmann和Muyldermans 2000(J.Immunol.Methods 240:185-195；参见例如该出版物的图2)的文章中应用于骆驼科动物的VHH结构域的，并且相应地，免疫球蛋白单可变结构域的FR1包含在1-30位的氨基酸残基，免疫球蛋白单可变结构域的CDR1包含在31-35位的氨基酸残基，免疫球蛋白单可变结构域的FR2包含在36-49位的氨基酸，免疫球蛋白单可变结构域的CDR2包含在50-65位的氨基酸残基，免疫球蛋白单可变结构域的FR3包含在66-94位的氨基酸残基，免疫球蛋白单可变结构域的CDR3包含在95-102位的氨基酸残基，并且免疫球蛋白单可变结构域的FR4包含在103-113位的氨基酸残基。As further described in paragraph q) on pages 58 and 59 of WO 08/020079 (incorporated herein by reference), according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) Numbering the amino acid residues of immunoglobulin single variable domains , as applied to the VHH domains of camelids in the article by Riechmann and Muyldermans 2000 (J. Immunol. Methods 240:185-195; see e.g. Figure 2 of this publication), and correspondingly, the immunoglobulin single FR1 of the variable domain contains amino acid residues at positions 1-30, CDR1 of the immunoglobulin single variable domain contains amino acid residues at positions 31-35, and FR2 of the immunoglobulin single variable domain contains Amino acids at positions 36-49, the CDR2 of the immunoglobulin single variable domain contains amino acid residues at positions 50-65, the FR3 of the immunoglobulin single variable domain contains amino acid residues at positions 66-94, and the immune The CDR3 of a globulin single variable domain comprises amino acid residues at positions 95-102, and the FR4 of an immunoglobulin single variable domain comprises amino acid residues at positions 103-113.

基于在本文中以及在WO 08/020079中、在WO 06/040153中和在另外的关于免疫球蛋白单可变结构域的在其中引用的参考文献中给出的免疫球蛋白单可变结构域序列的实例，将会清楚的是，氨基酸残基的精确数量还将会取决于在免疫球蛋白单可变结构域中存在的特定CDR的长度。就CDR而言，如在本领域中公知的，存在多种定义并且描述VH或VHH片段的CDR的习惯，如Kabat定义(其基于序列可变性并且是最常用的)和Chothia定义(其基于结构环区域的位置)。例如，参考网站http://www.bioinf.org.uk/abs/。对于本说明书和权利要求的目的来说，即使也可以提及根据Kabat的CDR，最优选基于Abm定义(其基于OxfordMolecular的AbM抗体建模软件)来定义CDR，因为认为这是在Kabat和Chothia定义之间的最佳折衷。再次参考网站http://www.bioinf.org.uk/abs/)。Based on the immunoglobulin single variable domains given herein and in WO 08/020079, in WO 06/040153 and in the additional references cited therein for immunoglobulin single variable domains Examples of sequences, it will be clear that the exact number of amino acid residues will also depend on the length of the particular CDRs present in an immunoglobulin single variable domain. In terms of CDRs, as is well known in the art, there are various definitions and conventions for describing the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the structure location of the ring region). For example, refer to the website http://www.bioinf.org.uk/abs/. For the purposes of this specification and claims, even though reference may also be made to CDRs according to Kabat, it is most preferred to define CDRs based on the Abm definition (which is based on Oxford Molecular's AbM antibody modeling software), as this is considered to be the definition in Kabat and Chothia the best compromise between. Refer again to the website http://www.bioinf.org.uk/abs/).

在一个实施方案中，FR4包含C端氨基酸序列VTVSS，即109、110、111、112和113位中的每一个。本发明还包括在109、110、111或112位终止的ISVD。在本发明的一个方面中，FR4以C端氨基酸序列VTVS(109-112位)终止，FR4以C端氨基酸序列VTV(109-111位)终止，FR4以C端氨基酸序列VT(109-110位)终止，或者FR4以C端氨基酸V(109位)终止。C端延伸可以存在于最后一个(位于最C端)的ISVD的FR4的最后一个氨基酸残基例如V109、T110、V111、S112或S113的C端，其中本发明的半胱氨酸部分优选存在于或者位于C端延伸的C末端。在一个实施方案中，FR4包含C端氨基酸序列VTVSS并且C端延伸是半胱氨酸(例如，本发明的多肽以VTVSSC终止)。在一个实施方案中，FR4包含C端氨基酸序列VTVS并且C端延伸是半胱氨酸(例如，本发明的多肽以VTVSC终止)。在一个实施方案中，FR4包含C端氨基酸序列VTV并且C端延伸是半胱氨酸(例如，本发明的多肽以VTVC终止)。在一个实施方案中，FR4包含C端氨基酸序列VT并且C端延伸是半胱氨酸(例如，本发明的多肽以VTC终止)。在一个实施方案中，FR4包含C端氨基酸V并且C端延伸是半胱氨酸(例如，本发明的多肽以VC终止)。In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS, ie each of positions 109, 110, 111, 112 and 113. The invention also includes ISVDs that terminate at position 109, 110, 111 or 112. In one aspect of the present invention, FR4 terminates with the C-terminal amino acid sequence VTVS (position 109-112), FR4 terminates with the C-terminal amino acid sequence VTV (position 109-111), and FR4 terminates with the C-terminal amino acid sequence VT (position 109-110). ) termination, or FR4 terminates with C-terminal amino acid V (position 109). The C-terminal extension may be present at the C-terminus of the last (most C-terminal) FR4 of the ISVD, e.g. V109, T110, V111, S112 or S113, wherein the cysteine moiety of the present invention is preferably present at Or at the C-terminus of the C-terminal extension. In one embodiment, FR4 comprises a C-terminal amino acid sequence VTVSS and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTVSSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVS and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTVSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTV and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTVC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VT and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VTC). In one embodiment, FR4 comprises a C-terminal amino acid V and the C-terminal extension is a cysteine (eg, a polypeptide of the invention terminates with VC).

在一个实施方案中，本发明涉及如在本文中所描述的二聚体，其中所述ISVD选自由下列各项组成的组：单一结构域抗体、结构域抗体、适合用作单一结构域抗体的氨基酸序列、适合用作结构域抗体的氨基酸序列、dAb、适合用作dAb的氨基酸序列、纳米抗体、VHH、人源化VHH、和骆驼源化VH。优选地，ISVD包含100至140个之间的氨基酸，如110-130个之间的氨基酸。In one embodiment, the invention relates to a dimer as described herein, wherein said ISVD is selected from the group consisting of single domain antibodies, domain antibodies, suitable for use as single domain antibodies Amino acid sequences, amino acid sequences suitable for use as domain antibodies, dAbs, amino acid sequences suitable for use as dAbs, Nanobodies, VHH, humanized VHH, and camelized VH. Preferably, the ISVD comprises between 100 and 140 amino acids, such as between 110-130 amino acids.

术语“噬菌体展示文库”是指外源性肽或蛋白质在其表面上表达的噬菌体的“文库”。外来肽或多肽被展示在噬菌体衣壳外表面上。外来肽可展示为作为噬菌体外壳蛋白的一部分并入的重组融合蛋白；通常不是噬菌体外壳蛋白，但能够变得并入衣壳外表面中的重组融合蛋白；或变得共价或非共价连接于所述蛋白质的蛋白质或肽。这是通过将外源性核酸序列插入可被包装至噬菌体粒子中的核酸中来达成。所述外源性核酸序列可例如被插入噬菌体外壳蛋白基因的编码序列中。如果外来序列是框内克隆，那么它编码的蛋白质将表达为外壳蛋白的一部分。因此，核酸序列的文库，如由编码一个或多个个体的整个B细胞谱系的基因区段制得的抗体谱系的文库，可被如此插入噬菌体中以产生“噬菌体文库”。当代表由核酸文库编码的肽和蛋白质的那些肽和蛋白质由噬菌体展示时，产生“肽展示文库”。尽管多种噬菌体用于所述文库构建中，但通常使用丝状噬菌体(Dunn(1996)Curr.Opin.Biotechnol.7:547-553)。参见例如以下对噬菌体展示文库的描述。The term "phage display library" refers to a "library" of phage on the surface of which exogenous peptides or proteins are expressed. Foreign peptides or polypeptides are displayed on the outer surface of the phage capsid. The foreign peptide can be displayed as a recombinant fusion protein incorporated as part of the phage coat protein; a recombinant fusion protein that is not normally a phage coat protein but can become incorporated into the outer surface of the capsid; or become covalently or non-covalently linked A protein or peptide based on said protein. This is achieved by inserting exogenous nucleic acid sequences into nucleic acids that can be packaged into phage particles. Said exogenous nucleic acid sequence may for example be inserted into the coding sequence of the phage coat protein gene. If the foreign sequence is cloned in frame, then the protein it encodes will be expressed as part of the coat protein. Thus, a library of nucleic acid sequences, such as a library of antibody repertoires made from gene segments encoding the entire B-cell repertoire of one or more individuals, can thus be inserted into phage to generate a "phage library". A "peptide display library" is produced when those peptides and proteins representative of those encoded by the nucleic acid library are displayed by phage. Although a variety of phages are used in such library construction, filamentous phages are commonly used (Dunn (1996) Curr. Opin. Biotechnol. 7:547-553). See, eg, the description of phage display libraries below.

术语“嵌合抗体”是指其中免疫球蛋白分子的氨基酸序列源于两个或更多个物种的抗体。通常，轻链与重链两者的可变区均对应于抗体的源于一个哺乳动物物种(例如小鼠、大鼠、兔等)的具有所需特异性、亲和力和能力的可变区，而恒定区与抗体中源于另一物种(通常是人)的序列同源以避免在那个物种中引发免疫应答。The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity and capacity, Instead, the constant regions are homologous to sequences in the antibody derived from another species, usually a human, to avoid eliciting an immune response in that species.

术语“嵌合抗体”是指重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性(美国专利No.4,816,567；Morrison SL等,Proc.Natl.Acad.Sci.USA,81:6851-6855,1984)。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。The term "chimeric antibody" refers to a portion of the heavy and/or light chain that is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to that derived from another antibody class or subclass. The corresponding sequences in antibodies belonging to one species or belonging to another antibody class or subclass are identical or homologous, and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison SL et al., Proc. . Natl. Acad. Sci. USA, 81:6851-6855, 1984). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).

术语“多特异性抗体”，本发明的抗体可以是单特异性、双特异性或多特异性的。多特异性抗体可对目标多肽的不同表位具特异性或可以含有对超过一种目标多肽具特异性的抗原结合域。参见例如Tutt等人，1991，《免疫学杂志》147：60-69；Kufer等人，2004，《生物技术趋势(TrendsBiotechnol.)》22：238-244。本发明的抗体可以键联到另一功能分子(例如另一肽或蛋白质)或与其共同表达。举例来说，抗体或其片段可以功能上键联(例如通过化学偶合、基因融合、非共价缔合或以其它方式)到一个或多个其它分子实体，如另一抗体或抗体片段，以制备具有第二结合特异性的双特异性或多特异性抗体。举例来说，本发明包括双特异性抗体，其中免疫球蛋白的一条臂对溶血素A的N端区或其片段具特异性，而免疫球蛋白的另一条臂对溶血素A的C端区或第二治疗剂靶标具特异性，或结合于治疗剂部分。可以用于本发明的背景中的示例性双特异性抗体格式涉及使用第一免疫球蛋白(Ig)CH3结构域和第二Ig CH3结构域，其中第一和第二Ig CH3结构域因至少一个氨基酸而彼此不同，且其中至少一个氨基酸差异使双特异性抗体与蛋白质A的结合与缺乏所述氨基酸差异的双特异性抗体相比有所减少。在一个实施例中，第一Ig CH3结构域结合蛋白质A且第二Ig CH3结构域含有减少或消除蛋白质A结合的突变，如H95R修饰(根据IMGT外显子编号；根据EU编号是H435R)。第二CH3可以进一步包含Y96F修饰(根据IMGT；根据EU是Y436F)。第二CH3内可发现的其它修饰包括：在IgG1抗体的情况下，D16E、L18M、N44S、K52N、V57M和V82I(根据IMGT；根据EU是D356E、L358M、N384S、K392N、V397M和V422I)；在IgG2抗体的情况下，N44S、K52N和V82I(IMGT；根据EU是N384S、K392N和V422I)；和在IgG4抗体的情况下，Q15R、N44S、K52N、V57M、R69K、E79Q和V82I(根据IMGT；根据EU是Q355R、N384S、K392N、V397M、R409K、E419Q和V422I)。关于上文所描述的双特异性抗体格式的变化涵盖于本发明范围内。可以用于本发明的背景中的其它示例性双特异性格式包括但不限于例如基于scFv或双功能抗体双特异性格式、IgG-scFv融合物、双重可变域(DVD)-Ig、四体杂交瘤(Quadroma)、杵入臼结构、常见轻链(例如具有杵入臼结构的常见轻链等)、CrossMab、CrossFab、(SEED)体、白氨酸拉链、Duobody、IgG1/IgG2、双重作用Fab(DAF)-IgG和Mab2双特异性格式(关于前述格式的综述参见例如Klein等人2012，mAbs 4：6，1-11和其中所引用的参考文献)。双特异性抗体也可以使用肽/核酸结合来构筑，例如其中具有正交化学反应性的非天然氨基酸用于产生位点特异性抗体-寡核苷酸结合物，其然后自组装成具有所规定的组成、价数和几何形状的多聚复合物。(参见例如Kazane等人，《美国化学学会杂志(J.Am.Chem.Soc.)》[电子出版：2012年12月4日)。By the term "multispecific antibody", an antibody of the invention may be monospecific, bispecific or multispecific. Multispecific antibodies can be specific for different epitopes of a target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide. See, eg, Tutt et al., 1991, Journal of Immunology 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. An antibody of the invention may be linked to or co-expressed with another functional molecule (eg, another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to Bispecific or multispecific antibodies are produced with a second binding specificity. For example, the invention includes bispecific antibodies in which one arm of the immunoglobulin is specific for the N-terminal region of hemolysin A or a fragment thereof and the other arm of the immunoglobulin is specific for the C-terminal region of hemolysin A Either the second therapeutic agent is target specific, or binds to a therapeutic agent moiety. An exemplary bispecific antibody format that may be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains are due to at least one Amino acids differ from each other, and wherein at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking said amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). Other modifications that can be found within the second CH3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; according to EU are D356E, L358M, N384S, K392N, V397M and V422I); In the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); EU is Q355R, N384S, K392N, V397M, R409K, E419Q and V422I). Variations on the bispecific antibody formats described above are encompassed within the scope of the invention. Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, e.g. scFv or bifunctional antibody based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetramers Hybridoma (Quadroma), Knob-into-Hole Structure, Common Light Chain (e.g. Common Light Chain with Knob-in-Hole Structure, etc.), CrossMab, CrossFab, (SEED) Body, Leucine Zipper, Duobody, IgG1/IgG2, Dual Action Fab(DAF)-IgG and Mab2 bispecific formats (for a review of the aforementioned formats see eg Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into molecules with defined The composition, valence and geometry of multimeric complexes. (See eg, Kazane et al., J. Am. Chem. Soc. [Epub: 4 Dec. 2012).

术语“单域抗体”用基因工程方法获得，主要有3类，第一类是从骆驼科动物HCAb获得的重链可变区，为单一的折叠单元，保留了完整的抗原结合活性，是最小的天然抗体片段。第二类是从鲨鱼等软骨鱼IgNAR获得的重链可变区，用VNAR表示。第三类是从人源或鼠源单抗获得的重链或轻链可变区，保留了抗原结合活性，但亲和性和可溶性大为下降。The term "single-domain antibody" is obtained by genetic engineering methods. There are mainly three types. The first type is the heavy chain variable region obtained from camelid HCAb. It is a single folding unit that retains complete antigen-binding activity and is the smallest natural antibody fragments. The second category is the heavy chain variable region obtained from cartilaginous fish IgNARs such as sharks, denoted by VNAR. The third type is the heavy chain or light chain variable region obtained from human or mouse monoclonal antibody, which retains the antigen binding activity, but the affinity and solubility are greatly reduced.

术语“Fc区”或“Fc”是指免疫球蛋白重链的C端区，其含有铰链区的至少一部分、CH2结构域和CH3结构域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫系统的各种细胞(例如，效应细胞)上的Fc受体结合或与经典补体系统的第一组分(例如C1q)结合，包括天然序列Fc区和变异Fc区。通常，人IgG重链Fc区为自其Cys226或Pro230位置的氨基酸残基至羧基末端的区段，但其边界可能有变化。Fc区的C-末端赖氨酸(残基447，依照EU编号系统)可以存在或可以不存在。Fc还可以指隔离的这一区域，或在包含Fc的蛋白多肽的情况下，例如“包含Fc区的结合蛋白”，还称为“Fc融合蛋白”(例如，抗体或免疫粘合素)。本发明的抗体中天然序列Fc区包括人IgG1，IgG2(IgG2A，IgG2B)，IgG3和IgG4。在IgG、IgA和IgD抗体同种型中，Fc区包含抗体的两条重链的每一条的CH2和CH3恒定结构域；IgM和IgEFc区包含在每条多肽链中的三个重链恒定结构域(CH结构域2-4)。The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain and the CH3 domain, which mediate the binding of the immunoglobulin to host tissues or factors , including binding to Fc receptors located on various cells of the immune system (eg, effector cells) or binding to the first component of the classical complement system (eg, C1q), including native sequence Fc regions and variant Fc regions. Typically, the human IgG heavy chain Fc region is the segment from its amino acid residue at position Cys226 or Pro230 to the carboxy-terminus, although its boundaries may vary. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may or may not be present. Fc may also refer to this region in isolation, or in the case of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein", also referred to as an "Fc fusion protein" (eg, an antibody or an immunoadhesin). The native sequence Fc region of the antibody of the present invention includes human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. In IgG, IgA, and IgD antibody isotypes, the Fc region contains the CH2 and CH3 constant domains of each of the antibody's two heavy chains; the IgM and IgEFc regions contain the three heavy chain constant domains in each polypeptide chain domain (CH domains 2-4).

术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。术语“免疫结合”是指发生在抗体分子和抗原(对于该抗原而言抗体为特异性的)之间的特异性结合反应。免疫结合相互作用的强度或亲和力可以相互作用的平衡解离常数(KD)表示，其中KD值越小，表示亲和力越高。两分子间的的免疫结合性质可使用本领域中公知的方法定量。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。指特定抗体-抗原相互作用的“结合速率常数”(Ka或Kon)和“解离速率常数”(Kd或Koff)两者都可通过浓度及缔合和解离的实际速率而计算得出，参见Malmqvist M,1993,Nature,361:186-187。Kd/Ka的比率等于解离常数KD，参见Davies DR等,1990,Annual Rev Biochem.,59:439-473。可用任何有效的方法测量KD、Ka和Kd值。在优选的实施方案中，用生物发光干涉测量法来测量解离常数。在其它优选的实施方案中，可用表面等离子共振技术(例如Biacore)或KinExa来测量解离常数。The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of the immune binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, wherein the smaller the KD value, the higher the affinity. The immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) referring to a particular antibody-antigen interaction can be calculated from the concentration and the actual rates of association and dissociation, see Malmqvist M, 1993, Nature, 361:186-187. The ratio of Kd/Ka is equal to the dissociation constant KD, see Davies DR et al., 1990, Annual Rev Biochem., 59:439-473. KD, Ka and Kd values can be measured by any effective method. In a preferred embodiment, the dissociation constant is measured using bioluminescence interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (eg Biacore) or KinExa.

术语“抗体药物偶联物”、“免疫偶联物”，由抗体、连接子和药物组成，所述连接子是可断裂连接子组合或不可断裂连接子。抗体是球状蛋白，含有一系列氨基酸位点可用于偶联药物-连接子。由于其三级和四级结构，只有溶剂可及的氨基酸可供偶联。事实上，高收率的偶联通常发生在赖氨酸残基的ε-氨基基团或半胱氨酸残基的巯基基团上。抗体蛋白表面的大量赖氨酸侧链导致大量的位点可供药物偶联，从而导致生成的抗体药物偶联物是混合物，含有不同的药物偶联数量(药物/抗体比值，DAR)和偶联位点。本发明提供的偶联产品，尽管仍然是混合物，但与传统方式偶联得到的抗体药物偶联物相比，其DAR分布范围很窄。其平均DAR值接近4，接近最佳抗体药物偶联物平均DAR值(2-4)范围。此外，偶联产品极少不含有裸抗(DAR＝0)，这一组分对细胞毒杀不起作用。同时，偶联产品也不含有重度偶联产品(DAR＝8),这一组分在体内的清除速度很快，相对于低DAR的组分而言。因此，本发明提供的抗体药物偶联物产品非均一性得到很大的改善。The term "antibody drug conjugate", "immunoconjugate", consists of an antibody, a linker and a drug, and the linker is a combination of cleavable linkers or a non-cleavable linker. Antibodies are globular proteins that contain a series of amino acid sites for drug-linker conjugation. Due to their tertiary and quaternary structures, only solvent-accessible amino acids are available for coupling. In fact, high-yield couplings usually occur at the ε-amino group of a lysine residue or the sulfhydryl group of a cysteine residue. A large number of lysine side chains on the surface of the antibody protein lead to a large number of sites available for drug conjugation, resulting in a mixture of antibody-drug conjugates containing different drug-conjugated quantities (drug/antibody ratio, DAR) and conjugates. joint point. Although the conjugated product provided by the present invention is still a mixture, compared with the antibody-drug conjugate obtained by conjugating in a traditional way, its DAR distribution range is very narrow. Its average DAR value is close to 4, which is close to the average DAR value (2-4) range of the best antibody-drug conjugates. In addition, very few conjugated products do not contain naked antibody (DAR=0), which is ineffective for cytotoxicity. At the same time, the conjugated product does not contain a heavily conjugated product (DAR=8), and this component has a fast clearance rate in the body, compared with the low DAR component. Therefore, the heterogeneity of the antibody drug conjugate product provided by the present invention is greatly improved.

本发明中组成抗体药物偶联物的抗体最好保持其原有野生状态时的抗原结合能力。因此，本发明中的抗体能够，最好专一性地，与抗原结合。为了开发可用于癌症诊断与治疗的有效的细胞水平目标物，研究人员力图找寻跨膜或其他肿瘤相关多肽。这些目标物能够特异性地表达在一种或多种癌症细胞表面，而在一种或多种非癌细胞表面表达很少或不表达。通常，相对于非癌细胞表面而言,这样的肿瘤相关多肽在癌细胞表面更加过度表达。确认这样的肿瘤相关因子，可大大提高基于抗体治疗癌症的专一靶向特性。The antibody constituting the antibody-drug conjugate in the present invention preferably maintains its original antigen-binding ability in the wild state. Thus, the antibodies of the invention are capable of, preferably specifically, binding to an antigen. In order to develop effective cellular-level targets that can be used in cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated peptides. These targets can be specifically expressed on the surface of one or more cancer cells, with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells than on the surface of non-cancer cells. Identification of such tumor-associated factors could greatly improve the specific targeting properties of antibody-based therapies for cancer.

术语“缀合”、“连接”、“偶联”是指两个或更多个分子的缔合。连接也可以是遗传的(即重组融合)。在具体上下文中，所述术语包括提及连接配体(例如抗体部分)与效应分子。所述连接这种连接可以使用多种本领域公认的技术来实现，例如可通过化学或重组的方式。“化学方式”是指所述抗体部分与效应分子之间的反应，使所述两个分子之间形成共价键以形成一个分子。The terms "conjugate", "link", "couple" refer to the association of two or more molecules. Linking can also be genetic (ie recombinant fusion). In specific contexts, the term includes reference to linking a ligand (eg, an antibody moiety) to an effector molecule. The linking can be achieved using a variety of art-recognized techniques, such as chemical or recombinant means. "Chemical means" refers to the reaction between the antibody moiety and the effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

术语“细胞毒剂”用于本文时指抑制或防止细胞的功能和/或引起细胞破坏的物质。该术语意图包括放射性同位素(例如I131、I125、Y90、Re186)、化学治疗剂、和毒素(诸如细菌、真菌、植物或动物起源的酶活毒素)，或其片段。The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioisotopes (eg, I131, I125, Y90, Re186), chemotherapeutic agents, and toxins (such as enzymatic toxins of bacterial, fungal, plant or animal origin), or fragments thereof.

术语“载体”、“核酸构建体”是指能够运输与其连接的另一种核酸的核酸分子。一种类型的载体是“质粒”，其是指其中可以连接另外的DNA区段的环状双链DNA环。另一种类型的载体是病毒载体，其中额外的DNA区段可以连接到病毒基因组中。某些载体能够在它们被导入的宿主细胞中自主复制(例如，具有细菌复制起点和游离型哺乳动物载体的细菌载体)。其他载体(例如非附加型哺乳动物载体)可以在导入宿主细胞后整合到宿主细胞的基因组中，并由此与宿主基因组一起复制。此外，某些载体能够指导它们有效连接的基因的表达。这种载体在本文中被称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常以质粒的形式存在。然而，也包括其他形式的表达载体，如病毒载体(例如，复制缺陷型逆转录病毒，腺病毒和腺伴随病毒)，其起到等同的功能。The terms "vector", "nucleic acid construct" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell after introduction into the host cell and thereby replicate along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually in the form of plasmids. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, are also included.

术语“核酸分子”旨在包括DNA分子和RNA分子。核酸分子可以是单链或双链的，并且可以是cDNA。The term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single- or double-stranded, and can be cDNA.

术语“多肽”是指包含至少两个连续连接的氨基酸残基的链，对链的长度没有上限。蛋白质中的一个或多个氨基酸残基可含有修饰，例如但不限于糖基化，磷酸化或二硫键。“蛋白质”可以包含一种或多种多肽。The term "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may comprise one or more polypeptides.

术语“宿主细胞”在其中载体可以增殖并且其DNA可以表达的细胞，所述细胞可以是原核细胞或者真核细胞。该术语还包括受试宿主细胞的任何后代。应理解，并不是所有的后代都与亲本细胞相同，因为在复制过程中可能会发生突变，这类后代被包括在内。The term "host cell" is a cell in which a vector can be propagated and its DNA can be expressed, which can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that not all progeny will be identical to the parental cells as mutations may occur during replication and such progeny are included.

本发明取得了以下有益的技术效果：The present invention has obtained following beneficial technical effect:

第一、在骆驼VHH噬菌体表面展示文库技术的基础上，制备获得了经过人源化的抗HSA纳米抗体，其能够结合HSA、cyno SA，表面本发明人源化的抗HSA纳米抗体不仅保持了骆驼VHH抗体的可溶性，而且显著降低了对人体的异源免疫反应。First, on the basis of the camel VHH phage surface display library technology, a humanized anti-HSA nanobody was prepared, which can bind to HSA and cyno SA. The surface humanized anti-HSA nanobody of the present invention not only maintains Camelid VHH antibodies are soluble and significantly reduce the heterologous immune response to humans.

第二、本发明对人源化的VHH进行了序列优化，获得了亲和力高、线性良好的人源化纳米抗体。采用免疫噬菌体库筛选骆驼VHH，对其相应的人源化抗体进行序列优化，产物纳米抗体经历了骆驼体内亲和力成熟、人源化后体外序列优化等过程，确保了产物纳米抗体的适当亲和力和结构及性质稳定。结果表明，其对HSA、cyno SA的结合能力与阳性对照相当或更优，将其融合于抗CD73mFab抗体上能够显著改善抗CD73mFab抗体在体内的药物动力学、增强其半衰期。Second, the present invention optimizes the sequence of the humanized VHH to obtain a humanized nanobody with high affinity and good linearity. The immune phage library was used to screen camel VHH, and the corresponding humanized antibody sequence was optimized. The product nanobody underwent affinity maturation in vivo and in vitro sequence optimization after humanization to ensure the appropriate affinity and structure of the product nanobody. and stable in nature. The results showed that its binding ability to HSA and cyno SA was equivalent to or better than that of the positive control, and its fusion to the anti-CD73mFab antibody could significantly improve the pharmacokinetics of the anti-CD73mFab antibody in vivo and enhance its half-life.

第三、本发明的抗HSA纳米抗体具有通用性，由于其分子量小、序列结构清楚，因此对于非肠道施用途径的药物均可采用共价键(二硫键、肽键、酰胺键等)、连接子(具有双功能基团)、或非共价键(氢键、离子键、疏水作用力等)的方式连接至本发明的抗HSA纳米抗体，并进而负载到HSA上，具有广阔的应用场景。Third, the anti-HSA nanobody of the present invention has versatility. Because of its small molecular weight and clear sequence structure, covalent bonds (disulfide bonds, peptide bonds, amide bonds, etc.) can be used for drugs in the parenteral administration route. , a linker (with a bifunctional group), or a non-covalent bond (hydrogen bond, ionic bond, hydrophobic force, etc.) is connected to the anti-HSA Nanobody of the present invention, and then loaded onto HSA, which has broad Application scenarios.

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

附图说明Description of drawings

本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

图1：ELISA检测噬菌体上清与HSA和cyno SA抗原的结合Figure 1: ELISA detection of binding of phage supernatant to HSA and cyno SA antigens

横坐标为克隆号，纵坐标为OD值The abscissa is the clone number, and the ordinate is the OD value

图2：ELISA检测抗HSA嵌合抗体与HSA的结合Figure 2: ELISA to detect the binding of anti-HSA chimeric antibody to HSA

图3：ELISA检测抗HSA嵌合抗体与cyno SA的结合Figure 3: ELISA detection of binding of anti-HSA chimeric antibody to cyno SA

图4：ELISA检测抗HSA嵌合抗体与MSA的结合Figure 4: ELISA to detect the binding of anti-HSA chimeric antibody to MSA

图5：ELISA检测抗HSA人源化抗体与HSA的结合Figure 5: Detection of binding of anti-HSA humanized antibody to HSA by ELISA

图6：ELISA检测抗HSA人源化抗体与cyno SA的结合Figure 6: ELISA detection of binding of anti-HSA humanized antibody to cyno SA

图7：ELISA检测抗HSA人源化抗体与MSA的结合Figure 7: Detection of binding of anti-HSA humanized antibody to MSA by ELISA

图8：具有HSA结合结构域的受试重组蛋白在Balb/C小鼠体内代谢血药浓度的变化曲线Figure 8: The change curve of the blood drug concentration of the tested recombinant protein with HSA binding domain in the body of Balb/C mice

发明详细描述Detailed description of the invention

下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals designate the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific techniques or conditions are indicated in the examples, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, third edition, Science Press) or follow the product instructions. The reagents or instruments used, whose manufacturers are not indicated, are conventional products that can be purchased from the market, for example, they can be purchased from Illumina Corporation.

实施例1：骆驼纳米抗体免疫噬菌体库构建Example 1: Camelid Nanobody Immune Phage Library Construction

利用HSA抗原免疫骆驼，分离外周血单核细胞(PBMC)并提取总RNA进行反转录，以反转录产物为模板扩增纳米抗体重链可变区(variable domain of the heavy-chain of heavy chain antibody,VHH)并连入噬菌体展示载体，电转入大肠杆菌TG1感受态细胞，构建骆驼免疫库。Camels were immunized with HSA antigen, peripheral blood mononuclear cells (PBMC) were isolated and total RNA was extracted for reverse transcription, and the variable domain of the heavy-chain of the nanobody was amplified using the reverse transcription product as a template chain antibody, VHH) and connected to the phage display vector, electroporated into Escherichia coli TG1 competent cells, and camel immune library was constructed.

具体地，骆驼免疫两周一次，共4次。每次注射0.8mg HSA胞外区重组蛋白(购于华兰生物工程股份有限公司，产品批号：201405030)，佐以弗氏完全/不完全佐剂(Sigma，F5881,F5506)，采取皮下多点注射的方式。每次免疫后2周采集1mL血分离血清，用免疫原作为测定抗原，通过ELISA法分别测定血清中全抗体(IgG)和重链抗体(heavy chain antibody，HcAb)的滴度。当血清滴度达到建库要求后，采集100mL骆驼外周血并用分离试剂盒(天津灏洋，Cat:TBD2011CM)分离PBMC，提取PBMC的总RNA反转获得cDNA，作为后续扩增VHH片段的模板。根据相关文献和数据库中检索到驼源VHH抗体的基因，设计并合成VHH抗体库构建引物，PCR扩增出抗体可变区基因序列。随后利用核酸内切酶对载体和扩增的抗体片段进行酶切。采用T4连接酶的连接方式构建连接产物，利用电转染技术将连接产物转入到TG1菌种之中。最终构建了一个1.8×10⁸骆驼抗人HSA VHH抗体免疫库，用于特异性抗人HSA纳米抗体的筛选。为检测文库的正确率，随机选取50个克隆进行菌落PCR，结果显示插入率达到100％。Specifically, camels are immunized once every two weeks, a total of 4 times. Each injection of 0.8mg HSA extracellular region recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030), accompanied by Freund's complete/incomplete adjuvant (Sigma, F5881, F5506), was taken subcutaneously at multiple points The way of injection. Two weeks after each immunization, 1 mL of serum was collected, and immunogen was used as the assay antigen, and the titers of full antibody (IgG) and heavy chain antibody (heavy chain antibody, HcAb) in the serum were measured by ELISA method. When the serum titer reached the requirement for library construction, 100 mL of camel peripheral blood was collected and PBMCs were separated with an isolation kit (Tianjin Haoyang, Cat: TBD2011CM). The total RNA of PBMCs was extracted and reversed to obtain cDNA, which was used as a template for subsequent amplification of VHH fragments. According to the genes of camel-derived VHH antibodies retrieved from relevant literature and databases, primers were designed and synthesized for the VHH antibody library construction, and the antibody variable region gene sequences were amplified by PCR. The vector and amplified antibody fragments are then digested with endonucleases. T4 ligase was used to construct the ligation product, and the ligation product was transferred into TG1 strains by electroporation technology. Finally, an immune library of 1.8×10⁸ camel anti-human HSA VHH antibodies was constructed for the screening of specific anti-human HSA nanobodies. To test the accuracy of the library, 50 clones were randomly selected for colony PCR, and the results showed that the insertion rate reached 100%.

实施例2：抗HSA特异性纳米抗体筛选Example 2: Anti-HSA specific nanobody screening

通过固相筛选的方法对所构建的骆驼免疫库进行筛选，获得特异性噬菌体展示纳米抗体。The constructed camel immune library was screened by solid-phase screening to obtain specific phage-displayed nanobodies.

(1)原始库呈现。骆驼免疫库转接至含有氨苄青霉素和四环素的2YT培养基，培养至对数生长期，加入M13辅助噬菌体，然后加入卡那霉素，在较低温度条件下呈现过夜。第二天收取培养上清，PEG沉淀对噬菌体进行浓缩，获得高滴度的抗体库呈现产物用于后续筛选。(1) The original library is presented. The camel immune library was transferred to 2YT medium containing ampicillin and tetracycline, cultivated to the logarithmic growth phase, added M13 helper phage, and then added kanamycin, and presented overnight at a lower temperature. The culture supernatant was collected the next day, and the phages were concentrated by PEG precipitation to obtain high-titer antibody library display products for subsequent screening.

(2)筛选。采用固相法进行特异性抗体的筛选。将特异性抗原HSA包被在免疫管表面，用封闭剂分别封闭免疫管和抗体库后，将抗体库加至免疫管中孵育，然后进行反复洗涤，最后用pH2.2的酸进行洗脱，洗脱物中和至中性后与对数生长期的XL-Blue混合孵育进行感染，进一步进行噬菌体展示，回收特异性噬菌体颗粒。筛选2-3轮后进行单克隆鉴定。(2) Screening. Screening of specific antibodies was carried out by solid-phase method. Coat the specific antigen HSA on the surface of the immune tube, block the immune tube and the antibody library with a blocking agent, add the antibody library to the immune tube for incubation, then wash repeatedly, and finally elute with pH 2.2 acid, After the eluate was neutralized to neutral, it was mixed with XL-Blue in the logarithmic growth phase and incubated for infection, and further phage display was performed to recover specific phage particles. Monoclonal identification was performed after 2-3 rounds of screening.

(3)鉴定。感染回收特异性噬菌体颗粒的XL-Blue涂布平板，长出克隆后进行单克隆鉴定。将单克隆挑取后进行培养，培养至对数增长期，加入M13辅助噬菌体感染，然后入卡那霉素，在30℃培养过夜。第二天取上清，分别用包被了HSA、cyno SA和BSA的酶联板进行ELISA反应，结果如图1所示。将对HAS、cyno SA反应呈阳性，对BSA反应呈阴性的噬菌体克隆提取噬粒(含有抗体基因的噬菌体展示载体)进行测序，确定VHH抗体基因序列。通过筛选获得了4株可同时结合HSA及cyno SA重组蛋白的噬菌体展示的纳米抗体：2H1，2H2，2H4，2MG6，其测序结果如下：(3) Identification. Infect and recover the XL-Blue coated plate with specific phage particles, and identify the monoclonal after the clones grow out. The single clones were picked and cultured, cultured to the logarithmic growth phase, added M13 helper phage for infection, then added kanamycin, and cultured overnight at 30°C. The next day, the supernatant was taken, and the ELISA reaction was carried out with enzyme-linked plates coated with HSA, cyno SA and BSA respectively. The results are shown in Figure 1. Phagemids (phage display vectors containing antibody genes) were extracted from phage clones that were positive for HAS and cyno SA and negative for BSA, and sequenced to determine the VHH antibody gene sequence. Four phage-displayed nanobodies that can simultaneously bind HSA and cyno SA recombinant proteins were obtained through screening: 2H1, 2H2, 2H4, 2MG6, and the sequencing results are as follows:

克隆2H1可变区氨基酸序列如SEQ ID NO:1所示，可变区核苷酸序列如SEQ ID NO:2所示；The amino acid sequence of the variable region of clone 2H1 is shown in SEQ ID NO:1, and the nucleotide sequence of the variable region is shown in SEQ ID NO:2;

克隆2H2可变区氨基酸序列如SEQ ID NO:3所示，可变区核苷酸序列如SEQ ID NO:4所示；The amino acid sequence of the variable region of clone 2H2 is shown in SEQ ID NO:3, and the nucleotide sequence of the variable region is shown in SEQ ID NO:4;

克隆2H4可变区氨基酸序列如SEQ ID NO:5所示，可变区核苷酸序列如SEQ ID NO:6所示；The amino acid sequence of the variable region of clone 2H4 is shown in SEQ ID NO:5, and the nucleotide sequence of the variable region is shown in SEQ ID NO:6;

克隆2MG6可变区氨基酸序列如SEQ ID NO:7所示，可变区核苷酸序列如SEQ ID NO:8所示。The amino acid sequence of the variable region of clone 2MG6 is shown in SEQ ID NO:7, and the nucleotide sequence of the variable region is shown in SEQ ID NO:8.

实施例3：抗人HSA纳米抗体及对照抗体的制备Embodiment 3: Preparation of anti-human HSA nanobody and control antibody

将同靶点对照纳米抗体ALB8的进行VHH基因全合成，并克隆至入含有人Fc(hFc)编码基因的真核瞬时表达载体中，并在HEK293细胞中重组表达。细胞转染5-6天后，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得ALB8重组蛋白。其中，对照抗体ALB8氨基酸序列来源于US 2014/0228546A1，可变区氨基酸序列如SEQ ID NO:9所示，可变区核苷酸序列如SEQ ID NO:10所示。hFc氨基酸序列如SEQ ID NO:11所示，核苷酸序列如SEQ ID NO:12所示。The VHH gene of the same target control Nanobody ALB8 was fully synthesized, cloned into a eukaryotic transient expression vector containing the human Fc (hFc) coding gene, and expressed recombinantly in HEK293 cells. 5-6 days after the cells were transfected, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain the ALB8 recombinant protein. Among them, the amino acid sequence of the control antibody ALB8 is derived from US 2014/0228546A1, the amino acid sequence of the variable region is shown in SEQ ID NO:9, and the nucleotide sequence of the variable region is shown in SEQ ID NO:10. The amino acid sequence of hFc is shown in SEQ ID NO:11, and the nucleotide sequence is shown in SEQ ID NO:12.

将测序正确的2H1，2H2，2H4，2MG6VHH基因，克隆至含有人Fc(hFc)编码基因的真核表达载体上。获得序列正确的表达质粒后，转染293F细胞进行瞬时表达，细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得嵌合抗体ch2H1， ch2H2，ch2H4，ch2MG6。The correctly sequenced 2H1, 2H2, 2H4, 2MG6 VHH genes were cloned into the eukaryotic expression vector containing the human Fc (hFc) coding gene. After obtaining the expression plasmid with the correct sequence, transfect 293F cells for transient expression, 5-6 days after cell transfection, take the culture supernatant, use ProA affinity chromatography to purify the expression supernatant, and obtain the chimeric antibody ch2H1, ch2H2, ch2H4, ch2MG6.

实施例4：嵌合抗体亲和力分析Example 4: Chimeric antibody affinity analysis

利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。测定时将ch2H1,ch2H2,ch2H4,ch2MG6嵌合抗体及对照抗体ALB8，用PBS缓冲液稀释至4μg/ml，流经AHC探针(Cat:18-0015，PALL)表面，时间为120s。HSA重组蛋白(购于华兰生物工程股份有限公司，产品批号：201405030)(60nM)作为流动相，结合时间为300s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数，如表1所示。Using the Octet QKe system instrument of Fortebio Company, the antibody affinity was determined by using the capture antibody (AHC) biological probe of the anti-human antibody Fc segment to capture the Fc segment of the antibody. During the measurement, the ch2H1, ch2H2, ch2H4, ch2MG6 chimeric antibody and the control antibody ALB8 were diluted to 4 μg/ml with PBS buffer, and flowed over the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s. HSA recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030) (60nM) was used as the mobile phase, and the binding time was 300s, and the dissociation time was 300s. After the experiment was completed, the response value of the blank control was subtracted, and the software was used to perform 1:1 Langmuir binding model fitting, and the kinetic constants of antigen-antibody binding were calculated, as shown in Table 1.

表1.嵌合抗体与HSA重组蛋白的亲和力测定结果Table 1. Affinity determination results of chimeric antibody and HSA recombinant protein

样本sample	KD(M)KD(M)
Ch2H1Ch2H1	2.79E-082.79E-08
Ch2H2Ch2H2	1.62E-081.62E-08
Ch2H4Ch2H4	7.79E-097.79E-09
Ch2MG6Ch2MG6	5.79E-105.79E-10
ALB8ALB8	6.76E-096.76E-09

表1的结果表明所有嵌合抗体均能够正确装配，均具有与HSA重组蛋白特异性结合的能力。The results in Table 1 show that all chimeric antibodies can be assembled correctly and have the ability to specifically bind to HSA recombinant protein.

实施例5：ELISA检测抗HSA嵌合抗体的种属特异性分析Example 5: Species Specific Analysis of ELISA Detection of Anti-HSA Chimeric Antibodies

将HSA重组蛋白(购于华兰生物工程股份有限公司，产品批号：201405030)、食蟹猴SA重组蛋白(Cat:#CSA-C52H4，lot.#3445-9C5F1-QD,ACRO)、小鼠SA重组蛋白(Cat:#MSA-M52H8，lot.#3441a-9CBF1-QF,ACRO)，4℃包被过夜，包被浓度1μg/mL；PBS洗板3次后，加入5％BSA PBS，37℃封闭60min，PBST洗板3次；加入不同稀释倍数的ch2H1，ch2H2，ch2H4，ch2MG6，以及对照抗体ALB8(起始浓度为1μg/mL，3倍梯度依次稀释12个浓度)，37℃孵育60min，PBST洗板4次；加入1:5000稀释的HRP-anti-human Fc(Cat：109-035-098，Jackson Immuno Research)，37℃孵育45min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm，计算EC50值。结果如图2-4所示。HSA recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030), cynomolgus monkey SA recombinant protein (Cat:#CSA-C52H4, lot.#3445-9C5F1-QD, ACRO), mouse SA Recombinant protein (Cat:#MSA-M52H8, lot.#3441a-9CBF1-QF, ACRO), coated overnight at 4°C, coating concentration 1μg/mL; after washing the plate with PBS 3 times, add 5% BSA PBS, 37°C Block for 60 min, wash the plate 3 times with PBST; add different dilutions of ch2H1, ch2H2, ch2H4, ch2MG6, and control antibody ALB8 (initial concentration is 1 μg/mL, 3-fold serial dilution of 12 concentrations), incubate at 37°C for 60 min, Wash the plate 4 times with PBST; add 1:5000 diluted HRP-anti-human Fc (Cat: 109-035-098, Jackson Immuno Research), incubate at 37°C for 45 min, wash the plate 4 times with PBST; add TMB substrate for color development, After incubating at 37°C for 10 minutes, add 2M HCl to terminate the reaction; take 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at a wavelength of 450nm, and calculate the EC50 value. The results are shown in Figure 2-4.

图2-3表明ch2H1，ch2H2，ch2H4，ch2MG6及对照抗体均可以特异性的与人、食蟹猴SA重组蛋白结合，其对应的半数有效结合浓度EC50值如表2所示。表2、图4表明Ch2H1、Ch2MG6和对照抗体ALB8还可与小鼠SA重组蛋白结合。Figure 2-3 shows that ch2H1, ch2H2, ch2H4, ch2MG6 and the control antibody can specifically bind to human and cynomolgus monkey SA recombinant proteins, and the corresponding half maximal effective concentration EC50 values are shown in Table 2. Table 2 and Figure 4 show that Ch2H1, Ch2MG6 and the control antibody ALB8 can also bind to mouse SA recombinant protein.

表2.ELISA检测抗HSA嵌合抗体对不同种属血清白蛋白结合的EC50Table 2. ELISA detection of anti-HSA chimeric antibody binding to serum albumin of different species EC50

实施例6：抗HSA纳米抗体的人源化及重组表达分析Example 6: Humanization and recombinant expression analysis of anti-HSA nanobody

6.1驼源纳米抗体2MG6的人源化6.1 Humanization of camel-derived nanobody 2MG6

1、CDR移植1. CDR transplantation

对驼源抗体2MG6的VHH序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)。随后根据同源性比对结果，选择最近似人源抗体template VH3(3-07)为基础模板，结合全序列blast结果，进行CDR移植，实现了2MG6可变区(VHH)在Framework区的人源化。2MG6抗体CDR移植(CDR Grafted)的人源化后氨基酸序列SEQ ID NO:13所示；核苷酸序列如SEQ ID NO:14所示。The VHH sequence of the camel-derived antibody 2MG6 was comprehensively analyzed to determine the complementarity determinant (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conservative three-dimensional conformation of the antibody. Then, according to the results of homology comparison, the most similar human antibody template VH3 (3-07) was selected as the basic template, and combined with the results of the full sequence blast, CDR transplantation was carried out to realize the humanization of the 2MG6 variable region (VHH) in the Framework region. source. The humanized amino acid sequence of the 2MG6 antibody CDR grafted (CDR Grafted) is shown in SEQ ID NO:13; the nucleotide sequence is shown in SEQ ID NO:14.

2、突变体设计2. Mutant design

对hz2MG6序列进行突变设计，突变位点如表3所示。Mutation design was carried out on the hz2MG6 sequence, and the mutation sites are shown in Table 3.

表3.Hz2MG6突变序列设计信息Table 3. Hz2MG6 mutant sequence design information

注：A14P表示将SEQ ID NO:13所示Hz2MG6的第14位氨基酸A突变为P，依次类推。Note: A14P means that the 14th amino acid A of Hz2MG6 shown in SEQ ID NO: 13 is mutated to P, and so on.

3、人源化抗体hz2MG6的重组表达及亲和力检测3. Recombinant expression and affinity detection of humanized antibody hz2MG6

将人源化设计的hz2MG6抗体可变区序列进行全合成，克隆至含有人Fc(hFc)编码基因的真核表达载体上。获得序列正确的表达质粒后，并根据突变设计，在表达质粒上进行定点突变，转入大肠杆菌扩增，获得hz2MG6突变表达质粒；转入HEK293细胞中重组表达，细胞转染5-6天后，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得的人源化抗体.及其突变体重组蛋白。The variable region sequence of the humanized designed hz2MG6 antibody was fully synthesized and cloned into a eukaryotic expression vector containing the gene encoding human Fc (hFc). After obtaining the expression plasmid with the correct sequence, and according to the mutation design, perform site-directed mutation on the expression plasmid, transfer it into Escherichia coli for amplification, and obtain the hz2MG6 mutant expression plasmid; transfer it into HEK293 cells for recombinant expression, and after 5-6 days of cell transfection, Take the culture supernatant, use the ProA affinity chromatography column to purify the expression supernatant, and obtain the humanized antibody and its mutant recombinant protein.

利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。测定时将hz2MG6抗体及嵌合抗体用PBS缓冲液稀释至4ug/mL，流经AHC探针(Cat:18-0015，PALL)表面，时间为300S。HSA重组蛋白(购于华兰生物工程股份有限公司，产品批号：201405030)作为流动相，HSA重组蛋白浓度为60nM。结合时间为300s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。Using the Octet QKe system instrument of Fortebio Company, the antibody affinity was determined by using the capture antibody (AHC) biological probe of the anti-human antibody Fc segment to capture the Fc segment of the antibody. During the measurement, hz2MG6 antibody and chimeric antibody were diluted to 4ug/mL with PBS buffer, and flowed on the surface of AHC probe (Cat: 18-0015, PALL) for 300 seconds. HSA recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030) was used as the mobile phase, and the concentration of HSA recombinant protein was 60 nM. The binding time is 300s and the dissociation time is 300s. After the experiment was completed, the response value of the blank control was deducted, and the 1:1 Langmuir binding model was fitted by software to calculate the kinetic constant of antigen-antibody binding.

通过ForteBio测定hz2MG6及其突变体与HSA重组蛋白的亲和力，KD结果如表4所示，人源化后的2MG亲和力从10^-8M至10^-10M，不同亲和力的突变体可为与不同重组蛋白组成融合蛋白提供更多选择，从而更有针对性调节融合蛋白的代谢特征。The affinity of hz2MG6 and its mutants to HSA recombinant protein was determined by ForteBio. The KD results are shown in Table 4. The affinity of 2MG after humanization ranges from 10^-8 M to 10^-10 M. Mutants with different affinity can be different from Composition of recombinant proteins into fusion proteins provides more options to regulate the metabolic characteristics of fusion proteins in a more targeted manner.

表4.hz2MG6突变体亲和力测定结果Table 4. Results of affinity determination of hz2MG6 mutants

名称name	KD值(M)KD value (M)	名称name	KD值(M)KD value (M)
hz2MG6hz2MG6	1.72E-091.72E-09	hz2MG6m11hz2MG6m11	7.36E-107.36E-10
hz2MG6m1hz2MG6m1	1.56E-091.56E-09	hz2MG6m12hz2MG6m12	6.83E-106.83E-10
hz2MG6m2hz2MG6m2	2.41E-102.41E-10	hz2MG6m13hz2MG6m13	5.13E-085.13E-08
hz2MG6m3hz2MG6m3	1.64E-091.64E-09	hz2MG6m14hz2MG6m14	2.54E-082.54E-08
hz2MG6m4hz2MG6m4	2.49E-102.49E-10	hz2MG6m15hz2MG6m15	6.63E-086.63E-08
hz2MG6m5hz2MG6m5	4.51E-094.51E-09	hz2MG6m16hz2MG6m16	3.61E-083.61E-08
hz2MG6m6hz2MG6m6	2.91E-092.91E-09	hz2MG6m17hz2MG6m17	4.40E-104.40E-10
hz2MG6m7hz2MG6m7	5.12E-075.12E-07	hz2MG6m18hz2MG6m18	5.72E-095.72E-09
hz2MG6m8hz2MG6m8	5.76E-095.76E-09	hz2MG6m19hz2MG6m19	9.85E-099.85E-09
hz2MG6m9hz2MG6m9	4.71E-074.71E-07	hz2MG6m20hz2MG6m20	7.10E-097.10E-09

hz2MG6m10hz2MG6m10

5.63E-095.63E-09

hz2MG6m21hz2MG6m21

3.42E-093.42E-09

6.2驼源纳米抗体2H4的人源化6.2 Humanization of camel-derived nanobody 2H4

1、CDR移植1. CDR transplantation

首先对驼源抗体2H4的VHH序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)。随后根据同源性比对结果，选择最相似人源抗体template VH3(3-07)为基础模板，结合全序列blast结果，进行CDR移植，实现了2H4可变区(VHH)在Framework区的人源化。重新名为hzH4；hzH4抗体CDR移植(CDR Grafted)的人源化可变区氨基酸序列如SEQ ID NO:36所示；人源化可变区核苷酸序列如SEQ ID NO:37所示。Firstly, the VHH sequence of the camel antibody 2H4 was comprehensively analyzed to determine the complementarity determinant (CDR) region where the antibody binds to the antigen and the framework region (framework) that supports the conservative three-dimensional conformation of the antibody. Then, according to the homology comparison results, the most similar human antibody template VH3 (3-07) was selected as the basic template, combined with the results of the full sequence blast, CDR transplantation was performed, and the human 2H4 variable region (VHH) in the Framework region was realized. source. Renamed as hzH4; the amino acid sequence of the humanized variable region of hzH4 antibody CDR grafted (CDR Grafted) is shown in SEQ ID NO:36; the nucleotide sequence of the humanized variable region is shown in SEQ ID NO:37.

2、突变体设计2. Mutant design

对hzH4序列进行突变设计，突变位点如表5所示。Mutation design was carried out on the hzH4 sequence, and the mutation sites are shown in Table 5.

表5.hzH4人源化突变序列设计Table 5. hzH4 humanized mutant sequence design

3、人源化抗体hzH4的重组表达及亲和力检测3. Recombinant expression and affinity detection of humanized antibody hzH4

将人源化设计的hzH4抗体可变区序列进行全合成，克隆至含有人Fc(hFc)编码基因的真核表达载体上。获得序列正确的表达质粒后，并根据突变设计，在表达质粒上进行定点突变，转入大肠杆菌扩增，获得hzH4突变表达质粒；转入HEK293细胞中重组表达，细胞转染5-6天后，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得的人源化抗体及突变体重组蛋白。The variable region sequence of the humanized designed hzH4 antibody was fully synthesized and cloned into a eukaryotic expression vector containing the gene encoding human Fc (hFc). After obtaining the expression plasmid with the correct sequence, and according to the mutation design, perform site-directed mutation on the expression plasmid, transfer it into Escherichia coli for amplification, and obtain the hzH4 mutant expression plasmid; transfer it into HEK293 cells for recombinant expression, and after 5-6 days of cell transfection, Take the culture supernatant, use ProA affinity chromatography column to purify the expression supernatant, and obtain the humanized antibody and mutant recombinant protein.

利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。测定时将hzH4抗体及突变体用PBS缓冲液稀释至4ug/mL，流经AHC探针(Cat:18-0015，PALL)表面，时间为300S。HSA重组蛋白(购于华兰生物工程股份有限公司，产品批号：201405030)作为流动相，HSA重组蛋白浓度为60nM。结合时间为300s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。Using the Octet QKe system instrument of Fortebio Company, the antibody affinity was determined by using the capture antibody (AHC) biological probe of the anti-human antibody Fc segment to capture the Fc segment of the antibody. During the measurement, the hzH4 antibody and mutants were diluted to 4ug/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat: 18-0015, PALL) for 300 seconds. HSA recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030) was used as the mobile phase, and the concentration of HSA recombinant protein was 60 nM. The binding time is 300s and the dissociation time is 300s. After the experiment was completed, the response value of the blank control was deducted, and the 1:1 Langmuir binding model was fitted by software to calculate the kinetic constant of antigen-antibody binding.

通过ForteBio测定hzH4及其突变体与HSA重组蛋白的亲和力，结果如表6所示，人源化后的H4亲和力从10^-8M至10^-9M，不同亲和力的突变体可为与不同重组蛋白组成融合蛋白提供更多选择，从而更有针对性调节融合蛋白的代谢特征。The affinity of hzH4 and its mutants to HSA recombinant protein was determined by ForteBio. The results are shown in Table 6. The affinity of humanized H4 ranges from 10^-8 M to 10^-9 M. Mutants with different affinity can be combined with different recombinant proteins. The protein composition fusion protein provides more choices, so that the metabolic characteristics of the fusion protein can be adjusted more specifically.

表6.HzH4突变体亲和力测定结果Table 6. HzH4 mutant affinity determination results

名称name	KD值(M)KD value (M)
hzH4wxya	8.61E-098.61E-09
hzH4-2hzH4-2	9.68E-099.68E-09
hzH4-3hzH4-3	7.32E-087.32E-08
hzH4-4hzH4-4	3.74E-083.74E-08

实施例7：ELISA检测抗HSA人源化抗体与血清白蛋白结合的种属交叉反应Example 7: ELISA detection of species cross-reactivity of anti-HSA humanized antibody binding to serum albumin

将HSA重组蛋白(购于华兰生物工程股份有限公司，产品批号：201405030)、食蟹猴SA重组蛋白(Cat:#CSA-C52H4，lot.#3445-9C5F1-QD,ACRO)、小鼠SA重组蛋白(Cat:#MSA-M52H8，lot.#3441a-9CBF1-QF,ACRO)，4℃包被过夜，包被浓度1μg/mL；PBS洗板3次后，加入5％BSA的PBS，37℃封闭60min，PBST洗板3次；加入不同稀释倍数的hz2MG6，hzH4，和对照抗体ALB8(起始浓度为1μg/mL，3倍梯度依次稀释12个浓度)，37℃孵育60min，PBST洗板4次；加入1:5000稀释的HRP-anti-human Fc(Cat：109-035-098，Jackson Immuno Research)，37℃孵育45min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。HSA recombinant protein (purchased from Hualan Bioengineering Co., Ltd., product batch number: 201405030), cynomolgus monkey SA recombinant protein (Cat:#CSA-C52H4, lot.#3445-9C5F1-QD, ACRO), mouse SA Recombinant protein (Cat:#MSA-M52H8, lot.#3441a-9CBF1-QF, ACRO), coated overnight at 4°C, with a coating concentration of 1 μg/mL; after washing the plate with PBS three times, add 5% BSA in PBS, 37 Block at ℃ for 60 min, wash the plate three times with PBST; add hz2MG6, hzH4, and control antibody ALB8 at different dilutions (initial concentration is 1 μg/mL, 3-fold serially diluted to 12 concentrations), incubate at 37 °C for 60 min, and wash the plate with PBST 4 times; add 1:5000 diluted HRP-anti-human Fc (Cat: 109-035-098, Jackson Immuno Research), incubate at 37°C for 45min, wash the plate 4 times with PBST; add TMB substrate for color development, and incubate at 37°C After 10 minutes, add 2M HCl to terminate the reaction; take 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the orifice plate at a wavelength of 450nm.

实验结果表明hz2MG6及对照抗体均可以特异性的与人、食蟹猴SA及小鼠SA重组蛋白结合，结果如图5-7所示，计算EC50值如表7所示。图5-7和表7的结果表明hzH4与人及食蟹猴SA特异性结合，但与小鼠SA重组蛋白则无结合活性。The experimental results showed that both hz2MG6 and the control antibody could specifically bind to human, cynomolgus monkey SA and mouse SA recombinant proteins, the results are shown in Figure 5-7, and the calculated EC50 values are shown in Table 7. The results in Figures 5-7 and Table 7 show that hzH4 specifically binds to human and cynomolgus monkey SA, but has no binding activity to mouse SA recombinant protein.

表7.ELISA检测抗HSA人源化抗体对不同种属血清白蛋白结合的EC50Table 7. EC50 of ELISA detection of anti-HSA humanized antibody binding to serum albumin of different species

实施例8：HSA抗体延长抗体药物在小鼠体内的半衰期Example 8: HSA antibody prolongs the half-life of antibody drug in mice

选择与鼠交叉的抗HSA纳米抗体hz2MG6和ALB8，分别构建抗CD73抗体TJD5-mFab(专利号：CN109476755A)，TJD5-mFab-hz2MG6(SEQ ID NO.41)及TJD5-mFab-ALB8(SEQ ID NO.42)的重组表达载体，并在HEK293细胞中进行瞬时表达，纯化制备重组蛋白样品。通过对上述3个重组蛋白在Balb/C小鼠体内进行药代动力学检测，证实抗HSA纳米抗体可以延长重组蛋白在动物体内的半衰期。Select anti-HSA nanobodies hz2MG6 and ALB8 crossed with mice to construct anti-CD73 antibodies TJD5-mFab (Patent No.: CN109476755A), TJD5-mFab-hz2MG6 (SEQ ID NO.41) and TJD5-mFab-ALB8 (SEQ ID NO. .42), and perform transient expression in HEK293 cells, and purify and prepare recombinant protein samples. Through the pharmacokinetic detection of the above three recombinant proteins in Balb/C mice, it was confirmed that the anti-HSA nanobody can prolong the half-life of the recombinant proteins in animals.

实验设计：雌性6周龄Balb/c小鼠(维通利华)尾静脉给予250ug/只的受试样品，6只小鼠分为两个亚组，3只/亚组，交叉采血，于0.5h、1h、2h、3h、4h、5h、6h、8h、24h、30h、48h、72h断尾采血，收集血清并于-20℃保存。Experimental design: Female 6-week-old Balb/c mice (Victoria) were given 250ug/test sample through the tail vein, and the 6 mice were divided into two subgroups, 3 mice/subgroup, and blood was collected crossover. Blood was collected at 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 24h, 30h, 48h, and 72h, and serum was collected and stored at -20°C.

采血全部完成后，包被human CD73-His(序列号：NP_002517.1)于96孔酶联板， 0.5ug/ml，100ul/孔，4℃过夜，PBS洗板3次后，加入5％BSA PBS，37℃封闭60min，PBST洗板3次；加入待检测血清样本(10000，20000倍稀释)，设置TJD5-Fab和标准曲线孔(起始浓度为0.05μg/mL，2倍连续稀释，12个梯度)，37℃孵育60min，PBST洗板4次；加入1:5000稀释的HRP-羊抗鼠Fab(Cat:A2304-1ML，lot#019M4758V，Sigma)，37℃孵育40min，PBST洗板4次；加入TMB底物(Cat：ME142，北京泰天河生物技术有限公司)显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。以标准抗体的浓度为Y轴，OD值为X轴，绘制时间——抗体浓度曲线图，进行线性拟合，并根据公式T_1/2＝|0.693/k|，计算药物半衰期T_1/2。各蛋白代谢曲线图如图8所示，拟合半衰期结果如表8所示。结果显示，当前条件下，融合了HSA纳米抗体的TJD5-mFab相比于未融合纳米抗体的TJD5-mFab同时间点的血药浓度更高，代谢T_1/2明显延长，表明抗HSA纳米抗体具有延长与之融合或偶联的分子半衰期的作用。After all the blood collection is completed, coat human CD73-His (serial number: NP_002517.1) on a 96-well enzyme-linked plate, 0.5ug/ml, 100ul/well, overnight at 4°C, wash theplate 3 times with PBS, and then add 5% BSA PBS, block at 37°C for 60 min, wash the plate three times with PBST; add serum samples to be tested (10000, 20000-fold dilution), set TJD5-Fab and standard curve wells (initial concentration is 0.05 μg/mL, 2-fold serial dilution, 12 gradient), incubate at 37°C for 60min, wash the plate 4 times with PBST; add 1:5000 diluted HRP-goat anti-mouse Fab (Cat:A2304-1ML, lot#019M4758V, Sigma), incubate at 37°C for 40min, wash the plate with PBST for 4 Second; add TMB substrate (Cat: ME142, Beijing Taitianhe Biotechnology Co., Ltd.) to develop color, after incubating at 37°C for 10 minutes, add 2M HCl to terminate the reaction; take 630nm as the reference wavelength, read and record the wavelength in the well plate at 450nm Absorbance A450nm-630nm. Take the standard antibody concentration as the Y axis and the OD value as the X axis, draw the time-antibody concentration curve, perform linear fitting, and calculate the drug half-life T_1/2 according to the formula T_1/2 =|0.693/k| . The metabolic curves of each protein are shown in Figure 8, and the results of the fitted half-life are shown in Table 8. The results showed that under the current conditions, TJD5-mFab fused with HSA Nanobody had a higher plasma drug concentration at the same time point than TJD5-mFab without fused Nanobody, and the metabolic T_1/2 was significantly prolonged, indicating that anti-HSA Nanobody It has the effect of prolonging the half-life of the molecule fused or coupled with it.

表8.受试重组蛋白在Balb/C小鼠体内代谢的半衰期Table 8. Metabolism half-life of tested recombinant proteins in Balb/C mice

组别group	半衰期(hrs)Half-life (hrs)
TJD5-mFab(n＝3)TJD5-mFab (n=3)	T1/2＝1.5T1/2=1.5
TJD5-mFab-hz2MG6(n＝3)TJD5-mFab-hz2MG6 (n=3)	T1/2＝23.6T1/2=23.6
TJD5-mFab-ALB8(n＝3)TJD5-mFab-ALB8 (n=3)	T1/2＝15.8T1/2=15.8

尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。In the description of this specification, references to the terms "one embodiment," "some embodiments," "exemplary embodiments," "example," "specific examples," or "some examples" are intended to mean that the implementation A specific feature, structure, material, or characteristic described by an embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Claims

一种具有白蛋白结合功能的分子，其特征在于，包括至少一个白蛋白结合结构域，所述白蛋白结合域包括互补决定区(CDRs)1、2和3，所述CDRs 1、2和3具有所选VHH或其突变体的CDRs1、2和3所示的氨基酸序列，其中，所述突变体是在所述所选VHH的CDRs1、2和3的基础上进行一个或几个氨基酸突变获得的，优选所述一个或几个氨基酸突变为一个、两个、三个、四个或五个氨基酸的替换突变，A molecule with albumin binding function, characterized in that it comprises at least one albumin binding domain, said albumin binding domain comprising complementarity determining regions (CDRs) 1, 2 and 3, said CDRs 1, 2 and 3 Have the amino acid sequence shown in CDRs1, 2 and 3 of the selected VHH or its mutant, wherein the mutant is obtained by performing one or several amino acid mutations on the basis of the CDRs1, 2 and 3 of the selected VHH Yes, preferably the one or several amino acid mutations are one, two, three, four or five amino acid substitution mutations,
其中，所述所选VHH的CDRs1、2和3的氨基酸序列为下列之一：Wherein, the amino acid sequence of CDRs1, 2 and 3 of the selected VHH is one of the following:
(1)所述所选VHH的CDRs1、2和3的氨基酸序列分别如SEQ ID NO.43、44和45所示；(1) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID NO.43, 44 and 45 respectively;
(2)所述所选VHH的CDRs1、2和3的氨基酸序列分别如SEQ ID NO.46、47和48所示；(2) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID NO.46, 47 and 48, respectively;
(3)所述所选VHH的CDRs1、2和3的氨基酸序列分别如SEQ ID NO.43、49和50所示；(3) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID NO.43, 49 and 50 respectively;
(4)所述所选VHH的CDRs1、2和3的氨基酸序列分别如SEQ ID NO.51、52和53所示。(4) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID NO.51, 52 and 53 respectively.
如权利要求1所述具有白蛋白结合功能的分子，其特征在于，所述所选VHH的所述突变体的CDRs1、2和3的氨基酸序列为下列之一：The molecule with albumin binding function according to claim 1, wherein the amino acid sequence of CDRs1, 2 and 3 of the mutant of the selected VHH is one of the following:
(1)所述所选VHH的所述突变体的CDR1的氨基酸序列如SEQ ID NO.51所示，CDR2的氨基酸序列如GIYTVGGSTFYTDX₁₀VKG所示，CDR3的氨基酸序列如GX₁TDTAX₂LX₃X₄PX₅SFGY所示；(1) The amino acid sequence of CDR1 of the mutant of the selected VHH is shown in SEQ ID NO.51, the amino acid sequence of CDR2 is shown in GIYTVGGSTFYTDX₁₀ VKG, and the amino acid sequence of CDR3 is shown in GX₁ TDTAX₂ LX₃ X₄ PX₅ SFGY shown;
(2)所述所选VHH的所述突变体的CDR1的氨基酸序列如SEQ ID NO.71所示，CDR2的氨基酸序列如AIYTX₆SGLSGTTYYADX₇VKG所示，CDR3的氨基酸序列如DWKYX₈STRCGLEVEYDX₉所示，(2) The amino acid sequence of CDR1 of the mutant of the selected VHH is shown in SEQ ID NO.71, the amino acid sequence of CDR2 is shown in AIYTX₆ SGLSGTTYYADX₇ VKG, and the amino acid sequence of CDR3 is shown in DWKYX₈ STRCGLEVEYDX₉ Show,
其中，in,
X₁为R、E或A；_X1 is R, E or A;
X₂为K、E或A；_X2 is K, E or A;
X₃为R、E或A；_X3 is R, E or A;
X₄为D或E；X₄ is D or E;
X₅为R、E或A；X₅ is R, E or A;
X₆为D或S；X₆ is D or S;
X₇为S或T；X₇ is S or T;
X₈为D或E；X₈ is D or E;
X₉为S或A；X₉ is S or A;
X₁₀为S或T。X₁₀ is S or T.
如权利要求1所述具有白蛋白结合功能的分子，其特征在于，所述所选VHH或其所述突变体包括重链可变区，其中，The molecule with albumin binding function according to claim 1, wherein said selected VHH or said mutant thereof comprises a heavy chain variable region, wherein,
(1)所述重链可变区包含SEQ ID NO:1所示的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；(1) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 1 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or 99% identical sequences;
(2)所述重链可变区包含SEQ ID NO:3所示的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；(2) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or 99% identical sequences;
(3)重链可变区包含SEQ ID NO:5所示的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；(3) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence;
(4)重链可变区包含SEQ ID NO:7所示的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。(4) The heavy chain variable region comprises the sequence shown in SEQ ID NO: 7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence.
如权利要求3所述具有白蛋白结合功能的分子，其特征在于，所述突变体的所述重链可变区的氨基酸序列是在SEQ ID NO:5所示VHH的基础上进行选自以下任一组合的突变：The molecule with albumin binding function as claimed in claim 3, wherein the amino acid sequence of the heavy chain variable region of the mutant is selected from the following on the basis of VHH shown in SEQ ID NO:5 Any combination of mutations:
V27F+S28T，V27F+S28T,
D54S+S66T，D54S+S66T,
D54E+S66A。D54E+S66A.
如权利要求4所述具有白蛋白结合功能的分子，其特征在于，所述突变体的所述重链可变区的氨基酸序列如SEQ ID NO:36、38、39或40所示。The molecule with albumin binding function according to claim 4, wherein the amino acid sequence of the heavy chain variable region of the mutant is as shown in SEQ ID NO: 36, 38, 39 or 40.
如权利要求3所述具有白蛋白结合功能的分子，其特征在于，所述突变体的所述重链可变区的氨基酸序列是在SEQ ID NO:7所示VHH的基础上进行选自以下任一组合的突变：The molecule with albumin binding function as claimed in claim 3, wherein the amino acid sequence of the heavy chain variable region of the mutant is selected from the following on the basis of VHH shown in SEQ ID NO:7 Any combination of mutations:
A14P，A14P,
A14P+S27F，A14P+S27F,
A14P+S26T，A14P+S26T,
A14P+S27F+S62T，A14P+S27F+S62T,
A14P+S27F+S62T+R99E，A14P+S27F+S62T+R99E,
A14P+S27F+S62T+R99A，A14P+S27F+S62T+R99A,
A14P+S27F+S62T+K104E，A14P+S27F+S62T+K104E,
A14P+S27F+S62T+K104A，A14P+S27F+S62T+K104A,
A14P+S27F+S62T+R106E，A14P+S27F+S62T+R106E,
A14P+S27F+S62T+R106A，A14P+S27F+S62T+R106A,
A14P+S27F+S62T+R109E，A14P+S27F+S62T+R109E,
A14P+S27F+S62T+R109A，A14P+S27F+S62T+R109A,
A14P+S27F+S62T+R106A+R99E，A14P+S27F+S62T+R106A+R99E,
A14P+S27F+S62T+R106A+R99A，A14P+S27F+S62T+R106A+R99A,
A14P+S27F+S62T+K104E+R99E，A14P+S27F+S62T+K104E+R99E,
A14P+S27F+S62T+K104E+R99A，A14P+S27F+S62T+K104E+R99A,
A14P+S27F+S62T+D107E，A14P+S27F+S62T+D107E,
A14P+S27F+S62T+R109E+R99E，A14P+S27F+S62T+R109E+R99E,
A14P+S27F+S62T+R109E+K104A，A14P+S27F+S62T+R109E+K104A,
A14P+S27F+S62T+R109E+R106A，A14P+S27F+S62T+R109E+R106A,
A14P+S27F+S62T+R109E+R99A。A14P+S27F+S62T+R109E+R99A.
如权利要求6所述具有白蛋白结合功能的分子，其特征在于，所述突变体的所述重链可变区的氨基酸序列如SEQ ID NO:13、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或35所示。The molecule with albumin binding function according to claim 6, wherein the amino acid sequence of the heavy chain variable region of the mutant is as SEQ ID NO: 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35.
如权利要求1所述具有白蛋白结合功能的分子，其特征在于，所述白蛋白结合结构域能够特异性结合人血清白蛋白(HSA)和食蟹猴血清白蛋白(cyno SA)，而对牛血清白蛋白(BSA)无特异性结合；且所述白蛋白结合结构域是以白蛋白为饵蛋白筛选纳米抗体库获得的纳米抗体或其功能衍生物。The molecule with albumin binding function as claimed in claim 1, is characterized in that, described albumin binding domain can specifically bind human serum albumin (HSA) and cynomolgus serum albumin (cyno SA), and bovine Serum albumin (BSA) has no specific binding; and the albumin binding domain is a nanobody or a functional derivative thereof obtained by screening a nanobody library with albumin as a bait protein.
如权利要求1所述具有白蛋白结合功能的分子，其特征在于，所述白蛋白结合结构域还能够特异性结合鼠血清白蛋白(MSA)。The molecule with albumin binding function according to claim 1, characterized in that the albumin binding domain can also specifically bind mouse serum albumin (MSA).
如权利要求1-9中任一项所述具有白蛋白结合功能的分子，其特征在于，包括两个或多个白蛋白结合结构域，所述两个或多个白蛋白结合结构域具有相同或不同的氨基酸序列。The molecule with albumin binding function according to any one of claims 1-9, characterized in that it comprises two or more albumin binding domains, and the two or more albumin binding domains have the same or different amino acid sequences.
如权利要求1-10中任一项所述具有白蛋白结合功能的分子，其特征在于，还包括至少一种具有生物活性的功能域或功能片段，所述具有生物活性的功能域或功能片段包括载体蛋白、活性肽、标签肽或伴侣蛋白组件，其与所述白蛋白结合结构域通过共价键、非共价键或连接子连接；The molecule with albumin binding function according to any one of claims 1-10, further comprising at least one functional domain or functional fragment with biological activity, said functional domain or functional fragment with biological activity comprising a carrier protein, an active peptide, a tag peptide or a chaperone module linked to the albumin binding domain by a covalent bond, a non-covalent bond or a linker;
优选的，所述载体蛋白包括BSA、OVA、HSA、KLH和Fc；所述活性肽包括线性肽或环形肽，且具有酶活性、细胞毒活性和抗原结合活性；所述标签肽包括纯化标签肽和示踪标签肽；所述伴侣蛋白包括HSP60、HSP70和HSP90。Preferably, the carrier protein includes BSA, OVA, HSA, KLH and Fc; the active peptide includes a linear peptide or a cyclic peptide, and has enzyme activity, cytotoxic activity and antigen binding activity; the tag peptide includes a purified tag peptide and tracer tag peptides; the chaperones include HSP60, HSP70 and HSP90.
如权利要求11所述具有白蛋白结合功能的分子，其特征在于，所述具有生物活性的功能域或功能片段包括多肽类、合成小分子、天然活性分子，并且所述白蛋白结合功能的分子以单体、同源多聚体、异源多聚体的形式存在。The molecule with albumin binding function according to claim 11, characterized in that, the functional domain or functional fragment with biological activity includes polypeptides, synthetic small molecules, natural active molecules, and the molecule with albumin binding function It exists in the form of monomer, homopolymer and heteromultimer.
如权利要求11或12所述具有白蛋白结合功能的分子，其特征在于，所述具有白蛋白结合功能的分子为纳米抗体、重链抗体、VHH-Fc融合蛋白、单价抗体、双价抗体、多价抗体、单特异性抗体、双特异性抗体、多特异性抗体、嵌合抗体、免疫偶联物。The molecule with albumin binding function according to claim 11 or 12, characterized in that, the molecule with albumin binding function is nanobody, heavy chain antibody, VHH-Fc fusion protein, monovalent antibody, bivalent antibody, Multivalent antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, immunoconjugates.
一种白蛋白结合结构域的制备方法，包括：A method for preparing an albumin-binding domain, comprising:
(1)利用HSA免疫能产生天然重链抗体的动物；(1) Using HSA to immunize animals that can produce natural heavy chain antibodies;
(2)分离PBMC扩增重链可变区编码核酸，构建噬菌体重链抗体库；(2) isolate PBMC to amplify the nucleic acid encoding the heavy chain variable region, and construct a phage heavy chain antibody library;
(3)利用HSA包被固相载体进行亲和淘选，去除未结合的噬菌体；(3) Use HSA to coat the solid phase carrier for affinity panning to remove unbound phage;
(4)从固相上将结合的噬菌体洗脱、扩增；(4) eluting and amplifying the bound phage from the solid phase;
(5)重复步骤(3)-(4)二至四次；(5) Repeat steps (3)-(4) two to four times;
(6)对特异性结合的噬菌体进行噬菌粒救援，进行噬菌体ELISA鉴定；(6) Perform phagemid rescue on specifically bound phages, and perform phage ELISA identification;
(7)对噬菌体ELISA鉴定阳性的噬菌体克隆进行测序获得白蛋白结合结构域的编码核酸。(7) Sequencing the positive phage clones identified by phage ELISA to obtain the encoding nucleic acid of the albumin binding domain.
根据权利要求14所述的方法，其特征在于，所述方法进一步包括：The method according to claim 14, characterized in that the method further comprises:
(8)对白蛋白结合结构域进行人源化改造；(8) Humanize the albumin binding domain;
(9)对人源化改造的白蛋白结合结构域进行序列优化。(9) Sequence optimization of the humanized transformed albumin binding domain.
一种组合物，其特征在于，包含权利要求1-13中任一项所述的具有白蛋白结合功能的分子；以及A composition, characterized in that it comprises the molecule with albumin binding function according to any one of claims 1-13; and
可选的药学上可接受的辅料。Optional pharmaceutically acceptable excipients.
一种多核苷酸，其特征在于，编码权利要求1-13中任一项所述的具有白蛋白结合功能的分子。A polynucleotide, characterized in that it encodes the molecule with albumin binding function according to any one of claims 1-13.
一种核酸构建体，其特征在于，包含权利要求17所述的多核苷酸。A nucleic acid construct, characterized in that it comprises the polynucleotide according to claim 17.
宿主细胞，其特征在于，包含权利要求17所述的多核苷酸或权利要求18所述的核酸构建体。A host cell comprising the polynucleotide of claim 17 or the nucleic acid construct of claim 18.
一种制备具有白蛋白结合功能的分子的方法，其特征在于，包括：A method for preparing a molecule with albumin binding function, characterized in that it comprises:
(1)在适合表达重组外源蛋白的条件下培养权利要求19所述的宿主细胞，(1) cultivating the host cell according to claim 19 under conditions suitable for expressing the recombinant foreign protein,
(2)可选的，从细胞培养物中分离纯化具有白蛋白结合功能的分子。(2) Optionally, isolate and purify molecules with albumin binding function from cell culture.
权利要求1-13中任一项所述的具有白蛋白结合功能的分子中的白蛋白结合域。The albumin binding domain in the molecule having albumin binding function according to any one of claims 1-13.
权利要求1-13中任一项所述的具有白蛋白结合功能的分子或权利要求21所述白蛋白结合域在制备药物和/或延长药物半衰期中的应用。Use of the molecule with albumin-binding function according to any one of claims 1-13 or the albumin-binding domain according to claim 21 in the preparation of drugs and/or in prolonging the half-life of drugs.
如权利要求22所述的应用，其特征在于，将具有预防或治疗作用的活性分子通过共价键、非公价键、或接头分子与所述具有白蛋白结合功能的分子或所述白蛋白结合域结合。The application according to claim 22, characterized in that the active molecule with preventive or therapeutic effect is combined with the molecule with albumin binding function or the albumin through a covalent bond, a non-public bond, or a linker molecule Binding Domain Binding.