CN101084014A - Single domain antibodies against tumor necrosis factor receptor 1 and methods of use thereof - Google Patents
Single domain antibodies against tumor necrosis factor receptor 1 and methods of use thereofDownload PDFInfo
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Abstract
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Translated fromChinese相关申请related application
本申请是于2004年11月10日申请的美国专利申请号10/985,847的部分继续申请，它是：This application is a continuation-in-part of U.S. Patent Application Serial No. 10/985,847, filed November 10, 2004, which is:
1)于2004年10月8日申请的国际专利申请号PCT/GB2004/004253(指定国为美国)的部分继续申请；和1) A continuation-in-part of International Patent Application No. PCT/GB2004/004253 (designated country is the United States) filed on October 8, 2004; and
2)于2003年12月24日申请的国际专利申请号PCT/GB2003/005646(指定国为美国)的部分继续申请，并且要求于2002年12月27日申请的英国申请号GB 0230202.4和于2003年11月28日申请的英国申请号GB 0327706.8的优先权，它是2) Part of the continuation application of the international patent application number PCT/GB2003/005646 (the designated country is the United States) filed on December 24, 2003, and the UK application number GB 0230202.4 filed on December 27, 2002 and filed in 2003 Priority of UK Application No. GB 0327706.8 filed on 28 November 2018, which is
于2003年6月30日申请的国际专利申请号PCT/GB2003/002804(指定国为美国)的部分继续申请，并且要求于2002年12月27日申请的英国申请GB 0230202.4的优先权，它是A continuation-in-part of International Patent Application No. PCT/GB2003/002804 (designated country is the United States), filed 30 June 2003, and claiming priority to British application GB 0230202.4, filed 27 December 2002, which is
于2002年6月28日申请的国际专利申请号PCT/GB02/03014(指定国为美国)的部分继续申请。A continuation-in-part of International Patent Application No. PCT/GB02/03014 (designated country is the United States) filed on June 28, 2002.
上述申请的全部公开内容都通过引用结合到本文中。The entire disclosures of the above applications are incorporated herein by reference.
发明背景Background of the invention
抗体的抗原结合域包含两个单独的区：重链可变区(VH)和轻链可变区(VL：可以是Vκ或Vλ)。抗原结合位点本身由六个多肽环构成：3个来自VH区(H1、H2和H3)，3个来自VL区(L1、L2和L3)。编码VH区和VL区的V基因的多变初级库(diverse primary repertoire)是由基因区段组合重排而产生的。VH基因由VH、D和JH三个基因区段重组产生。在人类，约有51个功能性VH区段(Cook和Tomlinson(1995)Immunol Today，16：237)、25个功能性D区段(Corbett等(1997)J.Mol.Biol.，268：69)和6个功能性JH区段(Ravetch等(1981)Cell，27：583)，这取决于单元型(haplotype)。VH区段编码多肽链区，构成VH区的第一和第二抗原结合环(H1和H2)，而VH、D和JH区段组合形成VH区的第三抗原结合环(H3)。VL基因仅由VL和JL两个基因区段重组产生。在人类，约有40个功能性Vκ区段(Schble和Zachau(1993)Biol.Chem.Hoppe-Seyler，374：1001)、31个功能性Vλ区段(Williams等(1996)J.Mol.Biol.，264：220；Kawasaki等(1997)Genome Res.，7：250)、5个功能性Jκ区段(Hieter等(1982)J.Biol.Chem.，257：1516)和4个功能性Jλ区段(Vasicek和Leder(1990)J.Exp.Med.，172：609)，这取决于单元型。VL区段编码多肽链区，构成VL区的第一和第二抗原结合环(L1和L2)，而VL和JL区段组合形成VL区的第三抗原结合环(L3)。据信，从该初级库中选出的抗体具有足够的多样性，使得能以至少中等亲和力结合几乎所有的抗原。高亲和力抗体由重排基因的“亲和力成熟”产生，其中产生点突变并通过免疫系统根据改进的结合进行选择。The antigen-binding domain of an antibody consists of two separate regions: a heavy chain variable region (VH ) and a light chain variable region (VL : either VK or Vλ). The antigen binding site itself is composed of six polypeptide loops: 3 from theVH region (H1, H2 and H3) and 3 from theVL region (L1, L2 and L3). The diverse primary repertoire of V genes encodingVH andVL regions is generated by combinatorial rearrangement of gene segments. The VH gene is produced by recombination of three gene segments VH , D and JH . In humans, there are about 51 functionalVH segments (Cook and Tomlinson (1995) Immunol Today, 16:237), 25 functional D segments (Corbett et al. (1997) J.Mol.Biol., 268: 69) and six functionalJH segments (Ravetch et al. (1981) Cell, 27:583), depending on the haplotype. TheVH segment encodes a polypeptide chain region that constitutes the first and second antigen-binding loops (H1 and H2) of theVH region, while theVH , D, andJH segments combine to form the third antigen-binding loop of theVH region ( H3). The VL gene is only produced by recombination of two gene segments, VL and JL. In humans, there are about 40 functional Vκ segments (Schble and Zachau (1993) Biol. Chem. Hoppe-Seyler, 374:1001), 31 functional Vλ segments (Williams et al. (1996) J. Mol .Biol., 264:220; Kawasaki et al. (1997) Genome Res., 7:250), 5 functional Jκ segments (Hieter et al. (1982) J.Biol.Chem., 257:1516) and 4 functional Sexual Jλ segments (Vasicek and Leder (1990) J. Exp. Med., 172:609), depending on the haplotype. TheVL segment encodes a polypeptide chain region that constitutes the first and second antigen-binding loops (L1 and L2) of theVL region, while theVL andJL segments combine to form the third antigen-binding loop (L3) of theVL region . The antibodies selected from this primary library are believed to be of sufficient diversity to bind with at least moderate affinity to virtually all antigens. High-affinity antibodies result from "affinity maturation" of rearranged genes, in which point mutations are created and selected for improved binding by the immune system.
对抗体结构和序列的分析已经表明，6个抗原结合环中有5个(H1、H2、L1、L2、L3)具有数目有限的主链构象或正则结构(Chothia和Lesk(1987)J.Mol.Biol.，196：901；Chothia等(1989)Nature，342：877)。主链构象由以下两点来确定：(i)抗原结合环的长度，和(ii)抗原结合环和抗体构架中某个关键位置的特定残基或残基种类。对环长度和关键残基进行分析，已经让我们能够预测由大多数人类抗体序列所编码的H1、H2、L1、L2和L3的主链构象(Chothia等(1992)J.Mol.Biol.，227：799；Tomlinson等(1995)EMBO J.，14：4628；Williams等(1996)J.Mol.Biol.，264：220)。尽管H3区在序列、长度和结构上多样性高得多(因为使用了D区段)，但是它也构成数目有限的短环长度的主链构象，这取决于环及抗体构架的关键位置上特定残基的长度和存在与否，或残基种类(Martin等(1996)J.Mol.Biol.，263：800；Shirai等(1996)FEBS Letters，399：1)。Analysis of antibody structure and sequence has shown that five of the six antigen-binding loops (H1, H2, L1, L2, L3) have a limited number of main-chain conformations or canonical structures (Chothia and Lesk (1987) J. Mol Biol., 196:901; Chothia et al. (1989) Nature, 342:877). The main-chain conformation is determined by (i) the length of the antigen-binding loop, and (ii) specific residues or residue classes at a key position in the antigen-binding loop and antibody framework. Analysis of loop lengths and key residues has allowed us to predict the main-chain conformations of H1, H2, L1, L2 and L3 encoded by most human antibody sequences (Chothia et al. (1992) J. Mol. Biol., 227:799; Tomlinson et al. (1995) EMBO J., 14:4628; Williams et al. (1996) J. Mol. Biol., 264:220). Although the H3 region is much more diverse in sequence, length, and structure (due to the use of the D segment), it also constitutes a limited number of short loop-length backbone conformations, depending on the key positions of the loop and antibody framework. The length and presence or absence of a particular residue, or type of residue (Martin et al. (1996) J. Mol. Biol., 263:800; Shirai et al. (1996) FEBS Letters, 399:1).
包含VH区和VL区互补对的双特异性抗体是本领域已知的。这些双特异性抗体必定包含两对VH和VL，每对VH/VL结合一个抗原或表位。该方法涉及杂种杂交瘤(Milstein和Cuello AC，Nature 305：537-40)、微型抗体(minibody)(Hu等(1996)Cancer Res 56：3055-3061)、双链抗体(diabody)(Holliger等(1993)Proc.Natl.Acad.Sci.U.S.A.，90，6444-6448；WO 94/13804)、螯合重组抗体(CRAb；(Neri等(1995)J.Mol.Biol.246，367-373)、biscFv(例如Atwell等(1996)Mol.Immunol.33，1301-1312)、“杵臼结构(knobs in holes)”稳定的抗体(Carter等(1997)Protein Sci.6，781-788)。在多种情况下，每种抗体都包含两个抗原结合位点，各自由VH区和VL区互补对形成。因此，各抗体能同时结合两个不同抗原或表位，而与EACH抗原或表位的结合是由VH及其互补VL区介导的。这些技术各有各的缺点；例如就杂种杂交瘤而论，无活性VH/VL对可极大地减少双特异性IgG部分。此外，大多数双特异性方法依赖于不同VH/VL对的缔合或VH链与VL链的缔合，以得到两个不同VH/VL结合位点。因此，在装配分子中不可能控制结合位点与各抗原或表位的比例，所以许多装配分子能结合一个抗原或表位，但不结合其它抗原或表位。在某些情况下，可以改造重链或轻链的亚单位界面(Carter等，1997)，以便改善对两个抗原或表位具有结合位点的分子数量，但是这却不能使所有分子都能结合两个抗原或表位。Bispecific antibodies comprising complementary pairs ofVH andVL domains are known in the art. These bispecific antibodies necessarily comprise two pairs ofVH andVL , each pair ofVH /VL binding an antigen or epitope. This method involves hybrid hybridoma (Milstein and Cuello AC, Nature 305: 537-40), minibody (minibody) (Hu et al. (1996) Cancer Res 56: 3055-3061), diabody (diabody) (Holliger et al. ( 1993) Proc.Natl.Acad.Sci.USA, 90,6444-6448; WO 94/13804), chelated recombinant antibody (CRAb; (Neri et al. (1995) J.Mol.Biol.246,367-373), biscFv (eg Atwell et al. (1996) Mol. Immunol.33, 1301-1312), "knobs in holes" stable antibody (Carter et al. (1997) Protein Sci.6, 781-788). In various Under normal circumstances, every kind of antibody all comprises two antigen-binding sites, each is formed by the complementary pair ofVH district andVL district.Therefore, each antibody can combine two different antigens or epitope simultaneously, and with EACH antigen or epitope The binding is mediated by theVH and its complementaryVL region. Each of these techniques has its own disadvantages; for example in the case of hybrid hybridomas, inactiveVH /VL pairs can greatly reduce the bispecific IgG fraction. Furthermore, most bispecific approaches rely on the association of differentVH /VL pairs or the association of aVH chain with aVL chain to obtain two distinctVH /VL binding sites. Thus, in assembly It is not possible to control the ratio of binding sites to each antigen or epitope in the molecule, so many assembled molecules can bind one antigen or epitope but not others. In some cases, heavy chain or light chain can be engineered subunit interface of the chain (Carter et al., 1997) in order to improve the number of molecules with binding sites for two antigens or epitopes, but this does not allow all molecules to bind both antigens or epitopes.
有些证据表明，两个不同的抗体结合特异性可以结合到同一结合位点，但是这些一般代表两个或更多个特异性，所述特异性对应于结构相关的抗原或表位或者具有广泛交叉反应的抗体。例如交叉反应性抗体已有描述，通常，其中两个抗原在序列和结构上相关(例如鸡卵清溶菌酶和火鸡溶菌酶(McCafferty等，WO 92/01047))，或者对应于游离半抗原和缀合在载体上的半抗原(Griffiths AD等，EMBO J.1994，13：14 3245-60)。在另一个实例中，WO 02/02773(AbbottLaboratories)描述了具有“双特异性”的抗体分子。所述抗体分子是指针对多种抗原而产生或选择的抗体，使得它们的特异性跨越不止一种抗原。WO 02/02773的抗体中的各互补VH/VL对，针对两个或更多个结构相关抗原具有单结合特异性；这样的互补对中的VH区和VL区各自并不具有单独的特异性。因此，抗体具有针对两个结构相关抗原的广泛的单特异性。此外，多反应性的天然自身抗体已有描述(Casali和Notkins，Ann.Rev.Immunol.，7，515-531)，可与至少两个(通常更多)结构无关的不同抗原或表位反应。也已经知道，采用噬菌体展示技术对单克隆抗体进行随机肽库的选择，将会鉴定出一系列适合抗原结合位点的肽序列。某些序列是高度相关的，适合共有序列，而其它序列则十分不同，称为模拟表位(mimotope)(Lane和Stephen，Current Opinion in Immunology，1993，5，268-271)。因此，显然天然四链抗体，包括连接和互补的VH区和VL区在内，具有与许多不同抗原结合的潜力，这些抗原来自各种各样的已知抗原。尚不太清楚在同一抗体中怎样产生针对两个给定抗原的结合位点，尤其是当它们并不一定是结构相关时。There is some evidence that two different antibody binding specificities can bind to the same binding site, but these generally represent two or more specificities that correspond to structurally related antigens or epitopes or have extensive overlap. Reactive antibodies. For example, cross-reactive antibodies have been described, usually where the two antigens are related in sequence and structure (e.g. chicken egg white lysozyme and turkey lysozyme (McCafferty et al., WO 92/01047)), or correspond to free haptens and a hapten conjugated to a carrier (Griffiths AD et al., EMBO J. 1994, 13:14 3245-60). In another example, WO 02/02773 (Abbott Laboratories) describes antibody molecules with "bispecificity". The antibody molecules refer to antibodies raised or selected against multiple antigens such that their specificity spans more than one antigen. Each complementaryVH /VL pair in the antibody of WO 02/02773 has a single binding specificity for two or more structurally related antigens; each of theVH andVL regions in such complementary pairs does not have individual specificity. Thus, antibodies have broad monospecificity against two structurally related antigens. In addition, polyreactive natural autoantibodies have been described (Casali and Notkins, Ann. Rev. Immunol., 7, 515-531), reacting with at least two (usually more) different antigens or epitopes that are not structurally related . It is also known that selection of random peptide libraries of monoclonal antibodies using phage display technology will identify a series of peptide sequences suitable for the antigen binding site. Certain sequences are highly related and fit into a consensus sequence, while others are quite different and are called mimotopes (Lane and Stephen, Current Opinion in Immunology, 1993, 5, 268-271). Thus, it is clear that native four-chain antibodies, including linked and complementaryVH andVL domains, have the potential to bind many different antigens from a wide variety of known antigens. It is less clear how binding sites for two given antigens are created in the same antibody, especially when they are not necessarily structurally related.
蛋白质工程方法已经表明与这些方面有关。例如，也已知道可以产生催化性抗体，其具有通过一个可变区而与金属离子结合的活性，并具有通过与金属离子和互补可变区接触而与半抗原(底物)结合的活性(Barbas等，1993，Proc.Natl.Acad.Sci.U.S.A.，90，6385-6389)。然而在这种情况下，认为与底物(第一抗原)的结合和催化，需要与金属离子(第二抗原)的结合。因此，与VH/VL对的结合涉及单组分抗原而不是多组分抗原。Protein engineering methods have been shown to be involved in these aspects. For example, it is also known that catalytic antibodies can be produced that have the activity of binding a metal ion through one variable domain and the activity of binding a hapten (substrate) through contact with a metal ion and a complementary variable domain ( Barbas et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6385-6389). In this case, however, it is believed that binding and catalysis to the substrate (primary antigen) requires binding to the metal ion (second antigen). Thus, binding toVH /VL pairs involves single-component rather than multicomponent antigens.
从骆驼抗体重链单域产生双特异性抗体的方法已有描述，该方法中在一个可变区产生对第一抗原的结合接触，在第二可变区产生对第二抗原的结合接触。然而，可变区并不互补。因此，针对第一抗原选择第一重链可变区，针对第二抗原选择第二重链可变区，再将这两个区一起连接在同一链上，得到双特异性抗体片段(Conrath等，J.Biol.Chem.270，27589-27594)。然而，骆驼重链单域是异乎寻常的，因为它们来源于没有轻链的天然骆驼抗体，而且重链单域无法与骆驼轻链结合形成互补VH和VL对。Methods for generating bispecific antibodies from camelid heavy chain single domains have been described in which a binding contact for a first antigen is created in one variable domain and a binding contact for a second antigen is created in a second variable domain. However, the variable regions are not complementary. Thus, the first heavy chain variable region is selected against the first antigen, the second heavy chain variable region is selected against the second antigen, and these two regions are linked together on the same chain to obtain bispecific antibody fragments (Conrath et al. , J. Biol. Chem. 270, 27589-27594). However, camelid heavy chain single domains are unusual because they are derived from natural camelid antibodies without light chains, and heavy chain single domains cannot associate with camelid light chains to form complementaryVH andVL pairs.
也已描述了单个重链可变区，来源于天然抗体，通常与轻链相连接(来自单克隆抗体或来自结构域库；参见EP-A-0368684)。已经知道，这些重链可变区与一个或多个相关抗原具有特异性相互作用，但却不能结合其它重链或轻链可变区，以产生对两个或更多个不同抗原具有特异性的配体。此外，已经知道，这些单域的体内半衰期非常短。因此，这些结构域的治疗价值有限。Single heavy chain variable regions have also been described, derived from natural antibodies, usually linked to light chains (either from monoclonal antibodies or from domain repertoires; see EP-A-0368684). These heavy chain variable regions are known to specifically interact with one or more related antigens, but are incapable of binding other heavy or light chain variable regions to produce specificities for two or more different antigens ligand. Furthermore, it is known that these single domains have a very short half-life in vivo. Therefore, the therapeutic value of these domains is limited.
已经知道，通过将具有不同特异性的重链可变区连接在一起，可制备双特异性抗体片段(如上所述)。该方法的缺点就是：分离的抗体可变区可能具有疏水界面，该界面通常与轻链相互作用并且暴露给溶剂，而且是“粘性的”，使得单域能与疏水表面结合。此外，当配偶体轻链不存在时，两个或更多个不同重链可变区的组合及其结合(可能通过其疏水界面)，可阻止它们结合配体中的一个、而不是两个，而在分离时它们是可以结合两个配体的。此外，在这种情况下，重链可变区将不与互补轻链可变区结合，因此稳定性下降且容易解折叠(Worn和Pluckthun，1998，Biochemistry 37，13120-7)。It is known that bispecific antibody fragments can be prepared by linking together heavy chain variable regions of different specificities (as described above). The disadvantage of this approach is that the isolated antibody variable domain may have a hydrophobic interface, which normally interacts with the light chain and is exposed to solvent, and is "sticky", allowing single domains to bind to hydrophobic surfaces. Furthermore, when the partner light chain is absent, the combination of two or more different heavy chain variable domains and their association (possibly via their hydrophobic interface) may prevent them from binding one, but not both, of the ligands , and they can bind two ligands when separated. Furthermore, in this case the heavy chain variable region will not be associated with the complementary light chain variable region, thus being less stable and prone to unfolding (Worn and Pluckthun, 1998,
发明概述Summary of the invention
本发明涉及肿瘤坏死因子1(TNFR1、p55、CD120a、P60、TNF受体超家族成员1A、TNFRSF1A)拮抗剂以及所述拮抗剂的使用方法。优选的拮抗剂在治疗、抑制或预防慢性炎性疾病中有效，并且基本上不拮抗肿瘤坏死因子2(TNFR2、P75、P80、CD120b、TNF受体超家族成员1B、TNFRSF1B)。在某些实施方案中，所述拮抗剂是单价的。The present invention relates to tumor necrosis factor 1 (TNFR1, p55, CD120a, P60, TNF receptor superfamily member 1A, TNFRSF1A) antagonists and methods for using the antagonists. Preferred antagonists are effective in the treatment, inhibition or prevention of chronic inflammatory diseases and do not substantially antagonize tumor necrosis factor 2 (TNFR2, P75, P80, CD120b, TNF receptor superfamily member 1B, TNFRSF1B). In certain embodiments, the antagonist is monovalent.
在其它实施方案中，所述拮抗剂是抗体或其抗原结合片段，例如对TNFR1具有结合特异性的单价抗原结合片段(例如scFv、Fab、Fab′、dAb)。In other embodiments, the antagonist is an antibody or antigen-binding fragment thereof, eg, a monovalent antigen-binding fragment (eg, scFv, Fab, Fab', dAb) that has binding specificity for TNFRl.
其它优选的拮抗剂是本文所述的能结合TNFR1的配体。配体包含对TNFR1具有结合特异性的免疫球蛋白单可变区或域抗体(singlevariable domain or domain antibody，dAb)，或所述dAb的合适形式的互补决定区。在某些实施方案中，所述配体是dAb单体，由对TNFR1具有结合特异性的免疫球蛋白单可变区或dAb组成，或者基本上由其组成。在其它实施方案中，所述配体是包含合适形式(例如抗体形式)的dAb(或dAb的CDR)的多肽。Other preferred antagonists are the ligands described herein that bind TNFRl. The ligand comprises an immunoglobulin single variable domain or domain antibody (dAb) having binding specificity for TNFR1, or a complementarity determining region of a suitable form of said dAb. In certain embodiments, the ligand is a dAb monomer consisting or consisting essentially of an immunoglobulin single variable domain or dAb having binding specificity for TNFRl. In other embodiments, the ligand is a polypeptide comprising a dAb (or a CDR of a dAb) in a suitable form (eg, an antibody form).
在某些实施方案中，所述配体是双特异性配体，它包含能结合TNFR1的第一dAb和具有与第一dAb不同的结合特异性的第二dAb。在一个实例中，双特异性配体包含能结合TNFR1上第一表位的第一dAb以及能结合不同靶上的表位的第二dAb。在另一实例中，第二dAb能结合血清白蛋白上的表位。In certain embodiments, the ligand is a dual specific ligand comprising a first dAb that binds TNFRl and a second dAb that has a different binding specificity than the first dAb. In one example, the dual specific ligand comprises a first dAb that binds a first epitope on TNFRl and a second dAb that binds an epitope on a different target. In another example, the second dAb binds an epitope on serum albumin.
在其它实施方案中，所述配体是多特异性配体，它包含对TNFR1具有结合特异性的第一表位结合域和至少一个其它表位结合域，后者具有不同于第一表位结合域的结合特异性。例如，第一表位结合域可以是结合TNFR1的dAb，或者可以是包含能结合TNFR1的dAb的CDR的结构域(例如移植到合适蛋白质支架或骨架上的CDR，例如亲和体(affibody)、SpA支架、LDL受体A类区或EGF区)，或者可以是结合TNFR1的结构域，其中所述结构域选自亲和体、SpA区、LDL受体A类区或EGF区。In other embodiments, the ligand is a multispecific ligand comprising a first epitope binding domain having binding specificity for TNFR1 and at least one other epitope binding domain having a different Binding specificity of the binding domain. For example, the first epitope binding domain may be a dAb that binds TNFR1, or may be a domain comprising the CDRs of a dAb that binds TNFR1 (e.g., CDRs grafted onto a suitable protein scaffold or backbone, such as an affibody, affibody, SpA scaffold, LDL receptor class A region or EGF region), or can be a domain that binds TNFR1, wherein the domain is selected from the group consisting of Affibody, SpA region, LDL receptor class A region or EGF region.
在某些实施方案中，所述配体或dAb单体具有一个或多个以下特征：1)从人TNFR1上解离下来的解离常数(Kd)为50nM～20pM，K解离速率常数为5×10-1s-1～1×10-7s-1；2)抑制肿瘤坏死因子α(TNFα)与TNFR1结合的IC50为500nM～50pM；3)在标准L929细胞测定中，中和人TNFR1的ND50为500nM～50pM；4)在标准细胞测定中，拮抗TNFR1活性的ND50≤100nM，在所述测定中，当浓度≤10μM时，激动TNFR1的活性≤5％；5)在小鼠LPS/D-半乳糖胺诱发的脓毒性休克模型中抑制致死率；6)抗聚集作用；7)当在大肠杆菌(E.coli)或毕赤酵母(例如巴斯德毕赤酵母(P.pastoris))中表达时，其分泌量至少约为0.5mg/L；8)能可逆地解折叠；9)在选自以下的慢性炎性疾病模型中有效：小鼠胶原诱发的关节炎模型、小鼠关节炎ΔARE模型、小鼠炎性肠病ΔARE模型、小鼠葡聚糖硫酸钠诱发的炎性肠病模型、小鼠吸烟所致慢性阻塞性肺病模型及合适的灵长类模型(例如灵长类胶原诱发的关节炎模型)；和/或10)在治疗、抑制或预防慢性炎性疾病中有效。In certain embodiments, the ligand or dAb monomer has one or more of the following characteristics: 1) The dissociation constant (Kd ) for dissociation from human TNFR1 is 50 nM to 20 pM, and the Kdissociation rate constant 5×10-1 s-1 ~1×10-7 s-1 ; 2) The IC50 for inhibiting the binding of tumor necrosis factor α (TNFα) to TNFR1 is 500nM ~ 50pM; 3) In the standard L929 cell assay, medium and the ND50 of human TNFR1 is 500nM～50pM; 4) In the standard cell assay, the ND50 of antagonizing TNFR1 activity ≤ 100nM, in said assay, when the concentration ≤ 10μM, the activity of agonizing TNFR1 ≤ 5%; 5) Inhibition of lethality in a mouse LPS/D-galactosamine-induced septic shock model; 6) anti-aggregation effect; 7) when tested in Escherichia coli (E.coli) or Pichia pastoris (P. pastoris)) secretes at least about 0.5 mg/L; 8) can reversibly unfold; 9) is effective in a chronic inflammatory disease model selected from: mouse collagen-induced joint Inflammation model, mouse arthritis ΔARE model, mouse inflammatory bowel disease ΔARE model, mouse dextran sodium sulfate-induced inflammatory bowel disease model, mouse smoking-induced chronic obstructive pulmonary disease model and suitable primates A model (eg, a primate collagen-induced arthritis model); and/or 10) is useful in treating, inhibiting or preventing chronic inflammatory diseases.
在具体的实施方案中，所述配体或dAb单体从人TNFR1上解离下来的解离常数(Kd)为50nM～20pM，K解离速率常数为5×10-1s-1～1×10-7s-1；抑制肿瘤坏死因子α(TNFα)与TNFR1结合的IC50为500nM～50pM；在标准L929细胞测定中，中和人TNFR1的ND50为500nM～50pM。在其它具体的实施方案中，所述配体或dAb单体从人TNFR1上解离下来的解离常数(Kd)为50nM～20pM，K解离速率常数为5×10-1s-1～1×10-7s-1；抑制肿瘤坏死因子α(TNFα)与TNFR1结合的IC50为500nM～50pM；在选自以下的慢性炎性疾病模型中有效：小鼠胶原诱发的关节炎模型、小鼠关节炎ΔARE模型、小鼠炎性肠病ΔARE模型、小鼠葡聚糖硫酸钠诱发的炎性肠病模型、小鼠吸烟所致慢性阻塞性肺病模型及合适的灵长类模型(例如灵长类胶原诱发的关节炎模型)。在其它具体的实施方案中，所述配体或dAb单体从人TNFR1上解离下来的解离常数(Kd)为50nM～20pM，K解离速率常数为5×10-1s-1～1×10-7s-1；在标准L929细胞测定中，中和人TNFR1的ND50为500nM～50pM；在标准细胞测定中，拮抗TNFR1活性的ND50≤100nM，在所述测定中，当浓度≤10μM时，激动TNFR1的活性≤5％。In a specific embodiment, the dissociation constant (Kd ) of the ligand or dAb monomer dissociated from human TNFR1 is 50 nM～20 pM, and the Kdissociation rate constant is 5×10-1 s-1 ～ 1×10-7 s-1 ; the IC50 for inhibiting the binding of tumor necrosis factor α (TNFα) to TNFR1 is 500nM-50pM; in the standard L929 cell assay, the ND50 for neutralizing human TNFR1 is 500nM-50pM. In other specific embodiments, the dissociation constant (Kd ) of the ligand or dAb monomer dissociated from human TNFR1 is 50 nM-20 pM, and the Kdissociation rate constant is 5×10-1 s-1 ～1×10-7 s-1 ; IC50 for inhibiting the binding of tumor necrosis factor α (TNFα) to TNFR1 is 500nM～50pM; effective in chronic inflammatory disease models selected from the following: mouse collagen-induced arthritis model , mouse arthritis ΔARE model, mouse inflammatory bowel disease ΔARE model, mouse dextran sodium sulfate-induced inflammatory bowel disease model, mouse smoking-induced chronic obstructive pulmonary disease model and a suitable primate model ( e.g. primate collagen-induced arthritis model). In other specific embodiments, the dissociation constant (Kd ) of the ligand or dAb monomer dissociated from human TNFR1 is 50 nM-20 pM, and the Kdissociation rate constant is 5×10-1 s-1 ~1×10-7 s-1 ; in a standard L929 cell assay, the ND50 of neutralizing human TNFR1 is 500nM ~ 50pM; in a standard cell assay, the ND50 of antagonizing TNFR1 activity is ≤100nM, in the assay, When the concentration is ≤10 μM, the activity of agonizing TNFR1 is ≤5%.
在更具体的实施方案中，所述配体或dAb单体的氨基酸序列与选自以下dAb的氨基酸序列具有至少约90％同源性：TAR2h-12(SEQID NO：32)、TAR2h-13(SEQ ID NO：33)、TAR2h-14(SEQ ID NO：34)、TAR2h-16(SEQ ID NO：35)、TAR2h-17(SEQ ID NO：36)、TAR2h-18(SEQIDNO：37)、TAR2h-19(SEQ ID NO：38)、TAR2h-20(SEQ ID NO：39)、TAR2h-21(SEQ ID NO：40)、TAR2h-22(SEQ ID NO：41)、TAR2h-23(SEQ ID NO：42)、TAR2h-24(SEQ ID NO：43)、TAR2h-25(SEQ ID NO：44)、TAR2h-26(SEQ ID NO：45)、TAR2h-27(SEQ ID NO：46)、TAR2h-29(SEQ ID NO：47)、TAR2h-30(SEQ ID NO：48)、TAR2h-32(SEQ ID NO：49)、TAR2h-33(SEQ ID NO：50)、TAR2h-10-1(SEQ ID NO：51)、TAR2h-10-2(SEQ ID NO：52)、TAR2h-10-3(SEQ IDNO：53)、TAR2h-10-4(SEQ ID NO：54)、TAR2h-10-5(SEQ ID NO：55)、TAR2h-10-6(SEQ ID NO：56)、TAR2h-10-7(SEQ ID NO：57)、TAR2h-10-8(SEQ ID NO：58)、TAR2h-10-9(SEQ ID NO：59)、TAR2h-10-10(SEQ ID NO：60)、TAR2h-10-11(SEQ ID NO：61)、TAR2h-10-12(SEQ ID NO：62)、TAR2h-10-13(SEQ ID NO：63)、TAR2h-10-14(SEQ ID NO：64)、TAR2h-10-15(SEQ ID NO：65)、TAR2h-10-16(SEQ ID NO：66)、TAR2h-10-17(SEQ ID NO：67)、TAR2h-10-18(SEQ ID NO：68)、TAR2h-10-19(SEQ ID NO：69)、TAR2h-10-20(SEQ ID NO：70)、TAR2h-10-21(SEQ ID NO：71)、TAR2h-10-22(SEQ ID NO：72)、TAR2h-10-27(SEQ ID NO：73)、TAR2h-10-29(SEQ ID NO：74)、TAR2h-10-31(SEQ ID NO：75)、TAR2h-10-35(SEQ ID NO：76)、TAR2h-10-36(SEQ ID NO：77)、TAR2h-10-37(SEQ ID NO：78)、TAR2h-10-38(SEQ ID NO：79)、TAR2h-10-45(SEQ ID NO：80)、TAR2h-10-47(SEQ ID NO：81)、TAR2h-10-48(SEQ ID NO：82)、TAR2h-10-57(SEQ ID NO：83)、TAR2h-10-56(SEQ ID NO：84)、TAR2h-10-58(SEQ ID NO：85)、TAR2h-10-66(SEQ ID NO：86)、TAR2h-10-64(SEQ ID NO：87)、TAR2h-10-65(SEQ ID NO：88)、TAR2h-10-68(SEQ ID NO：89)、TAR2h-10-69(SEQ ID NO：90)、TAR2h-10-67(SEQ ID NO：91)、TAR2h-10-61(SEQ ID NO：92)、TAR2h-10-62(SEQ ID NO：93)、TAR2h-10-63(SEQ ID NO：94)、TAR2h-10-60(SEQ ID NO：95)、TAR2h-10-55(SEQ ID NO：96)、TAR2h-10-59(SEQ ID NO：97)、TAR2h-10-70(SEQ ID NO：98)、TAR2h-34(SEQ ID NO：373)、TAR2h-35(SEQ ID NO：374)、TAR2h-36(SEQ ID NO：375)、TAR2h-37(SEQ ID NO：376)、TAR2h-38(SEQ ID NO：377)、TAR2h-39(SEQID NO：378)、TAR2h-40(SEQ ID NO：379)、TAR2h-41(SEQ ID NO：380)、TAR2h-42(SEQ ID NO：381)、TAR2h-43(SEQ ID NO：382)、TAR2h-44(SEQ ID NO：383)、TAR2h-45(SEQ ID NO：384)、TAR2h-47(SEQ ID NO：385)、TAR2h-48(SEQ ID NO：386)、TAR2h-50(SEQID NO：387)、TAR2h-51(SEQ ID NO：388)、TAR2h-66(SEQ ID NO：389)、TAR2h-67(SEQ ID NO：390)、TAR2h-68(SEQ ID NO：391)、TAR2h-70(SEQ ID NO：392)、TAR2h-71(SEQ ID NO：393)、TAR2h-72(SEQ ID NO：394)、TAR2h-73(SEQ ID NO：395)、TAR2h-74(SEQID NO：396)、TAR2h-75(SEQ ID NO：397)、TAR2h-76(SEQ ID NO：398)、TAR2h-77(SEQ ID NO：399)、TAR2h-78(SEQ ID NO：400)、TAR2h-79(SEQ ID NO：401)和TAR2h-15(SEQ ID NO：431)。In a more specific embodiment, the ligand or dAb monomer has an amino acid sequence that is at least about 90% homologous to the amino acid sequence of a dAb selected from the group consisting of: TAR2h-12 (SEQ ID NO: 32), TAR2h-13 ( SEQ ID NO: 33), TAR2h-14 (SEQ ID NO: 34), TAR2h-16 (SEQ ID NO: 35), TAR2h-17 (SEQ ID NO: 36), TAR2h-18 (SEQ ID NO: 37), TAR2h -19 (SEQ ID NO: 38), TAR2h-20 (SEQ ID NO: 39), TAR2h-21 (SEQ ID NO: 40), TAR2h-22 (SEQ ID NO: 41), TAR2h-23 (SEQ ID NO : 42), TAR2h-24 (SEQ ID NO: 43), TAR2h-25 (SEQ ID NO: 44), TAR2h-26 (SEQ ID NO: 45), TAR2h-27 (SEQ ID NO: 46), TAR2h- 29 (SEQ ID NO: 47), TAR2h-30 (SEQ ID NO: 48), TAR2h-32 (SEQ ID NO: 49), TAR2h-33 (SEQ ID NO: 50), TAR2h-10-1 (SEQ ID NO: 51), TAR2h-10-2 (SEQ ID NO: 52), TAR2h-10-3 (SEQ ID NO: 53), TAR2h-10-4 (SEQ ID NO: 54), TAR2h-10-5 (SEQ ID NO: 55), TAR2h-10-6 (SEQ ID NO: 56), TAR2h-10-7 (SEQ ID NO: 57), TAR2h-10-8 (SEQ ID NO: 58), TAR2h-10-9 (SEQ ID NO: 59), TAR2h-10-10 (SEQ ID NO: 60), TAR2h-10-11 (SEQ ID NO: 61), TAR2h-10-12 (SEQ ID NO: 62), TAR2h-10 -13 (SEQ ID NO: 63), TAR2h-10-14 (SEQ ID NO: 64), TAR2h-10-15 (SEQ ID NO: 65), TAR2h-10-16 (SEQ ID NO: 66), TAR2h -10-17 (SEQ ID NO: 67), TAR2h-10-18 (SEQ ID NO: 68), TAR2h-10-19 (SEQ ID NO: 69), TAR2h-10-20 (SEQ ID NO: 70) , TAR2h-10-21 (SEQ ID NO: 71), TAR2h-10-22 (SEQ ID NO: 72), TAR2h-10-27 (SEQ ID NO: 73), TAR2h-10-29 (SEQ ID NO: 74), TAR2h-10-31 (SEQ ID NO: 75), TAR2h-10-35 (SEQ ID NO: 76), TAR2h-10-36 (SEQ ID NO: 77), TAR2h-10-37 (SEQ ID NO: 78), TAR2h-10-38 (SEQ ID NO: 79), TAR2h-10-45 (SEQ ID NO: 80), TAR2h-10-47 (SEQ ID NO: 81), TAR2h-10-48 ( SEQ ID NO: 82), TAR2h-10-57 (SEQ ID NO: 83), TAR2h-10-56 (SEQ ID NO: 84), TAR2h-10-58 (SEQ ID NO: 85), TAR2h-10- 66 (SEQ ID NO: 86), TAR2h-10-64 (SEQ ID NO: 87), TAR2h-10-65 (SEQ ID NO: 88), TAR2h-10-68 (SEQ ID NO: 89), TAR2h- 10-69 (SEQ ID NO: 90), TAR2h-10-67 (SEQ ID NO: 91), TAR2h-10-61 (SEQ ID NO: 92), TAR2h-10-62 (SEQ ID NO: 93), TAR2h-10-63 (SEQ ID NO: 94), TAR2h-10-60 (SEQ ID NO: 95), TAR2h-10-55 (SEQ ID NO: 96), TAR2h-10-59 (SEQ ID NO: 97 ), TAR2h-10-70 (SEQ ID NO: 98), TAR2h-34 (SEQ ID NO: 373), TAR2h-35 (SEQ ID NO: 374), TAR2h-36 (SEQ ID NO: 375), TAR2h- 37 (SEQ ID NO: 376), TAR2h-38 (SEQ ID NO: 377), TAR2h-39 (SEQ ID NO: 378), TAR2h-40 (SEQ ID NO: 379), TAR2h-41 (SEQ ID NO: 380 ), TAR2h-42 (SEQ ID NO: 381), TAR2h-43 (SEQ ID NO: 382), TAR2h-44 (SEQ ID NO: 383), TAR2h-45 (SEQ ID NO: 384), TAR2h-47 ( SEQ ID NO: 385), TAR2h-48 (SEQ ID NO: 386), TAR2h-50 (SEQ ID NO: 387), TAR2h-51 (SEQ ID NO: 388), TAR2h-66 (SEQ ID NO: 389), TAR2h-67 (SEQ ID NO: 390), TAR2h-68 (SEQ ID NO: 391), TAR2h-70 (SEQ ID NO: 392), TAR2h-71 (SEQ ID NO: 393), TAR2h-72 (SEQ ID NO: 394), TAR2h-73 (SEQ ID NO: 395), TAR2h-74 (SEQ ID NO: 396), TAR2h-75 (SEQ ID NO: 397), TAR2h-76 (SEQ ID NO: 398), TAR2h- 77 (SEQ ID NO:399), TAR2h-78 (SEQ ID NO:400), TAR2h-79 (SEQ ID NO:401) and TAR2h-15 (SEQ ID NO:431).
在另外的实施方案中，所述配体或dAb单体的氨基酸序列与选自以下dAb的氨基酸序列具有至少约90％同源性：TAR2h-131-8(SEQID NO：433)、TAR2h-131-24(SEQ ID NO：434)、TAR2h-15-8(SEQ IDNO：435)、TAR2h-15-8-1 SEQ ID NO：436)、TAR2h-15-8-2(SEQ ID NO：437)、TAR2h-185-23(SEQ ID NO：438)、TAR2h-154-10-5(SEQ ID NO：439)、TAR2h-14-2(SEQ ID NO：440)、TAR2h-151-8(SEQ ID NO：441)、TAR2h-152-7(SEQ ID NO：442)、TAR2h-35-4(SEQ ID NO：443)、TAR2h-154-7(SEQ ID NO：444)、TAR2h-80(SEQ ID NO：445)、TAR2h-81(SEQ ID NO：446)、TAR2h-82(SEQ ID NO：447)、TAR2h-83(SEQ ID NO：448)、TAR2h-84(SEQ ID NO：449)、TAR2h-85(SEQID NO：450)、TAR2h-86(SEQ ID NO：451)、TAR2h-87(SEQ ID NO：452)、TAR2h-88(SEQ ID NO：453)、TAR2h-89(SEQ ID NO：454)、TAR2h-90(SEQ ID NO：455)、TAR2h-91(SEQ ID NO：456)、TAR2h-92(SEQ ID NO：457)、TAR2h-93(SEQ ID NO：458)、TAR2h-94(SEQID NO：459)、TAR2h-95(SEQ ID NO：460)、TAR2h-96(SEQ ID NO：461)、TAR2h-97(SEQ ID NO：462)、TAR2h-99(SEQ ID NO：463)、TAR2h-100(SEQ ID NO：464)、TAR2h-101(SEQ ID NO：465)、TAR2h-102(SEQ ID NO：466)、TAR2h-103(SEQ ID NO：467)、TAR2h-104(SEQ ID NO：468)、TAR2h-105(SEQ ID NO：469)、TAR2h-106(SEQ ID NO：470)、TAR2h-107(SEQ ID NO：471)、TAR2h-108(SEQ ID NO：472)、TAR2h-109(SEQ ID NO：473)、TAR2h-110(SEQ ID NO：474)、TAR2h-111(SEQ ID NO：475)、TAR2h-112(SEQ ID NO：476)、TAR2h-113(SEQ ID NO：477)、TAR2h-114(SEQ ID NO：478)、TAR2h-115(SEQ ID NO：479)、TAR2h-116(SEQ ID NO：480)、TAR2h-117(SEQ ID NO：481)、TAR2h-118(SEQ ID NO：482)、TAR2h-119(SEQ ID NO：483)、TAR2h-120(SEQ ID NO：484)、TAR2h-121(SEQ ID NO：485)、TAR2h-122(SEQ ID NO：486)、TAR2h-123(SEQ ID NO：487)、TAR2h-124(SEQ ID NO：488)、TAR2h-125(SEQ ID NO：489)、TAR2h-126(SEQ ID NO：490)、TAR2h-127(SEQ ID NO：490)、TAR2h-128(SEQ ID NO：492)、TAR2h-129(SEQ ID NO：493)、TAR2h-130(SEQ ID NO：494)、TAR2h-131(SEQ ID NO：495)、TAR2h-132(SEQ ID NO：496)、TAR2h-133(SEQ ID NO：497)、TAR2h-151(SEQ ID NO：498)、TAR2h-152(SEQ ID NO：499)、TAR2h-153(SEQ ID NO：500)、TAR2h-154(SEQ ID NO：501)、TAR2h-159(SEQ ID NO：502)、TAR2h-165(SEQ ID NO：503)、TAR2h-166(SEQ ID NO：504)、TAR2h-168(SEQ ID NO：505)、TAR2h-171(SEQ ID NO：506)、TAR2h-172(SEQ ID NO：507)、TAR2h-173(SEQ ID NO：508)、TAR2h-174(SEQ ID NO：509)、TAR2h-176(SEQ ID NO：510)、TAR2h-178(SEQ ID NO：511)、TAR2h-201(SEQ ID NO：512)、TAR2h-202(SEQ ID NO：513)、TAR2h-203(SEQ ID NO：514)、TAR2h-204(SEQ ID NO：515)、TAR2h-185-25(SEQ ID NO：516)、TAR2h-154-10(SEQ ID NO：517)和TAR2h-205(SEQ ID NO：627)。In additional embodiments, the ligand or dAb monomer has an amino acid sequence that is at least about 90% homologous to the amino acid sequence of a dAb selected from: TAR2h-131-8 (SEQ ID NO: 433), TAR2h-131 -24 (SEQ ID NO: 434), TAR2h-15-8 (SEQ ID NO: 435), TAR2h-15-8-1 (SEQ ID NO: 436), TAR2h-15-8-2 (SEQ ID NO: 437) , TAR2h-185-23 (SEQ ID NO: 438), TAR2h-154-10-5 (SEQ ID NO: 439), TAR2h-14-2 (SEQ ID NO: 440), TAR2h-151-8 (SEQ ID NO: 441), TAR2h-152-7 (SEQ ID NO: 442), TAR2h-35-4 (SEQ ID NO: 443), TAR2h-154-7 (SEQ ID NO: 444), TAR2h-80 (SEQ ID NO: 445), TAR2h-81 (SEQ ID NO: 446), TAR2h-82 (SEQ ID NO: 447), TAR2h-83 (SEQ ID NO: 448), TAR2h-84 (SEQ ID NO: 449), TAR2h -85 (SEQ ID NO: 450), TAR2h-86 (SEQ ID NO: 451), TAR2h-87 (SEQ ID NO: 452), TAR2h-88 (SEQ ID NO: 453), TAR2h-89 (SEQ ID NO: 454), TAR2h-90 (SEQ ID NO: 455), TAR2h-91 (SEQ ID NO: 456), TAR2h-92 (SEQ ID NO: 457), TAR2h-93 (SEQ ID NO: 458), TAR2h-94 (SEQ ID NO: 459), TAR2h-95 (SEQ ID NO: 460), TAR2h-96 (SEQ ID NO: 461), TAR2h-97 (SEQ ID NO: 462), TAR2h-99 (SEQ ID NO: 463) , TAR2h-100 (SEQ ID NO: 464), TAR2h-101 (SEQ ID NO: 465), TAR2h-102 (SEQ ID NO: 466), TAR2h-103 (SEQ ID NO: 467), TAR2h-104 (SEQ ID NO: 468), TAR2h-105 (SEQ ID NO: 469), TAR2h-106 (SEQ ID NO: 470), TAR2h-107 (SEQ ID NO: 471), TAR2h-108 (SEQ ID NO: 472), TAR2h-109 (SEQ ID NO: 473), TAR2h-110 (SEQ ID NO: 474), TAR2h-111 (SEQ ID NO: 475), TAR2h-112 (SEQ ID NO: 476), TAR2h-113 (SEQ ID NO: 477), TAR2h-114 (SEQ ID NO: 478), TAR2h-115 (SEQ ID NO: 479), TAR2h-116 (SEQ ID NO: 480), TAR2h-117 (SEQ ID NO: 481), TAR2h -118 (SEQ ID NO: 482), TAR2h-119 (SEQ ID NO: 483), TAR2h-120 (SEQ ID NO: 484), TAR2h-121 (SEQ ID NO: 485), TAR2h-122 (SEQ ID NO : 486), TAR2h-123 (SEQ ID NO: 487), TAR2h-124 (SEQ ID NO: 488), TAR2h-125 (SEQ ID NO: 489), TAR2h-126 (SEQ ID NO: 490), TAR2h- 127 (SEQ ID NO: 490), TAR2h-128 (SEQ ID NO: 492), TAR2h-129 (SEQ ID NO: 493), TAR2h-130 (SEQ ID NO: 494), TAR2h-131 (SEQ ID NO: 495), TAR2h-132 (SEQ ID NO: 496), TAR2h-133 (SEQ ID NO: 497), TAR2h-151 (SEQ ID NO: 498), TAR2h-152 (SEQ ID NO: 499), TAR2h-153 (SEQ ID NO: 500), TAR2h-154 (SEQ ID NO: 501), TAR2h-159 (SEQ ID NO: 502), TAR2h-165 (SEQ ID NO: 503), TAR2h-166 (SEQ ID NO: 504 ), TAR2h-168 (SEQ ID NO: 505), TAR2h-171 (SEQ ID NO: 506), TAR2h-172 (SEQ ID NO: 507), TAR2h-173 (SEQ ID NO: 508), TAR2h-174 ( SEQ ID NO:509), TAR2h-176 (SEQ ID NO:510), TAR2h-178 (SEQ ID NO:511), TAR2h-201 (SEQ ID NO:512), TAR2h-202 (SEQ ID NO:513) , TAR2h-203 (SEQ ID NO:514), TAR2h-204 (SEQ ID NO:515), TAR2h-185-25 (SEQ ID NO:516), TAR2h-154-10 (SEQ ID NO:517) and TAR2h -205 (SEQ ID NO: 627).
本发明涉及肿瘤坏死因子1(TNFR1)拮抗剂，所述拮抗剂能结合肿瘤坏死因子1(TNFR1)并抑制通过TNFR1的信号转导，其中所述拮抗剂不抑制TNFα与TNFR1的结合。在某些实施方案中，所述拮抗剂包含第一域抗体(dAb)单体和第二dAb单体，其中所述第一dAb单体能结合选自域1、域2、域3和域4的TNFR1域，所述第二dAb单体能结合选自域1、域2、域3和域4的TNFR1域，其中在标准L929细胞毒性测定或标准HeLa IL-8测定中，当浓度约为1μM时，所述拮抗剂不激动TNFR1。The present invention relates to tumor necrosis factor 1 (TNFR1) antagonists that bind tumor necrosis factor 1 (TNFR1) and inhibit signal transduction through TNFR1, wherein the antagonists do not inhibit the binding of TNFα to TNFR1. In certain embodiments, the antagonist comprises a first domain antibody (dAb) monomer and a second dAb monomer, wherein the first dAb monomer is capable of binding a domain selected from
在某些实施方案中，本发明是域抗体(dAb)单体或包含dAb的配体，其能结合肿瘤坏死因子1(TNFR1)并抑制通过TNFR1的信号转导，其中所述dAb单体不抑制TNFα与TNFR1的结合。In certain embodiments, the invention is a domain antibody (dAb) monomer or a ligand comprising a dAb that binds tumor necrosis factor 1 (TNFR1) and inhibits signal transduction through TNFR1, wherein the dAb monomer is not Inhibits the binding of TNFα to TNFR1.
在其它实施方案中，本发明是域抗体(dAb)单体或包含dAb的配体，其能结合肿瘤坏死因子1(TNFR1)，其中所述dAb能结合TNFR1的域1并与TAR2m-21-23竞争性结合小鼠TNFR1或者与TAR2h-205竞争性结合人TNFR1。In other embodiments, the invention is a domain antibody (dAb) monomer or ligand comprising a dAb that binds tumor necrosis factor 1 (TNFR1), wherein said dAb binds
在其它实施方案中，本发明是域抗体(dAb)单体或包含dAb的配体，其能结合肿瘤坏死因子1(TNFR1)，其中所述dAb能结合TNFR1的域3并与TAR2h-131-8、TAR2h-15-8、TAR2h-35-4、TAR2h-154-7、TAR2h-154-10或TAR2h-185-25竞争性结合人TNFR1。In other embodiments, the invention is a domain antibody (dAb) monomer or ligand comprising a dAb that binds tumor necrosis factor 1 (TNFR1), wherein said dAb binds
本发明也涉及抗体或其抗原结合片段，它们对TNFR1具有结合特异性并在治疗、抑制或预防慢性炎性疾病中有效。在某些实施方案中，所述抗体或抗原结合片段是单价抗原结合片段。The present invention also relates to antibodies or antigen-binding fragments thereof that have binding specificity for TNFR1 and are effective in treating, inhibiting or preventing chronic inflammatory diseases. In certain embodiments, the antibody or antigen-binding fragment is a monovalent antigen-binding fragment.
本发明也提供dAb单体、以及包含dAb单体的配体，其对TNFR1具有结合特异性并能抑制TNFR-1介导的信号传导，但基本上不抑制TNFα与TNFR1的结合。在某些实施方案中，dAb单体抑制由TNFα诱导的细胞表面上TNFR1的交联或成簇。The invention also provides dAb monomers, and ligands comprising dAb monomers, which have binding specificity for TNFR1 and are capable of inhibiting TNFR-1 mediated signaling, but do not substantially inhibit the binding of TNFα to TNFR1. In certain embodiments, the dAb monomer inhibits TNF[alpha]-induced crosslinking or clustering of TNFRl on the cell surface.
本发明也提供编码本发明配体的分离和/或重组的核酸分子、以及包含所述重组核酸分子的载体。也提供包含本发明重组核酸分子或载体的宿主细胞和用于制备配体的方法。The invention also provides isolated and/or recombinant nucleic acid molecules encoding the ligands of the invention, and vectors comprising said recombinant nucleic acid molecules. Also provided are host cells comprising recombinant nucleic acid molecules or vectors of the invention and methods for making the ligands.
本发明也涉及药物组合物，所述组合物包含本发明的拮抗剂或配体以及药理学上、生理上或药学上可接受的载体。The invention also relates to pharmaceutical compositions comprising the antagonist or ligand of the invention and a pharmacologically, physiologically or pharmaceutically acceptable carrier.
本发明也涉及治疗、抑制或预防疾病或障碍(例如慢性炎性疾病、自身免疫性疾病、炎性疾病、关节炎、多发性硬化、炎性肠病、慢性阻塞性肺病、肺炎、脓毒性休克)的方法，该方法包括给予有需要的哺乳动物治疗有效量的本发明拮抗剂或配体。The invention also relates to treating, inhibiting or preventing diseases or disorders (e.g. chronic inflammatory disease, autoimmune disease, inflammatory disease, arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, pneumonia, septic shock ) method comprising administering to a mammal in need thereof a therapeutically effective amount of an antagonist or ligand of the invention.
本发明也涉及用于治疗或诊断的本发明拮抗剂或配体，以及本发明拮抗剂或配体在制备用于治疗、抑制或预防如上所述的疾病或障碍(例如慢性炎性疾病、自身免疫性疾病、炎性疾病、关节炎、多发性硬化、炎性肠病、慢性阻塞性肺病、肺炎或脓毒性休克)的药物中的用途。在其它实施方案中，所述疾病可以是囊性纤维化或严重甾体抵抗型哮喘。The present invention also relates to antagonists or ligands of the invention for use in therapy or diagnosis, as well as antagonists or ligands of the invention in the preparation for use in the treatment, suppression or prevention of diseases or disorders as described above (e.g. chronic inflammatory diseases, autoimmune immune disease, inflammatory disease, arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, pneumonia or septic shock). In other embodiments, the disease may be cystic fibrosis or severe steroid-resistant asthma.
本发明还涉及用于治疗、抑制或预防本文所述的疾病或障碍(例如慢性炎性疾病、自身免疫性疾病、炎性疾病、关节炎、多发性硬化、炎性肠病、慢性阻塞性肺病、肺炎或脓毒性休克)的药物组合物，所述组合物包含本发明的拮抗剂或配体作为活性成分。在其它实施方案中，所述疾病可以是囊性纤维化或严重甾体抵抗型哮喘。The invention also relates to methods for treating, inhibiting or preventing the diseases or disorders described herein (e.g. chronic inflammatory disease, autoimmune disease, inflammatory disease, arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease) , pneumonia or septic shock) comprising the antagonist or ligand of the present invention as an active ingredient. In other embodiments, the disease may be cystic fibrosis or severe steroid-resistant asthma.
对TNFR1和配体具有结合特异性的单可变区或域抗体(dAb)以及包含这些单可变区或dAb的配体，具有若干优势。例如，本文所述的对TNFR1具有结合特异性的单可变区或dAb能拮抗TNFR1。因此，可以给予包含本发明抗TNFR1免疫球蛋白单可变区或dAb的治疗药(例如用于治疗、诊断或预防目的)，所述治疗药的副作用(例如免疫抑制)危险因为结合和/或拮抗TNFR2而基本降低。靶向TNFα的治疗药例如ENBREL(entarecept；Immunex Corporation)拮抗TNFR1和TNFR2，给予这些药物可产生免疫抑制及相关副作用(例如严重感染)。这些副作用限制了这类药物的使用，尤其是对于需要长期给药的慢性疾病。(Kollias G.和Kontoyiannis D.，Cytokine GrowthFactor Rev.，73(4-5)：315-321(2002))。相比之下，因为本发明的配体特异性拮抗TNFR1，所以对于慢性疾病而言，它们可以长期给予，其副作用的危险性降低，并能提供治疗炎性病症和慢性炎性病症(包括特征为静止期和活动性炎症期的长期疾病，例如炎性肠病和关节炎)的优势。Single variable region or domain antibodies (dAbs) having binding specificities for TNFRl and a ligand, and ligands comprising these single variable region or dAbs, have several advantages. For example, a single variable domain or dAb described herein that has binding specificity for TNFRl can antagonize TNFRl. Accordingly, therapeutics comprising an anti-TNFR1 immunoglobulin single variable domain or dAb of the invention can be administered (e.g., for therapeutic, diagnostic or prophylactic purposes) with a risk of side effects (e.g., immunosuppression) due to binding and/or Antagonized TNFR2 and basically decreased. Therapeutics targeting TNF[alpha] such as ENBREL(R) (entarecept; Immunex Corporation) antagonize TNFR1 and TNFR2 and administration of these drugs can produce immunosuppression and associated side effects (eg, severe infection). These side effects limit the use of this class of drugs, especially for chronic diseases that require long-term administration. (Kollias G. and Kontoyiannis D., Cytokine Growth Factor Rev., 73(4-5):315-321 (2002)). In contrast, because the ligands of the present invention specifically antagonize TNFR1, they can be administered chronically with reduced risk of side effects for chronic diseases and provide the ability to treat inflammatory and chronic inflammatory disorders (including characteristic Advantages for long-term diseases such as inflammatory bowel disease and arthritis) in both quiescent and active inflammatory phases.
附图简述Brief description of the drawings
图1显示VH/HAS在位置H50、H52、H52a、H53、H55、H56、H58、H95、H96、H97、H98的多样化(DVT或NNK分别编码)，它们在VH HAS的抗原结合位点中。(SEQ ID NO：1，核苷酸序列；SEQ IDNO：2，氨基酸序列)。Vκ序列在位置L50、L53具有多样性。Figure 1 shows the diversification ofVH /HAS at positions H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98 (encoded by DVT or NNK, respectively), which are at the antigen-binding site ofVH HAS hit. (SEQ ID NO: 1, nucleotide sequence; SEQ ID NO: 2, amino acid sequence). The Vκ sequence has diversity at positions L50, L53.
图2示意图显示用于制备单链Fv(scFv)文库的质粒pIT1/pIT2的结构，并显示跨越表达控制和克隆区的质粒核苷酸序列(SEQ ID NO：3)以及所编码的氨基酸序列(SEQ ID NO：4)。该质粒用于制备Figure 2 schematically shows the structure of the plasmid pIT1/pIT2 used to prepare the single-chain Fv (scFv) library, and shows the plasmid nucleotide sequence (SEQ ID NO: 3) and the encoded amino acid sequence ( SEQ ID NO: 4). This plasmid is used to prepare
文库1：种系Vκ/DVT VH，Library 1: Germline Vκ/DVTVH ,
文库2：种系Vκ/NNK VH，Library 2: Germline Vκ/NNKVH ,
文库3：种系VH/DVT Vκ，和Library 3: GermlineVH /DVT Vκ, and
文库4：种系VH/NNK Vκ，以噬菌体展示/ScFv形式。Library 4: GermlineVH /NNK VK in phage display/ScFv format.
根据结合蛋白A和蛋白L通用配体，对这些文库进行预筛选，使得大部分克隆和所选文库是功能性的。针对HSA(第一轮)和β-gal(第二轮)或HSA β-gal选择或者针对β-gal(第一轮)和HSA(第二轮)β-gal HSA选择，对文库进行选择。扩增序列中这些来自PCR克隆的可溶性scFv。选出一个编码双特异性抗体K8的克隆进行进一步工作。These libraries were pre-screened against binding protein A and protein L universal ligands such that the majority of clones and selected libraries were functional. Libraries were selected against HSA (first round) and β-gal (second round) or HSA β-gal selection or against β-gal (first round) and HSA (second round) β-gal HSA selection. These soluble scFvs were derived from PCR clones in the amplified sequence. A clone encoding the bispecific antibody K8 was selected for further work.
图3显示以下序列的比对：VH链(VH dummy(SEQ ID NO：5)、K8(SEQ ID NO：6)、VH2(SEQ ID NO：7)、VH4(SEQ ID NO：8)、VHC11(SEQ ID NO：9)、VHA10sd(SEQ ID NO：10)、VHAlsd(SEQ ID NO：11)、VHA5sd(SEQ ID NO：12)、VHC5sd(SEQ ID NO：13)、VHCllsd(SEQ ID NO：14)、VHCllsd(SEQ ID NO：15))和Vκ链(Vk dummy(SEQ ID NO：16)、K8(SEQ ID NO：17)、E5sc(SEQ ID NO：18)、C3(SEQ ID NO：19))。Figure 3 shows the alignment of the following sequences:VH chain (VH dummy (SEQ ID NO: 5), K8 (SEQ ID NO: 6), VH2 (SEQ ID NO: 7), VH4 (SEQ ID NO: 8) , VHC11 (SEQ ID NO: 9), VHA10sd (SEQ ID NO: 10), VHAlsd (SEQ ID NO: 11), VHA5sd (SEQ ID NO: 12), VHC5sd (SEQ ID NO: 13), VHCllsd (SEQ ID NO: 14), VHCllsd (SEQ ID NO: 15)) and Vκ chain (Vk dummy (SEQ ID NO: 16), K8 (SEQ ID NO: 17), E5sc (SEQ ID NO: 18), C3 (SEQ ID NO: 19)).
图4显示K8抗体的结合特性的表征，K8抗体的结合特性经单克隆噬菌体ELISA而表征，发现双特异性K8抗体能结合HSA和β-gal并展示在噬菌体表面，其吸收信号大于1.0。没有检测到与其它蛋白质的交叉反应性。Figure 4 shows the characterization of the binding properties of the K8 antibody. The binding properties of the K8 antibody were characterized by monoclonal phage ELISA. It was found that the bispecific K8 antibody can bind to HSA and β-gal and display on the surface of the phage, and its absorption signal is greater than 1.0. No cross-reactivity with other proteins was detected.
图5显示可溶性scFv ELISA的结果，采用已知浓度的K8抗体片段。96孔板用浓度100μg HSA、10μg/ml BSA和100μg/ml β-gal和1μg/ml蛋白A包被。采用50μg系列稀释的K8 scFv，用蛋白L-HRP检测结合抗体片段。ELISA结果证实了K8抗体的双特异性。Figure 5 shows the results of a soluble scFv ELISA using known concentrations of the K8 antibody fragment. A 96-well plate was coated with concentrations of 100 μg HSA, 10 μg/ml BSA and 100 μg/ml β-gal and 1 μg/ml protein A. Bound antibody fragments were detected with protein L-HRP using 50 μg of serially diluted K8 scFv. ELISA results confirmed the bispecificity of the K8 antibody.
图6显示克隆K8Vκ/dummy VH的结合特征，分析采用可溶性scFvELISA。可溶性scFv片段通过IPTG诱导产生，参见Harrison等，Methods Enzymol.1996；267：83-109，然后直接测定含有scFv的上清液。按照实施例1所述，进行可溶性scFv ELISA，用蛋白L-HRP测定结合scFv。ELISA结果表明，该克隆仍能结合β-gal，而结合BSA则被消除掉。Figure 6 shows the binding profile of clone K8VK/dummyVH , analyzed by soluble scFvELISA. Soluble scFv fragments were induced by IPTG, see Harrison et al., Methods Enzymol. 1996;267:83-109, and the scFv-containing supernatants were then directly assayed. Soluble scFv ELISA was performed as described in Example 1, and bound scFv was determined using protein L-HRP. The results of ELISA showed that the clone could still bind β-gal, but the binding to BSA was eliminated.
图7显示可变区载体1和2的序列(SEQ ID NO：2和SEQ ID NO：3)。Figure 7 shows the sequences of
图8是用于构建VH1/VH2多特异性配体的CH载体图谱。Fig. 8 is a map ofCH carriers used to constructVH1 /VH2 multispecific ligands.
图9是用于构建Vκ1/Vκ2多特异性配体的Cκ载体图谱。Figure 9 is a map of the Cκ vector used to construct the Vκ1/Vκ2 multispecific ligand.
图10显示TNF受体测定，与TAR1-5二聚体4、TAR1-5-19二聚体4和TAR1-5-19单体进行比较。Figure 10 shows TNF receptor assays compared to TAR1-5
图11显示比较TAR1-5二聚体1-6的TNF受体测定。所有二聚体都经FPLC纯化，显示优化二聚体种类的结果。Figure 11 shows a TNF receptor assay comparing TAR1-5 dimers 1-6. All dimers were purified by FPLC, showing results for optimized dimer species.
图12显示不同形式的TAR1-519同型二聚体的TNF受体测定：具有3U、5U或7U接头的dAb-接头-dAb形式，Fab形式和半胱氨酸铰链接头形式。Figure 12 shows TNF receptor assays of TAR1-519 homodimers in different formats: dAb-linker-dAb format with 3U, 5U or 7U linker, Fab format and cysteine hinge linker format.
图13显示文库1的Dummy VH序列。(氨基酸序列((SEQ ID NO：5；核苷酸序列：编码链(SEQ ID NO：20)，非编码链(SEQ ID NO：21)。VH构架序列基于种系序列DP47-JH4b。NNK随机化(N＝A或T或C或G核苷酸；K＝G或T核苷酸)结合到文库1中的位置用粗体加上下划线表示。Figure 13 shows the DummyVH sequences of
图14显示文库2的Dummy VH序列。(氨基酸序列((SEQ ID NO：22；核苷酸序列：编码链(SEQ ID NO：23)，非编码链(SEQ ID NO：24)。VH构架序列基于种系序列DP47-JH4b。NNK随机化(N＝A或T或C或G核苷酸；K＝G或T核苷酸)结合到文库2中的位置用粗体加上下划线表示。Figure 14 shows the DummyVH sequences of
图15显示文库3的Dummy Vκ序列。(氨基酸序列((SEQ ID NO：16；核苷酸序列：编码链(SEQ ID NO：25)，非编码链(SEQ ID NO：26)。Vκ构架序列基于种系序列DPκ9-Jκ1。NNK随机化(N＝A或T或C或G核苷酸；K＝G或T核苷酸)结合到文库3中的位置用粗体加上下划线表示。Figure 15 shows the Dummy VK sequences of
图16显示抗MSA dAb MSA 16的核苷酸序列和氨基酸序列(核苷酸序列(SEQ ID NO：27)、氨基酸序列(SEQ ID NO：28)和MSA 26(核苷酸序列(SEQ ID NO：29)、氨基酸序列(SEQ ID NO：30))。Figure 16 shows the nucleotide sequence and amino acid sequence (nucleotide sequence (SEQ ID NO: 27), amino acid sequence (SEQ ID NO: 28) and MSA 26 (nucleotide sequence (SEQ ID NO: 28) of anti-MSA dAb MSA 16 : 29), amino acid sequence (SEQ ID NO: 30)).
图17显示MSA16和26的抑制biacore。通过抑制biacore对纯化dAb MSA16和MSA26进行分析，得出Kd。简而言之，测定dAb，以求出在用高密度MSA包被的biacore CM5芯片上达到200RU响应所需的dAb浓度。一旦测出所需dAb浓度，将预期Kd左右浓度范围的MSA抗原与dAb预混合并孵育过夜。然后以30μl/分钟的高流速，测定各预混物中dAb与MSA包被的biacore芯片的结合。Figure 17 shows the inhibition biacore of MSA16 and 26. Purified dAbs MSA16 and MSA26 were analyzed by inhibition biacore for Kd . Briefly, dAbs were assayed to determine the concentration of dAb required to achieve a 200RU response on a biacore CM5 chip coated with high density MSA. Once the desired dAb concentration has been determined, MSA antigen in the concentration range around the expectedKd is premixed with the dAb and incubated overnight. Binding of dAbs in each premix to MSA-coated biacore chips was then assayed at a high flow rate of 30 μl/min.
图18显示注射后MSA16的血清水平。测定小鼠的dAb MSA16血清半衰期。给CD1小鼠单次静脉注射约1.5mg/kg MSA16。用2区室模型建模，显示MSA16的tα为0.98小时，tβ为36.5小时，AUC为913小时.mg/ml。与HEL4(一种抗鸡卵清溶菌酶dAb)相比，MSA16的半衰期相当长，tα为0.06小时，tβ为0.34小时。Figure 18 shows serum levels of MSA16 after injection. Determination of serum half-life of dAb MSA16 in mice. A single intravenous injection of approximately 1.5 mg/kg MSA16 was given to CD1 mice. Using a 2-compartment model, it showed that the tα of MSA16 was 0.98 hours, the tβ was 36.5 hours, and the AUC was 913 hours.mg/ml. Compared with HEL4 (an anti-chicken egg white lysozyme dAb), the half-life of MSA16 is quite long, with tα of 0.06 hours and tβ of 0.34 hours.
图19a-19c显示ELISA(图19a)和TNF受体测定(图19b、19c)，表明TNF与包含MSA26Ck和TAR1-5-19CH的Fab样片段的结合被抑制。加入带有Fab样片段的MSA可降低抑制水平。一个用1μg/mlTNFα包被的ELISA板用双特异性VκCH和VκCκ Fab样片段来探测，同时也用对照TNFα结合dAb来检测，其浓度为经计算在ELISA中给出相似信号的浓度。在2mg/ml MSA存在或不存在下，采用双特异性dAb和对照dAb来探测ELISA板。双特异性孔的信号下降超过50％，但dAb孔的信号却完全没有下降(参见图19a)。在有或没有MSA的情况下，将相同的双特异性蛋白也进行受体测定，并且也表现出与MSA竞争(参见图19c)。这证明MSA竞争性结合双特异性蛋白和TNFα。Figures 19a-19c show ELISA (Figure 19a) and TNF receptor assays (Figures 19b, 19c) showing that binding of TNF to a Fab-like fragment comprising MSA26Ck and TAR1-5-19CH is inhibited. Addition of MSA with Fab-like fragments reduces the level of inhibition. An ELISA plate coated with 1 μg/ml TNFα was probed with bispecific VκCH and VκCκ Fab-like fragments, and also with a control TNFα-binding dAb at a concentration calculated to give a similar signal in the ELISA. ELISA plates were probed with bispecific dAbs and control dAbs in the presence or absence of 2 mg/ml MSA. The signal for the bispecific wells decreased by more than 50%, but the signal for the dAb wells did not decrease at all (see Figure 19a). The same bispecific protein was also subjected to receptor assays with and without MSA and also appeared to compete with MSA (see Figure 19c). This demonstrates that MSA competitively binds the bispecific protein and TNFα.
图20显示TNF受体测定，表明TNF与二硫键键合的TAR1-5-19dAb和MSA16 dAb异型二聚体的结合被抑制。加入带有二聚体的MSA，以剂量依赖性方式降低抑制水平。在恒定浓度的异型二聚体(18nM)和系列稀释的MSA和HAS的存在下，进行TNF受体测定(图19(b))。一定浓度范围(最多2mg/ml)的HAS的存在不引起二聚体抑制TNFα的能力下降。然而加入MSA，剂量依赖性地降低了二聚体抑制TNFα的能力(图19a)。这证明MSA和TNFα竞争性结合cys键合的TAR1-5-19、MSA16二聚体。在测定中，MSA和HAS单用对TNF结合水平没有影响。Figure 20 shows a TNF receptor assay showing inhibition of TNF binding to disulfide-bonded TAR1-5-19 dAb and MSA16 dAb heterodimers. Addition of MSA with dimer decreased the level of inhibition in a dose-dependent manner. TNF receptor assays were performed in the presence of a constant concentration of heterodimer (18 nM) and serial dilutions of MSA and HAS (Figure 19(b)). The presence of HAS in a range of concentrations (up to 2 mg/ml) did not cause a decrease in the ability of the dimer to inhibit TNFα. Addition of MSA, however, dose-dependently reduced the ability of the dimer to inhibit TNF[alpha] (Fig. 19a). This demonstrates that MSA and TNF[alpha] compete for binding to the cys-linked TAR1-5-19, MSA16 dimer. MSA and HAS alone had no effect on the level of TNF binding in the assay.
图21A-21M显示一些对人TNFR1具有结合特异性的人免疫球蛋白可变区的氨基酸序列(SEQ ID NO：31-98和SEQ ID NO：373-401和431)。所示的氨基酸序列是连续的，没有空位；序列中插入了符号～，表示互补决定区(CDR)的位置。CDR1的两侧是～，CDR2的两侧是～～，CDR3的两侧是～～～。Figures 21A-21M show the amino acid sequences of some human immunoglobulin variable regions with binding specificity for human TNFRl (SEQ ID NOs: 31-98 and SEQ ID NOs: 373-401 and 431). The amino acid sequence shown is continuous without gaps; the symbol ~ is inserted in the sequence to indicate the position of the complementarity determining region (CDR). CDR1 is flanked by ~, CDR2 is flanked by ~~, and CDR3 is flanked by ~~~.
图22A-22T显示一些编码图21A-21M所示的人免疫球蛋白可变区的核酸的核苷酸序列(SEQ ID NO：99-166和SEQ ID NO：402-430和432)。所示的核苷酸序列是连续的，没有空位；序列中插入了符号～，表示编码CDR的序列的位置。编码CDR1的序列的两侧是～，编码CDR2的序列的两侧是～～，编码CDR3的序列的两侧是～～～。Figures 22A-22T show the nucleotide sequences (SEQ ID NOs: 99-166 and SEQ ID NOs: 402-430 and 432) of some of the nucleic acids encoding the human immunoglobulin variable regions shown in Figures 21A-21M. The nucleotide sequence shown is continuous without gaps; the symbol ~ is inserted in the sequence to indicate the position of the sequence encoding the CDR. The sequence encoding CDR1 is flanked by ~, the sequence encoding CDR2 is flanked by ~~, and the sequence encoding CDR3 is flanked by ~~~.
图23A-23B显示一些对小鼠TNFR1具有结合特异性的人免疫球蛋白可变区的氨基酸序列(SEQ ID NO：167-179)。所示的氨基酸序列是连续的，没有空位；在某些系列中插入了符号～，表示互补决定区(CDR)的位置。CDR1的两侧是～，CDR2的两侧是～～，CDR3的两侧是～～～。Figures 23A-23B show the amino acid sequences (SEQ ID NOs: 167-179) of some human immunoglobulin variable regions with binding specificity for mouse TNFR1. The amino acid sequences shown are contiguous without gaps; in some series the symbol ~ has been inserted to indicate the position of complementarity determining regions (CDRs). CDR1 is flanked by ~, CDR2 is flanked by ~~, and CDR3 is flanked by ~~~.
图24A-24C显示一些编码图23A-23B所示的人免疫球蛋白可变区的核酸的核苷酸序列(SEQ ID NO：180-192和626)。SEQ ID NO：186和SEQ ID NO：626都编码SEQ ID NO：173的氨基酸序列。编码CDR1的SEQ ID NO：626序列的两侧是～，编码CDR2的序列的两侧是～～，编码CDR3的序列的两侧是～～～。Figures 24A-24C show the nucleotide sequences (SEQ ID NOs: 180-192 and 626) of some of the nucleic acids encoding the human immunoglobulin variable regions shown in Figures 23A-23B. Both SEQ ID NO: 186 and SEQ ID NO: 626 encode the amino acid sequence of SEQ ID NO: 173. The sequence of SEQ ID NO:626 encoding CDR1 is flanked by ~, the sequence encoding CDR2 is flanked by ~~, and the sequence encoding CDR3 is flanked by ~~~.
图25A-25L显示编码一些人免疫球蛋白可变区的核苷酸序列以及所编码的人免疫球蛋白可变区的氨基酸序列(SEQ ID NO：193-198和200-295)。Figures 25A-25L show the nucleotide sequences encoding certain human immunoglobulin variable regions and the amino acid sequences of the encoded human immunoglobulin variable regions (SEQ ID NOS: 193-198 and 200-295).
图26曲线图显示在L929测定中抗TNFR1 dAb形式基本上不激动TNFR1。将L929细胞在含有以下成分的培养基中培养：不同浓度的抗TNFR1 dAb单体(TAR2m-21-23)、市售抗myc抗体(9E10)交联的TAR2m-21-23单体、双特异性抗TNFR1 dAb/抗SA dAb(TAR2m-21-23 3U TAR7m-16)或PEG化抗TNFR1 dAb单体(TAR2m-21-23 40KPEG)。就抗myc抗体交联TAR2m-21-23单体而论，在培养之前，将dAb和抗体按2∶1的比例混合并在室温下预孵育1小时，以模拟体内免疫交联效应。(TAR2m-21-23单体包含myc表位)。将浓度为3,000nM的TAR2m-21-23单体与L929细胞一起孵育。TAR2m-21-23单体和抗Myc抗体一起孵育，dAb浓度为3,000nM。将浓度为25nM、83.3nM、250nM、833nM和2,500nM的TAR2m-21-23 3U TAR7m-16与细胞一起孵育。将浓度为158.25nM、527.5nM、1582.5nM、5,275nM和15,825nM的TAR2m-21-23 40K PEG与细胞一起孵育。孵育过夜后，评价细胞存活率。结果表明，将L929细胞与10nM、1nM或0.1nM能交联和激动TNFR1的市售抗TNFR1 IgG抗体(目录号AF-425-PB；R&D系统，Minneapolis，MN)一起孵育，导致以剂量依赖方式增加无活力细胞，因此证明了这些细胞对TNFR1激动剂的敏感性。相比之下，与不同数量的抗TNFR1形式孵育，并不拮抗TNFR1，也不导致培养物中无活力细胞数量的增加，甚至当采用超过1000倍市售抗TNFR1 IgG抗体浓度时。Figure 26 is a graph showing that the anti-TNFRl dAb format does not substantially agonize TNFRl in the L929 assay. L929 cells were cultured in media containing the following components: different concentrations of anti-TNFR1 dAb monomer (TAR2m-21-23), commercially available anti-myc antibody (9E10) cross-linked TAR2m-21-23 monomer, bispecific Anti-TNFR1 dAb/anti-SA dAb (TAR2m-21-23 3U TAR7m-16) or PEGylated anti-TNFR1 dAb monomer (TAR2m-21-23 40KPEG). For cross-linking of TAR2m-21-23 monomers by anti-myc antibody, dAb and antibody were mixed in a 2:1 ratio and pre-incubated at room temperature for 1 hour prior to incubation to mimic the in vivo immune cross-linking effect. (TAR2m-21-23 monomer contains myc epitope). TAR2m-21-23 monomer at a concentration of 3,000 nM was incubated with L929 cells. TAR2m-21-23 monomer was incubated with anti-Myc antibody at a dAb concentration of 3,000 nM. TAR2m-21-23 3U TAR7m-16 at concentrations of 25nM, 83.3nM, 250nM, 833nM and 2,500nM were incubated with the cells. TAR2m-21-23 40K PEG at concentrations of 158.25nM, 527.5nM, 1582.5nM, 5,275nM and 15,825nM were incubated with the cells. After overnight incubation, cell viability was assessed. The results showed that incubation of L929 cells with 10 nM, 1 nM or 0.1 nM of a commercially available anti-TNFR1 IgG antibody (Cat. No. AF-425-PB; R&D Systems, Minneapolis, MN) capable of cross-linking and agonizing TNFR1 resulted in a dose-dependent manner Non-viable cells were increased, thus demonstrating the sensitivity of these cells to TNFR1 agonists. In contrast, incubation with varying amounts of anti-TNFR1 forms did not antagonize TNFR1, nor did it lead to an increase in the number of non-viable cells in culture, even when over 1000-fold concentrations of commercially available anti-TNFR1 IgG antibodies were employed.
图27A-27I显示一些对人TNFR1具有结合特异性的人免疫球蛋白可变区的氨基酸序列(SEQ ID NO：433-517和627)。所示的氨基酸序列是连续的，没有空位；序列中插入了符号～，表示互补决定区(CDR)的位置。CDR1的两侧是～，CDR2的两侧是～～，CDR3的两侧是～～～。Figures 27A-27I show the amino acid sequences of some human immunoglobulin variable regions with binding specificity for human TNFR1 (SEQ ID NOs: 433-517 and 627). The amino acid sequence shown is continuous without gaps; the symbol ~ is inserted in the sequence to indicate the position of the complementarity determining region (CDR). CDR1 is flanked by ~, CDR2 is flanked by ~~, and CDR3 is flanked by ~~~.
图28A-28O显示一些编码图27A-27H所示的人免疫球蛋白可变区的核酸的核苷酸序列(SEQ ID NO：518-602和628)。所示的核苷酸序列是连续的，没有空位；序列中插入了符号～，表示编码CDR的序列的位置。编码CDR1的序列的两侧是～，编码CDR2的序列的两侧是～～，编码CDR3的序列的两侧是～～～。Figures 28A-28O show the nucleotide sequences (SEQ ID NOs: 518-602 and 628) of some of the nucleic acids encoding the human immunoglobulin variable regions shown in Figures 27A-27H. The nucleotide sequence shown is continuous without gaps; the symbol ~ is inserted in the sequence to indicate the position of the sequence encoding the CDR. The sequence encoding CDR1 is flanked by ~, the sequence encoding CDR2 is flanked by ~~, and the sequence encoding CDR3 is flanked by ~~~.
发明详述Detailed description of the invention
在本发明说明书中描述了许多实施方案，撰写方式使得本发明说明书能够既清楚又简明，但是应当理解，在不偏离本发明的情况下，可以对这些实施方案进行各种组合或分开。In this specification of the invention many embodiments are described and written in such a manner that the description of the invention will be both clear and concise, but it should be understood that various combinations and divisions of these embodiments can be made without departing from the invention.
定义definition
“互补”，当两个免疫球蛋白结构域属于能构成关联对或关联组的结构家族时，或者它们来源于这样的家族并保留了该特征时，则它们是“互补”的。例如，抗体的VH区和VL区就是互补的；两个VH区则不互补，两个VL区也不互补。在免疫球蛋白超家族其它成员中也可发现互补区，例如T细胞受体的Vα和Vβ(或γ和δ)区。在本发明第二格局的情况下，非互补区并不协同结合靶分子，而是独立作用于相同或不同分子上的不同靶表位。人工结构域，例如基于蛋白质支架的结构域(它们不结合表位，除非经改造使它们结合表位)，是非互补的。同样，基于(例如)免疫球蛋白结构域和纤连蛋白结构域的两个结构域不是互补的。Two immunoglobulin domains are "complementary" when they belong to a structural family capable of constituting a cognate pair or cognate group, or when they are derived from such a family and retain this characteristic. For example, theVH andVL regions of an antibody are complementary; the twoVH regions are not complementary, and the twoVL regions are not complementary. Complementary regions are also found in other members of the immunoglobulin superfamily, such as theVα andVβ (or γ and δ) regions of the T cell receptor. In the case of the second configuration of the invention, the non-complementary regions do not bind target molecules cooperatively, but act independently on different target epitopes on the same or different molecules. Artificial domains, such as domains based on protein scaffolds (which do not bind epitopes unless engineered so that they do), are non-complementary. Likewise, two domains based on, for example, an immunoglobulin domain and a fibronectin domain are not complementary.
“免疫球蛋白”，是指这样的多肽家族：其保留抗体分子的免疫球蛋白折叠特征，含有两个β折叠，一般还含有一个保守二硫键。免疫球蛋白超家族成员在体内参与细胞和非细胞相互作用的许多方面，包括在免疫系统(例如抗体、T细胞受体分子等)中具有广泛作用，参与细胞粘附(例如ICAM分子)和胞内信号转导(例如受体分子，例如PDGF受体)。本发明适用于所有具有结合域的免疫球蛋白超家族分子。优选本发明涉及抗体。"Immunoglobulin" refers to a family of polypeptides that retain the immunoglobulin fold characteristic of antibody molecules, containing two beta sheets and generally a conserved disulfide bond. Members of the immunoglobulin superfamily are involved in many aspects of cellular and noncellular interactions in vivo, including broad roles in the immune system (e.g., antibodies, T cell receptor molecules, etc.), involvement in cell adhesion (e.g., ICAM molecules) and cellular Internal signal transduction (eg receptor molecules such as PDGF receptor). The present invention is applicable to all immunoglobulin superfamily molecules having binding domains. Preferably the invention relates to antibodies.
“结合”，本发明的各可变区结合在一起构成一组结构域；例如，互补区可以结合在一起，例如VL区与VH区结合在一起。非互补区也能结合在一起。结构域可以以大量方式结合在一起，包括各结构域通过共价或非共价方式连接。By "associated", the variable regions of the invention are combined to form a set of domains; for example, complementary regions can be combined, such asVL andVH regions. Non-complementary regions can also be joined together. Domains can be joined together in a number of ways, including covalent or non-covalent linking of domains.
“domain(域，结构域，区)”，是经过折叠的蛋白质结构，它保留了蛋白质三级结构，但独立于蛋白质的其余部分。通常，各结构域负责蛋白质的分离的功能性，并且在许多情况下可以添加、去除或转移给其它蛋白质，而不丧失该蛋白质和/或该结构域的其余部分的功能。单抗体可变区(single antibody variable domain)是指经过折叠的多肽区，包含特征为抗体可变区的序列。因此，它包含完整抗体可变区和修饰可变区(例如其中一个或多个环已经被不具有抗体可变区特征的序列所取代)，或者已被截短或包含N-端或C-端延伸的抗体可变区，以及保留全长结构域的至少部分结合活性和特异性的可变区的折叠片段。A "domain" is a folded protein structure that preserves the protein's tertiary structure but is independent of the rest of the protein. Typically, each domain is responsible for a protein's discrete functionality, and in many cases can be added, removed or transferred to other proteins without losing the function of the protein and/or the rest of the domain. A single antibody variable domain (single antibody variable domain) refers to a folded polypeptide region comprising sequences characteristic of antibody variable domains. Thus, it comprises both intact antibody variable regions and modified variable regions (for example in which one or more loops have been replaced by sequences not characteristic of antibody variable regions), or which have been truncated or contain N-terminal or C-terminal Extended antibody variable regions, as well as folded fragments of the variable regions that retain at least part of the binding activity and specificity of the full-length domain.
“库(repertoire)”，一级序列不同的多样化变异体(例如多肽变异体)的集合。本发明所用的文库可包括含有至少1000个成员的多肽库。A "repertoire", a collection of diverse variants (eg, polypeptide variants) that differ in primary sequence. Libraries used in the present invention may include polypeptide repertoires comprising at least 1000 members.
“文库(library)”，术语文库是指异源多肽或核酸的混合物。文库由成员组成，每个成员都具有一个多肽或核酸序列。就这一方面而言，文库(library)和库(repertoire)同义。文库成员间的序列差异造成文库的多样性。文库可以呈多肽或核酸的简单混合物的形式，或者可以呈用核酸文库转化的生物体或细胞的形式，例如细菌、病毒、动植物细胞等。优选各个生物体或细胞仅含有一个文库成员或数目有限的文库成员。最好将核酸掺入到表达载体中，以表达该核酸所编码的多肽。因此，在一个优选的方面，文库可以呈宿主生物体群体的形式，每个生物体含有一个或多个拷贝的表达载体，所述载体含有呈核酸形式的文库的一个成员，所述核酸可以表达而产生其相应多肽成员。因此，宿主生物体群体具有编码很大的具有遗传多样性的多肽变异体库的潜力。"Library", the term library refers to a mixture of heterologous polypeptides or nucleic acids. A library is composed of members, each member having a polypeptide or nucleic acid sequence. In this respect, library and repertoire are synonymous. Sequence differences among library members contribute to library diversity. Libraries may be in the form of simple mixtures of polypeptides or nucleic acids, or may be in the form of organisms or cells transformed with the library of nucleic acids, such as bacteria, viruses, animal or plant cells, and the like. Preferably each organism or cell contains only one or a limited number of library members. Preferably, the nucleic acid is incorporated into an expression vector to express the polypeptide encoded by the nucleic acid. Thus, in a preferred aspect, the library may be in the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a member of the library in the form of a nucleic acid capable of expressing And produce its corresponding polypeptide member. Thus, populations of host organisms have the potential to encode large repertoires of genetically diverse polypeptide variants.
“闭合构象多特异性配体(closed conformation multi-specificligand)”用于描述本文所定义的多特异性配体，包含至少两个本文所定义的表位结合域(epitope binding domain)。术语“闭合构象”(多特异性配体)是指配体的表位结合域是这样排列的：使得通过一个表位结合域的表位结合，与通过另一个表位结合域的表位结合发生竞争。也就是说，关联表位可以通过每个表位结合域单独结合，而不是同时结合。采用本文所述的方法，可得到配体的闭合构象。"Closed conformation multi-specific ligand" is used to describe a multi-specific ligand as defined herein, comprising at least two epitope binding domains as defined herein. The term "closed conformation" (multispecific ligand) means that the epitope binding domains of the ligand are arranged such that an epitope bound by one epitope binding domain binds an epitope bound by the other epitope binding domain Competition occurs. That is, cognate epitopes can be bound by each epitope-binding domain individually, rather than simultaneously. Using the methods described herein, the closed conformation of the ligand can be obtained.
“抗体”，抗体(例如IgG、IgM、IgA、IgD或IgE)或其片段(例如Fab、F(ab′)2、Fv、二硫键连接的Fv、scFv、闭合构象多特异性抗体、二硫键连接的scFv、双链抗体)，无论是来自任何物种的天然产生的抗体，还是由重组DNA技术产生的抗体；无论是从下列哪种样品中分离的：血清、B细胞、杂交瘤、转染瘤、酵母或细菌。"Antibody", an antibody (e.g. IgG, IgM, IgA, IgD or IgE) or a fragment thereof (e.g. Fab, F(ab')2 , Fv, disulfide-linked Fv, scFv, closed conformation multispecific antibody, di sulfur-bonded scFv, diabodies), whether naturally occurring antibodies from any species, or antibodies produced by recombinant DNA techniques; whether isolated from any of the following samples: serum, B cells, hybridomas, Transfectoma, yeast or bacteria.
“双特异性配体”，是指包含本文所定义的第一免疫球蛋白单可变区和第二免疫球蛋白单可变区的配体，其中可变区能结合两个不同抗原或同一抗原上的两个表位，而它们通常并不与单特异性免疫球蛋白结合。例如，两个表位可以在同一半抗原上，而不是同一表位或足够近以便结合单特异性配体。本发明的双特异性配体由具有不同特异性的可变区组成，但不包括具有相同特异性的相互互补的可变区对。"Dual specific ligand" means a ligand comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as defined herein, wherein the variable domains are capable of binding two different antigens or the same Two epitopes on an antigen that are not normally bound by monospecific immunoglobulins. For example, two epitopes may be on the same hapten, but not the same epitope or close enough to bind a monospecific ligand. The dual specific ligands of the invention consist of variable domains with different specificities, but do not include mutually complementary variable domain pairs with the same specificity.
“抗原”，一种能与本发明的配体结合的分子。通常，抗原与抗体配体结合并能在体内产生抗体反应。抗原可以是多肽、蛋白质、核酸或其它分子。一般而言，针对特定抗原的靶特异性而选择本发明双特异性配体。就常规抗体及其片段而言，由可变环(L1、L2、L3和H1、H2、H3)所限定的抗体结合位点能够结合抗原。"Antigen", a molecule capable of binding a ligand of the invention. Typically, an antigen binds to an antibody ligand and can generate an antibody response in vivo. Antigens can be polypeptides, proteins, nucleic acids or other molecules. In general, the dual specific ligands of the invention are selected for their target specificity for a particular antigen. In the case of conventional antibodies and fragments thereof, the antibody binding site defined by the variable loops (L1, L2, L3 and H1, H2, H3) is capable of binding antigen.
“表位”，与免疫球蛋白VH/VL对常规结合的结构单元。表位限定针对抗体的最少结合位点，因此代表了抗体特异性的靶。就单域抗体而言，表位代表了与分离的可变区结合的结构单元。"Epitope", the structural unit to which an immunoglobulinVH /VL pair is conventionally bound. An epitope defines the fewest binding sites for an antibody and thus represents the target for antibody specificity. In the case of single domain antibodies, an epitope represents a structural unit bound to an isolated variable domain.
“通用配体(generic ligand)”，能结合某一库中所有成员的配体。一般而言，并不通过上述抗原结合位点而结合。非限制性实例包括蛋白A、蛋白L和蛋白G。A "generic ligand" is a ligand that binds all members of a library. In general, binding is not via the antigen binding site described above. Non-limiting examples include Protein A, Protein L and Protein G.
“选择”，来自筛选或来自达尔文选择过程，其中在结构域与抗原或表位之间或者在抗体与抗原或表位之间发生结合相互作用。因此，在互补可变区存在或不存在下，可以选择结合抗原或表位的第一可变区。"Selection", from screening or from a Darwinian selection process in which a binding interaction occurs between a domain and an antigen or epitope or between an antibody and an antigen or epitope. Thus, the first variable domain can be selected for binding to an antigen or epitope, in the presence or absence of complementary variable domains.
“通用构架(universal framework)”，单抗体构架序列，对应于序列中的保守抗体区，根据Kabat(″Sequences of Proteins ofImmunologicalInterest″，US Department of Health and Human Services(美国健康和人类服务部))所定义；或者对应于人种系免疫球蛋白库或结构，根据Chothia和Lesk，(1987)J.Mol.Biol.196：910-917所定义。本发明提供单构架或一组这样的构架的用途，已经发现它们允许事实上任何结合特异性的来源，尽管变异仅在超变区内。"Universal framework (universal framework)", single antibody framework sequence, corresponding to the conserved antibody region in the sequence, according to Kabat ("Sequences of Proteins of Immunological Interest", US Department of Health and Human Services (US Department of Health and Human Services)) Definition; or corresponds to the human germline immunoglobulin repertoire or structure, as defined by Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917. The present invention provides the use of a single framework or a set of such frameworks, which have been found to allow virtually any source of binding specificity, although variations are only within the hypervariable regions.
“半衰期”，是指配体的血清浓度在体内降至50％所需要的时间，例如因为通过天然机制所致的配体降解和/或清除或螯合。本发明的配体在体内是稳定的，其半衰期因结合抗降解和/或清除或螯合的分子而延长。通常，这类分子是天然存在的蛋白质，它们本身在体内具有较长的半衰期。如果配体的功能活性在体内的持续时间比对延长半衰期的分子不具有特异性的类似配体更长的话，则配体半衰期延长。因此，将对HSA和靶分子具有特异性的配体，与对HAS无特异性且不结合HAS、而结合别的分子的相同配体进行比较。例如，它能结合靶分子上的第二表位。通常，半衰期延长10％、20％、30％、40％、50％以上。半衰期延长范围在2x、3x、4x、5x、10x、20x、30x、40x、50x以上是可能的。或者，另外，半衰期延长范围高达30x、40x、50x、60x、70x、80x、90x、100x、150x也是可能的。"Half-life" refers to the time required for the serum concentration of a ligand to drop to 50% in vivo, for example due to ligand degradation and/or clearance or sequestration by natural mechanisms. The ligands of the invention are stable in vivo and their half-life is extended by binding molecules that resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins, which themselves have long half-lives in the body. A ligand has an increased half-life if its functional activity lasts longer in vivo than a similar ligand that has no specificity for the half-life-extending molecule. Thus, a ligand that is specific for HSA and a target molecule is compared to the same ligand that is not specific for HAS and does not bind HAS, but binds another molecule. For example, it can bind a second epitope on the target molecule. Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50% or more. Half-life extensions in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x and more are possible. Alternatively, additionally, half-life extensions ranging up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x are also possible.
“均质免疫测定(homogeneous immunoassay)”，无需分离结合和未结合试剂的步骤就可检测被分析物的免疫测定。"Homogeneous immunoassay" is an immunoassay that detects an analyte without the need for a step of separating bound and unbound reagents.
“基本相同(或“基本同源”)”，第一氨基酸或核苷酸序列与第二氨基酸或核苷酸序列含有足够数量的相同或相当(例如具有相似侧链，例如保守氨基酸取代)氨基酸残基或核苷酸，以使第一和第二氨基酸或核苷酸序列具有相似活性。就抗体而言，第二抗体与第一抗体具有相同的结合特异性并且具有第一抗体亲和力的至少50％。"Substantially identical (or "substantially homologous")", a first amino acid or nucleotide sequence contains a sufficient number of identical or comparable (e.g., have similar side chains, e.g., conservative amino acid substitutions) amino acids to a second amino acid or nucleotide sequence residues or nucleotides such that the first and second amino acid or nucleotide sequences have similar activities. In the case of antibodies, the second antibody has the same binding specificity as the first antibody and has at least 50% of the affinity of the first antibody.
本文所用的术语“低严格性”、“中等严格性”、“高严格性”或“极高严格性条件”描述了核酸杂交和洗涤条件。进行杂交反应的指南可参见Current Protocols in Molecular Biology，John Wiley和Sons，N.Y.(1989)，6.3.1-6.3.6，所述文献通过引用全部结合到本文中。该参考文献中描述了含水方法和无水方法，都可以采用。本文所用的具体杂交条件如下：(1)低严格性杂交条件在6X氯化钠/柠檬酸钠(SSC)中，在约45℃，再在0.2X SSC、0.1％SDS中，在至少50℃(对于低严格性条件，洗涤温度可升高至55℃)洗涤两次；(2)中等严格性杂交条件在6X SSC中，在约45℃，再在0.2X SSC、0.1％SDS中，在60℃洗涤一次或多次；(3)高严格性杂交条件在6X SSC中，在约45℃，再在0.2X SSC、0.1％SDS中在65℃洗涤一次或多次；和优选(4)极高严格性杂交条件是0.5M磷酸钠、7％SDS中，在65℃，再在0.2X SSC、1％SDS中，在65℃洗涤一次或多次。极高严格性条件(4)是优选的条件，除非另有说明，否则应采用该条件。The terms "low stringency", "medium stringency", "high stringency" or "very high stringency conditions" as used herein describe nucleic acid hybridization and washing conditions. Guidelines for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6, which is hereby incorporated by reference in its entirety. Both aqueous and anhydrous methods are described in this reference and can be used. The specific hybridization conditions used herein are as follows: (1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC), at about 45 ° C, then in 0.2X SSC, 0.1% SDS, at least 50 ° C (For low stringency conditions, the washing temperature can be increased to 55°C) wash twice; (2) medium stringency hybridization conditions in 6X SSC, at about 45°C, then in 0.2X SSC, 0.1% SDS, at Wash one or more times at 60°C; (3) high stringency hybridization conditions are in 6X SSC, at about 45°C, then wash one or more times at 65°C in 0.2X SSC, 0.1% SDS; and preferably (4) Extremely stringent hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and should be used unless otherwise stated.
本文所用的术语“闭合构象”(多特异性配体)是指配体的表位结合域彼此连接或结合，任选通过蛋白质支架的方式，使得通过一个表位结合域的表位结合，与通过另一个表位结合域的表位结合发生竞争。也就是说，关联表位可以通过各自的表位结合域单独结合，而不是同时结合。采用本文所述的方法，可得到配体的闭合构象。The term "closed conformation" (multispecific ligand) as used herein means that the epitope binding domains of the ligand are linked or bound to each other, optionally by means of a protein scaffold, such that the epitope binding through one epitope binding domain, and Competition occurs for epitope binding by another epitope binding domain. That is, cognate epitopes can be bound individually, rather than simultaneously, by their respective epitope-binding domains. Using the methods described herein, the closed conformation of the ligand can be obtained.
“开放构象(open conformation)”，是指配体的表位结合域彼此连接或结合，任选通过蛋白质支架的方式，使得通过一个表位结合域的表位结合，与通过另一个表位结合域的表位结合不发生竞争。"Open conformation" means that the epitope-binding domains of a ligand are linked or bound to each other, optionally by means of a protein scaffold, such that an epitope bound by one epitope-binding domain is bound by another epitope. Domains do not compete for epitope binding.
本文所用的术语“竞争”是指当第二表位与其关联表位结合域结合时，第一表位与其关联表位结合域的结合受到抑制。例如，结合可以是空间上受到抑制，例如通过结合域的物理阻断或通过改变结合域的结构或环境，使其对表位的亲和力(affinity)或亲合力(avidity)降低。As used herein, the term "competes" means that the binding of a first epitope to its cognate epitope binding domain is inhibited when a second epitope binds to its cognate epitope binding domain. For example, binding can be sterically inhibited, eg, by physical blocking of the binding domain or by altering the structure or environment of the binding domain such that its affinity or avidity for the epitope is reduced.
术语“免疫球蛋白单可变区”，是指能独立于其它V区(V region，V domain)而特异性结合抗原或表位的抗体可变区(VH、VHH、VL)；然而，本文所用的术语免疫球蛋白单可变区可以与其它可变区(variableregion，variable domain)呈现例如同型多聚体或异型多聚体的形式，其中其它区(region，domain)不是免疫球蛋白单可变区的抗原结合所必需的(即其中免疫球蛋白单可变区与抗原的结合独立于另外的可变区)。“免疫球蛋白单可变区”不仅包括分离的抗体单可变区多肽，而且包括含有抗体单可变区多肽序列的一个或多个单体的较大多肽。本文所用的术语“域抗体”或“dAb”与本文所用的术语“免疫球蛋白单可变区”多肽同义。本文所用的免疫球蛋白单可变区多肽是指哺乳动物(优选人，但也包括啮齿动物)免疫球蛋白单可变区多肽(例如公开于WO 00/29004，所述文献的内容通过引用全部结合到本文中)或骆驼科动物(camelid，包括骆驼、骆马及相关种系)VHH dAb。骆驼科动物dAb是免疫球蛋白单可变区多肽，它来自骆驼(camel)、美洲驼(llama)、羊驼(alpaca)、单峰骆驼(dromedary)和栗色骆马(guanaco)等物种，并且包含天然缺乏轻链的重链抗体：VHH。VHH分子要比IgG分子小约10倍，作为单一多肽，它们非常稳定，能耐受极端pH和温度条件。The term "immunoglobulin single variable region" refers to an antibody variable region (V H ,VHH , VL ) that can specifically bind to an antigen or epitope independently of other V regions (V region, V domain); However, the term immunoglobulin single variable region as used herein may take the form of, for example, homomultimers or heteromultimers with other variable regions (variable domains) which are not immunoglobulin Required for antigen binding of a protein single variable domain (ie, wherein the immunoglobulin single variable domain binds antigen independently of the other variable domain). "Immunoglobulin single variable domain" includes not only isolated antibody single variable domain polypeptides, but also larger polypeptides comprising one or more monomers of an antibody single variable domain polypeptide sequence. The term "domain antibody" or "dAb" as used herein is synonymous with the term "immunoglobulin single variable domain" polypeptide as used herein. Immunoglobulin single variable domain polypeptides as used herein refers to mammalian (preferably human, but also rodent) immunoglobulin single variable domain polypeptides (for example as disclosed in WO 00/29004, the contents of which are incorporated by reference in their entirety) incorporated herein) or camelid (including camelids, vicuna and related strains) VHH dAbs. Camelid dAbs are immunoglobulin single variable domain polypeptides from species such as camel, llama, alpaca, dromedary and guanaco, and Antibodies containing heavy chains that naturally lack light chains:VHH .VHH molecules are about 10 times smaller than IgG molecules, and as single polypeptides, they are very stable and can withstand extreme pH and temperature conditions.
本文所用的术语“肿瘤坏死因子受体1(Tumor Necrosis FactorReceptor 1，TNFR1)拮抗剂”是指一种能结合TNFR1并能抑制TNFR1的一个或多个功能的药物(例如分子、化合物)。例如，TNFR1拮抗剂可抑制TNFα与TNFR1的结合和/或抑制通过TNFR1介导的信号转导。因此，TNFR1介导的过程和细胞应答(例如在标准L929细胞毒性测定中TNFα诱导的细胞死亡)可以被TNFR1拮抗剂所抑制。TNFR1拮抗剂可以是例如有机小分子、天然产物、蛋白质、肽或肽模拟物(peptidomimetic)。可以通过筛选本文所述的文库或分子集合(例如Chemical Repository of the National Cancer Institute)或采用其它合适方法，鉴定TNFR1拮抗剂。优选的TNFR1拮抗剂是本文所述的抗体、抗体的抗原结合片段、配体和dAb单体。The term "tumor necrosis factor receptor 1 (Tumor
与本文所公开的序列相似或同源(例如至少约70％序列同一性)的序列也是本发明的组成部分。在某些实施方案中，氨基酸水平上的序列同一性可以约为80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％以上。在核酸水平上，序列同一性可以约为70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％以上。或者，当核酸区段在所选杂交条件(例如极高严格性杂交条件)下与互补链杂交时，则存在基本的同一性。核酸可以存在于完整细胞中，存在于细胞裂解物中，或者呈部分纯化或基本纯化的形式。Sequences similar or homologous (eg, at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In certain embodiments, the sequence identity at the amino acid level may be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, More than 99%. At the nucleic acid level, sequence identity may be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, More than 99%. Alternatively, substantial identity exists when the nucleic acid segments hybridize to a complementary strand under selected hybridization conditions (eg, very high stringency hybridization conditions). Nucleic acids may be present in intact cells, in cell lysates, or in partially or substantially purified form.
两序列间“同源性”或“序列同一性”或“相似性”(这些术语在本文可互换使用)的计算如下进行。为了优化比较目的，将序列比对(例如为了比较目的，对于最佳比对，可以在第一和第二氨基酸或核酸序列中引入空位，非同源序列可以忽略不计)。在一个优选的实施方案中，为了比较目的，所比对的参考序列长度占参考序列总长度的至少30％、优选至少40％、更优选至少50％、甚至更优选至少60％、甚至更优选至少70％、80％、90％、100％。然后，对相应氨基酸位置或核苷酸位置的氨基酸残基或核苷酸进行比较。当第一序列中的某个位置被与第二序列相应位置的相同氨基酸残基或核苷酸占据时，那么分子在该位置上是相同的(本文所用的氨基酸或核酸“同源性”等同于氨基酸或核酸“同一性”)。两序列间的％同一性是考虑到空位数和各空位长度后的两序列共享的相同位置数的函数，需要引入这些空位以进行两序列的最佳比对。Calculations of "homology" or "sequence identity" or "similarity" (the terms are used interchangeably herein) between two sequences are performed as follows. The sequences are aligned for optimal comparison purposes (eg, for optimal alignment, gaps can be introduced in the first and second amino acid or nucleic acid sequences, non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the aligned reference sequence accounts for at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably of the total length of the reference sequence At least 70%, 80%, 90%, 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "homology" is equivalent to to amino acid or nucleic acid "identity"). The % identity between the two sequences is a function of the number of identical positions shared by the two sequences taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.
最好采用BLAST算法(2.0版)进行序列比对，参数设定到默认值。BLAST算法在美国政府(“.gov”)的国立健康研究院(“nih”)国立生物技术信息中心(National Center for Biotechnology Information，“.ncbi”)的万维网(“www”)上、在“/Blast/”目录下、在“blast_help.html”文件内有详细介绍。检索参数定义如下，最好是设定在已定好的默认参数。Preferably, the BLAST algorithm (version 2.0) is used for sequence alignment, with parameters set to default values. The BLAST algorithm is available on the World Wide Web ("www") at the National Center for Biotechnology Information (".ncbi") of the National Institutes of Health ("nih") of the U.S. Government (".gov") at "/ Blast/" directory, detailed introduction in the "blast_help.html" file. The search parameters are defined as follows, and it is best to set the default parameters that have been set.
BLAST(Basic Local Alignment Search Tool)是blastp、blastn、blastx、tblastn和tblastx程序所采用的启发式检索算法；这些程序都主要归功于采用Karlin和Altschul，1990，Proc.Natl.Acad.Sci.USA87(6)：2264-8(参见如上所述的“blast_help.html”文件)的统计学方法的发现，只有很少的强化。BLAST程序专门用于序列相似性检索，例如鉴定查询序列的同源物。这些程序一般不用于基序风格的检索。有关序列数据库相似性检索的基本问题的讨论，参见Altschul等(1994)。BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm used by the blastp, blastn, blastx, tblastn and tblastx programs; these programs are mainly attributed to the adoption of Karlin and Altschul, 1990, Proc.Natl.Acad.Sci.USA87( 6): 2264-8 (see "blast_help.html" file as above) found statistical methods with only minor enhancements. The BLAST program is designed for sequence similarity searching, eg, identifying homologues of a query sequence. These programs are generally not used for motif-style searches. For a discussion of fundamental issues in similarity searching in sequence databases, see Altschul et al. (1994).
在美国国立生物技术信息中心网站可获得的5个BLAST程序来完成以下任务：There are five BLAST programs available at the National Center for Biotechnology Information website to accomplish the following tasks:
“blastp”比较氨基酸查询序列和蛋白质序列数据库；"blastp" compares amino acid query sequences with protein sequence databases;
“blastn”比较核苷酸查询序列和核苷酸序列数据库；"blastn" compares a nucleotide query sequence to a nucleotide sequence database;
“blastx”比较核苷酸查询序列(两条链)的六个框概念翻译产物和蛋白质序列数据库；"blastx" compares six-box concept translation products of a nucleotide query sequence (both strands) and protein sequence databases;
“tblastn”比较蛋白质查询序列和所有六个读框(两条链)动态翻译的核苷酸序列数据库。"tblastn" compares the protein query sequence to a dynamically translated nucleotide sequence database for all six reading frames (both strands).
“tblastx”比较核苷酸查询序列的六个读框翻译和核苷酸序列数据库的六个读框翻译。"tblastx" compares the six reading frame translations of a nucleotide query sequence with the six reading frame translations of a nucleotide sequence database.
BLAST采用下列检索参数：BLAST takes the following search parameters:
HISTOGRAM显示每次检索得分的直方图；默认为yes(是)。(参见BLAST手册中的参数H)。HISTOGRAM displays a histogram of the scores for each retrieval; the default is yes (yes). (See parameter H in the BLAST manual).
DESCRIPTIONS限制对指定数量报告的匹配序列简短描述的数量；默认限制为100个描述。(参见手册的参数V)。另见EXPECT和CUTOFF。DESCRIPTIONS Limit the number of matching sequence short descriptions reported for the specified number; the default limit is 100 descriptions. (see parameter V in the manual). See also EXPECT and CUTOFF.
ALIGNMENTS将数据库序列限定在报告高打分区段对(high-scoring segment pairs，HSP)的指定数量；默认限制为50。如果数据库序列比这更多，为了满足统计显著性的报告阈值(参见以下EXPECT和CUTOFF)，仅报告归于最大统计显著性的匹配。(参见BLAST手册的参数B)。ALIGNMENTS limits database sequences to reporting the specified number of high-scoring segment pairs (HSPs); the default limit is 50. If there are more database sequences than this, in order to meet the reporting threshold for statistical significance (see EXPECT and CUTOFF below), only the hit attributed to the greatest statistical significance is reported. (See parameter B of the BLAST manual).
EXPECT针对数据库序列报告的匹配的统计显著性的阈值；默认值为10，使得预期仅偶然找到10个匹配，按照Karlin和Altschul(1990)的随机模型。如果归于匹配的统计显著性大于EXPECT阈值，则将不报告匹配。EXPECT阈值越低，严格性越高，导致报告的匹配机会越少。分数值是可接受的。(参见BLAST手册的参数E)。EXPECT Threshold for statistical significance of matches reported for database sequences; default is 10, such that only 10 matches are expected to be found by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance attributed to a match is greater than the EXPECT threshold, no match will be reported. Lower EXPECT thresholds are more stringent, resulting in fewer match opportunities being reported. Fractional values are acceptable. (See parameter E of the BLAST manual).
CUTOFF用于报告高打分区段对的截止(Cutoff)分值。默认值从EXPECT值(参见上文)求出。HSP是为数据库序列而报告的，仅当归于它们的统计显著性至少与具有等于CUTOFF值的分值的孤立HSP一样高时。CUTOFF值越高，严格性越高，导致报告的匹配机会越小。(参见BLAST手册的参数S)。通常，显著性阈值可以用EXPECT更直观地控制。CUTOFF is used to report the cutoff (Cutoff) score for high scoring segment pairs. The default value is derived from the EXPECT value (see above). HSPs are reported for database sequences only if the statistical significance ascribed to them is at least as high as the isolated HSP with a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, resulting in fewer chances of a match being reported. (See parameter S in the BLAST manual). Often, the significance threshold can be controlled more intuitively with EXPECT.
MATRIX指定替代打分矩阵，用于BLASTP、BLASTX、TBLASTN和TBLASTX。默认矩阵是BLOSUM62(Henikoff和Henikoff，1992，Proc.Natl.Aacad.Sci.USA 89(22)：10915-9)。有效替代选择包括：PAM40、PAM120、PAM250和IDENTITY。替代打分矩阵不适用于BLASTN；指定MATRIX在BLASTN请求中的方向返回错误响应。MATRIX specifies an alternative scoring matrix for BLASTP, BLASTX, TBLASTN, and TBLASTX. The default matrix is BLOSUM62 (Henikoff and Henikoff, 1992, Proc. Natl. Aacad. Sci. USA 89(22):10915-9). Valid alternatives include: PAM40, PAM120, PAM250, and IDENTITY. Alternative scoring matrices are not available for BLASTN; specifying the direction of MATRIX in a BLASTN request returns an error response.
STRAND将TBLASTN检索正好限制在数据库序列的顶链或底链；或将BLASTN、BLASTX或TBLASTX检索正好限制在查询序列的顶链或底链的读框。STRAND restricts a TBLASTN search to just the top or bottom strand of a database sequence; or restricts a BLASTN, BLASTX, or TBLASTX search to just the top or bottom strand of the query sequence in frame.
FILTER掩蔽具有低成分复杂性的查询序列区段(通过Wootton和Federhen(1993)Computers and Chemistry 17：149-163的SEG程序来确定)，或由短周期性内部重复序列组成的区段(通过Claverie和States，1993，Computers and Chemistry 17：191-201的XNU程序来确定，或者对于BLASTN，通过Tatusov和Lipman的DUST程序来确定(参见NCBI的互联网站点))。过滤可从blast输出中消除具有统计学显著性但没有生物学意义的报告(例如针对常见富含酸性、碱性或脯氨酸区的命中)，留下更具生物学意义的查询序列区，适用于针对数据库序列的特定匹配。FILTER masks query sequence segments with low compositional complexity (determined by the SEG program of Wootton and Federhen (1993) Computers and Chemistry 17:149-163), or segments consisting of short periodic internal repeats (by Claverie and States, 1993, Computers and Chemistry 17:191-201, or for BLASTN, the DUST program of Tatusov and Lipman (see NCBI's Internet site)). Filtering removes statistically significant but not biologically significant reports (such as hits for common acidic, basic, or proline-rich regions) from the blast output, leaving more biologically significant query sequence regions, Applies to specific matches against database sequences.
过滤程序中发现的低复杂性序列在核苷酸序列中用字母“N”替代(例如“N”重复13次)，在蛋白质序列中用字母“X”替代(例如“X”重复9次)。Low-complexity sequences found in the filter were replaced with the letter "N" in nucleotide sequences (eg, "N" repeated 13 times) and in protein sequences with the letter "X" (eg, "X" was repeated 9 times) .
过滤仅适用于查询序列(或其翻译产物)，而不适用于数据库序列。对于BLASTN，默认过滤是DUST，而对于其它程序是SEG。Filtering applies only to query sequences (or their translation products), not to database sequences. The default filter is DUST for BLASTN and SEG for other programs.
当用于SWISS-PROT中的序列时，SEG、XNU或这两者完全没有掩蔽什么，这并非是不常见的，所以不应当希望过滤总会有结果。此外，在某些情况下，序列完全被掩蔽，这表明任何针对未过滤查询序列而报告的匹配的统计学显著性应当受到怀疑。It is not uncommon for SEG, XNU, or both to mask nothing at all when used on sequences in SWISS-PROT, so filtering should not be expected to always have results. Furthermore, in some cases sequences were completely masked, suggesting that the statistical significance of any reported matches against unfiltered query sequences should be suspect.
NCBI-gi Causes NCBI gi标识符在输出中显示，除了检索号和/或位置名称之外。NCBI-gi Causes NCBI gi identifiers to appear in the output in addition to accession numbers and/or location names.
最优选采用上述NCBI网址的“/BLAST”目录下所提供的简单BLAST检索算法，进行序列比较。Most preferably, sequence comparisons are performed using the simple BLAST search algorithm provided under the "/BLAST" directory of the above-mentioned NCBI website.
除非另有说明，否则本文所用的所有科技术语都具有本领域(例如细胞培养、分子遗传学、核酸化学、杂交技术和生物化学)普通技术人员公知的相同含义。标准技术用于分子、遗传和生化方法(一般参见Sambrook等，Molecular Cloning：A Laboratory Manual，第2版(1989)Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.和Ausubel等，Short Protocols in Molecular Biology(1999)第4版，JohnWiley&Sons，Inc.，所述文献通过引用结合到本文中)和化学方法。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (eg, cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used in molecular, genetic and biochemical methods (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology ( 1999) 4th edition, John Wiley & Sons, Inc., which is incorporated herein by reference) and Chemical Methods.
TNFR1是跨膜受体，它含有胞外区和胞内区，其中胞外区能结合配体，胞内区缺乏内在信号转导活性、但能结合信号转导分子。具有结合TNF的TNFR1复合物含有3条TNFR1链和3条TNF链。(Banner等，Cell，73(3)431-445(1993))。TNF配体以三聚体形式存在，它与3条TNFR1链结合(出处同上)。这3条TNFR1链在受体-配体复合物中紧密簇集在一起，这种簇集是TNFR1介导的信号转导的先决条件。事实上，在TNF不存在时，结合TNFR1的多价物质(例如抗TNFR1抗体)可诱导TNFR1成簇和信号转导，并且通常用作TNFR1激动剂。(参见例如Belka等，EMBO，14(6)：1156-1165(1995)；Mandik-Nayak等，J.Immunol.，167：1920-1928(2001))。因此，与TNFR1结合的多价物质通常并非有效的TNFR1拮抗剂，甚至当它们阻断TNFα与TNFR1的结合时。TNFR1 is a transmembrane receptor, which contains an extracellular region and an intracellular region, wherein the extracellular region can bind ligands, and the intracellular region lacks intrinsic signal transduction activity, but can bind signal transduction molecules. The TNFR1 complex with bound TNF contains 3 TNFR1 chains and 3 TNF chains. (Banner et al., Cell, 73(3) 431-445 (1993)). TNF ligand exists as a trimer, which binds to three TNFR1 chains (supra). The three TNFR1 chains are tightly clustered together in the receptor-ligand complex, and this clustering is a prerequisite for TNFR1-mediated signal transduction. Indeed, polyvalent substances that bind TNFR1 (eg, anti-TNFR1 antibodies) can induce TNFR1 clustering and signaling in the absence of TNF and are commonly used as TNFR1 agonists. (See eg Belka et al., EMBO, 14(6):1156-1165 (1995); Mandik-Nayak et al., J. Immunol., 167:1920-1928 (2001 )). Thus, multivalent substances that bind to TNFR1 are generally not effective TNFR1 antagonists, even when they block the binding of TNFα to TNFR1.
TNFR1的胞外区包含13个氨基酸的氨基端区段(SEQ ID NO：603(人)的氨基酸1-13；SEQ ID NO：604(小鼠)的氨基酸1-13)、域1(SEQID NO：603(人)的氨基酸14-53；SEQ ID NO：604(小鼠)的氨基酸14-53)、域2(SEQ ID NO：603(人)的氨基酸54-97；SEQ ID NO：604(小鼠)的氨基酸54-97)、域3(SEQ ID NO：603(人)的氨基酸98-138；SEQED NO：604(小鼠)的氨基酸98-138)和域4(SEQ ID NO：603(人)的氨基酸139-167；SEQ ID NO：604(小鼠)的氨基酸139-167)，其后接着是膜近端区(SEQ ID NO：603(人)的氨基酸168-182；SEQ ID NO：604(小鼠)的氨基酸168-183)。(参见Banner等，Cell 73(3)431-445(1993)和Loetscher等，Cell 61(2)351-359(1990))。域2和域3接触结合配体(TNFβ、TNFα)。(Banner等，Cell，73(3)431-445(1993))。TNFR1胞外区也含有一个称为前配体结合装配区或PLAD区的区(SEQ ID NO：603(人)的氨基酸1-53；SEQ ID NO：604(小鼠)的氨基酸1-53)(美国政府，WO 01/58953；Deng等，Nature Medicine，doi：10.1038/nm1304(2005))。The extracellular region of TNFR1 comprises an amino-terminal segment of 13 amino acids (amino acids 1-13 of SEQ ID NO: 603 (human); amino acids 1-13 of SEQ ID NO: 604 (mouse)), domain 1 (SEQ ID NO : amino acids 14-53 of 603 (human); SEQ ID NO: amino acids 14-53 of 604 (mouse), domain 2 (amino acids 54-97 of SEQ ID NO: 603 (human); SEQ ID NO: 604 ( mouse), domain 3 (amino acids 98-138 of SEQ ID NO:603 (human); amino acids 98-138 of SEQED NO:604 (mouse)) and domain 4 (SEQ ID NO:603 (human) amino acids 139-167; SEQ ID NO:604 (mouse) amino acids 139-167), followed by the membrane proximal region (SEQ ID NO:603 (human) amino acids 168-182; SEQ ID NO: 604 (amino acids 168-183 of mouse). (See Banner et al., Cell 73(3) 431-445 (1993) and Loetscher et al., Cell 61(2) 351-359 (1990)).
TNFR1通过蛋白水解等过程在体内从细胞表面脱落，产生可溶性形式的TNFR1，所述过程包括TNFR1在域4或在膜近端区(SEQ IDNO：603的氨基酸168-182；SEQ ID NO：604的氨基酸168-183)的蛋白水解。可溶性TNFR1保留了结合TNFα的能力，因此起到TNFα活性的内源抑制剂的作用。TNFR1 is shed from the cell surface in vivo by processes such as proteolysis, which involves TNFR1 in
本发明涉及抗体或其抗原结合片段(例如dAb)或配体，其结合TNFR1，但不与TNF竞争性结合TNFR1。例如，抗体或其抗原结合片段(例如dAb)或配体能结合TNFR1的域1或TNFR1的域4。这样的抗体或其抗原结合片段(例如dAb)或配体具有作为诊断试剂的优势，因此可用于结合和检测、量化或测定样品中的TNFR1，但是不与样品中的TNF竞争性结合TNFR1。因此，可以准确测定样品中是否存在TNFR1以及样品中有多少TNFR1。在某些实施方案中，结合TNFR1、但不与TNF竞争性结合TNFR1的抗体或其抗原结合片段(例如dAb)或配体，就是本文所述的TNFR1拮抗剂。The invention relates to antibodies or antigen-binding fragments thereof (eg, dAbs) or ligands that bind TNFRl but do not compete with TNF for binding to TNFRl. For example, an antibody or antigen-binding fragment thereof (eg, a dAb) or a ligand can bind
本发明也涉及一种诊断试剂盒和使用说明书(例如测定样品中TNFR1的存在和/或数量)，用于确定样品中是否存在TNFR1以及样品中有多少TNFR1，所述试剂盒包括结合TNFR1、但不与TNF竞争性结合TNFR1的抗体或其抗原结合片段(例如dAb)或配体。在某些实施方案中，试剂盒还包括一种或多种辅助试剂，例如合适的缓冲剂或合适的检测试剂(例如可检测标记抗体或其抗原结合片段，其结合能结合TNFR1、但不与TNF竞争性结合TNFR1的抗体或其抗原结合片段(例如dAb)或配体)。The invention also relates to a diagnostic kit and instructions for use (e.g., to determine the presence and/or amount of TNFR1 in a sample) for determining whether and how much TNFR1 is present in a sample, said kit comprising binding to TNFR1, but An antibody or antigen-binding fragment (eg, dAb) or ligand thereof that does not compete with TNF for binding to TNFRl. In certain embodiments, the kit also includes one or more auxiliary reagents, such as a suitable buffer or a suitable detection reagent (such as a detectably labeled antibody or antigen-binding fragment thereof that binds to TNFR1 but does not bind to Antibodies or antigen-binding fragments thereof (eg, dAbs or ligands) that competitively bind TNF to TNFRl.
本发明也涉及一种装置，所述装置包括固体表面，其中把能结合TNFR1、但不与TNF竞争性结合TNFR1的抗体或其抗原结合片段(例如dAb)或配体固定在所述固体表面上，使得固定化抗体或其抗原结合片段(例如dAb)或配体能结合TNTR1。可以使用抗体或其抗原结合片段(例如dAb)或配体可固定在其上的任何合适固体表面，例如玻璃、塑料、碳水化合物(例如琼脂糖珠)。如有必要，支持物可含有所需官能团或者经过修饰以含有所需官能团，以便固定抗体或其抗原结合片段(例如dAb)或配体。装置和/或支持物可具有任何合适形状，例如片、小杆、带、板、玻片、小珠、小丸、盘、凝胶、试管、球、芯片、板或皿等。在某些实施方案中，装置是浸棒(dipstick)。The present invention also relates to a device comprising a solid surface wherein an antibody or antigen-binding fragment thereof (e.g. dAb) or ligand that binds TNFR1 but does not compete with TNF for binding to TNFR1 is immobilized on the solid surface , such that the immobilized antibody or antigen-binding fragment thereof (eg, dAb) or ligand can bind TNTR1. Any suitable solid surface on which antibodies or antigen-binding fragments thereof (eg dAbs) or ligands can be immobilized can be used, eg glass, plastic, carbohydrates (eg agarose beads). If necessary, the support may contain or be modified to contain desired functional groups for the immobilization of antibodies or antigen-binding fragments thereof (eg, dAbs) or ligands. The device and/or support may have any suitable shape, such as a sheet, rod, ribbon, plate, slide, bead, pellet, disc, gel, test tube, sphere, chip, plate or dish, and the like. In certain embodiments, the device is a dipstick.
本发明涉及对肿瘤坏死因子受体1(TNFR1；p55；CD120a)具有结合特异性的TNFR1拮抗剂(例如本文所述的配体)。优选本发明的拮抗剂对肿瘤坏死因子2(TNFR2)不具有结合特异性，或者基本上不拮抗TNFR2。在标准细胞测定中，当拮抗剂(1nM、10nM、100nM、1μM、10μM或100μM)导致不超过约5％的针对由TNFα(100pg/ml)诱导的TNFR2介导的活性的抑制时，TNFR1拮抗剂基本上不拮抗TNFR2。特别优选的TNFR1拮抗剂是用于治疗慢性炎性疾病的有效治疗药物(是有效的，具有疗效)。例如，在某些实施方案中，TNFR1拮抗剂在例如以下的慢性炎性疾病模型中有效：小鼠胶原诱发的关节炎模型、小鼠关节炎ΔARE模型、小鼠葡聚糖硫酸钠诱发的炎性肠病模型、小鼠炎性肠病ΔARE模型、小鼠吸烟所致慢性阻塞性肺病模型或合适的灵长类模型(例如灵长类胶原诱发的关节炎)。The present invention relates to antagonists of TNFRl (eg, ligands described herein) that have binding specificity for tumor necrosis factor receptor 1 (TNFRl; p55; CD120a). Preferably, the antagonists of the invention have no binding specificity for tumor necrosis factor 2 (TNFR2), or do not substantially antagonize TNFR2. TNFR1 antagonizes when the antagonist (1 nM, 10 nM, 100 nM, 1 μM, 10 μM or 100 μM) results in no more than about 5% inhibition of TNFR2-mediated activity induced by TNFα (100 pg/ml) in standard cellular assays The agent does not substantially antagonize TNFR2. Particularly preferred antagonists of TNFRl are effective therapeutic agents (are effective, have curative effect) for the treatment of chronic inflammatory diseases. For example, in certain embodiments, antagonists of TNFR1 are effective in models of chronic inflammatory diseases such as: mouse collagen-induced arthritis model, mouse arthritis ΔARE model, mouse dextran sodium sulfate-induced inflammatory disease Sexual bowel disease model, mouse inflammatory bowel disease ΔARE model, mouse smoking-induced chronic obstructive pulmonary disease model or a suitable primate model (such as primate collagen-induced arthritis).
TNFR1拮抗剂可以是单价或多价的。在某些实施方案中，所述拮抗剂是单价的，含有一个与TNFR1相互作用的结合位点。单价拮抗剂能结合一个TNFR1，而且不诱导细胞表面TNFR1的交联或成簇，这样的交联或成簇能导致受体活化和信号转导。在具体的实施方案中，TNFR1单价拮抗剂结合TNFR1的域1。在更具体的实施方案中，TNFR1单价拮抗剂结合TNFR1的域1，并与TAR2m-21-23竞争性结合小鼠TNFR1或与TAR2h-205竞争性结合人TNFR1。TNFRl antagonists can be monovalent or multivalent. In certain embodiments, the antagonist is monovalent and contains a binding site that interacts with TNFRl. Monovalent antagonists bind one TNFR1 without inducing cross-linking or clustering of TNFR1 on the cell surface, which can lead to receptor activation and signal transduction. In specific embodiments, the TNFRl monovalent antagonist binds
在其它实施方案中，TNFR1拮抗剂是多价的。TNFR1多价拮抗剂可含有两个或更多个拷贝的针对TNFR1的特定结合位点或者含有两个或更多个能结合TNFR1的不同结合位点。例如，本文所述的TNFR1拮抗剂可以是二聚体、三聚体或多聚体，其包含两个或更多个拷贝的能结合TNFR1的特定dAb或者两个或更多个能结合TNFR1的不同dAb。在标准细胞测定中，优选TNFR1多价拮抗剂基本上不激动TNFR1(起到TNFR1激动剂的作用)(即在测定中，当浓度为1nM、10nM、100nM、1μM、10μM、100μM、1000μM或5,000μM时，导致不超过约5％的由TNFα(100pg/ml)诱导的TNPR1介导的活性)。In other embodiments, the TNFRl antagonists are multivalent. Multivalent antagonists of TNFR1 may contain two or more copies of a specific binding site for TNFR1 or contain two or more different binding sites that bind TNFR1. For example, a TNFR1 antagonist described herein may be a dimer, trimer or multimer comprising two or more copies of a specific dAb that binds TNFR1 or two or more dAbs that bind TNFR1 Different dAbs. Preferably, the multivalent antagonist of TNFR1 does not substantially agonize TNFR1 (acts as a TNFR1 agonist) in a standard cellular assay (i.e., in the assay, when the concentration is 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1000 μM or 5,000 [mu]M resulted in no more than about 5% of the TNPR1-mediated activity induced by TNF[alpha] (100 pg/ml)).
在某些实施方案中，TNFR1多价拮抗剂含有两个或更多个针对TNFR1的所需表位或结构域的结合位点。例如，TNFR1多价拮抗剂可包含两个或更多个能结合TNFR1的域1中同一表位的结合位点。In certain embodiments, multivalent antagonists of TNFRl contain two or more binding sites for desired epitopes or domains of TNFRl. For example, a multivalent antagonist of TNFRl may comprise two or more binding sites that bind the same epitope in
在其它实施方案中，TNFR1多价拮抗剂含有两个或更多个能结合TNFR1的不同表位或结构域的结合位点。在一个实例中，TNFR1多价拮抗剂包含第一结合位点和第二结合位点，其中第一结合位点能结合TNFR1的域1中的第一表位，第二结合位点能结合域1中不同的第二表位。在其它实例中，TNFR1多价拮抗剂可包含能结合TNFR1的两个或更多个所需表位或结构域的结合位点。例如，TNFR1多价拮抗剂可包含针对以下TNFR1的结构域的结合位点：域1和域2、域1和域3、域1和域4、域2和域3、域2和域4、或域3和域4。例如，TNFR1多价拮抗剂可包含针对TNFR1的域1、域2和域3的结合位点，针对TNFR1的域1、域2和域4的结合位点，或针对TNFR1的域1、域3和域4的结合位点。在某些实施方案中，TNFR1拮抗剂是双特异性配体，其包含能结合TNFR1的域1的dAb以及能结合TNFR1的域3的dAb。在本文所述的标准L929细胞毒性测定或标准HeLa IL-8测定中，优选所述多价拮抗剂当浓度为约1nM、或约10nM、或约100nM、或约1μM、或约10μM时，不激动TNFR1。In other embodiments, multivalent antagonists of TNFRl contain two or more binding sites that bind different epitopes or domains of TNFRl. In one example, the multivalent antagonist of TNFR1 comprises a first binding site and a second binding site, wherein the first binding site binds a first epitope in
一些TNFR1拮抗剂能结合TNFR1并抑制TNFα与TNFR1的结合。在某些实施方案中，这样的TNFR1拮抗剂能结合TNFR1的域2和/或域3。在具体的实施方案中，所述拮抗剂与TAR2h-10-27、TAR2h-131-8、TAR2h-15-8、TAR2h-35-4、TAR2h-154-7、TAR2h-154-10或TAR2h-185-25竞争性结合TNFR1。Some TNFR1 antagonists bind TNFR1 and inhibit the binding of TNFα to TNFR1. In certain embodiments, such antagonists of
其它配体(其中优选的实施方案是TNFR1拮抗剂)不抑制TNFα与TNFR1的结合。这类配体(和拮抗剂)具有作为诊断试剂的优势，因为它们可用于结合和检测、量化或测定样品中的TNFR1，但是不与样品中的TNF竞争性结合TNFR1。因此，可以准确测定样品中是否存在TNFR1以及有多少TNFR1。Other ligands, of which preferred embodiments are TNFRl antagonists, do not inhibit the binding of TNF[alpha] to TNFRl. Such ligands (and antagonists) have advantages as diagnostic reagents because they can be used to bind and detect, quantify or measure TNFRl in a sample, but do not compete with TNF in the sample for binding to TNFRl. Thus, it is possible to accurately determine whether and how much TNFR1 is present in a sample.
一些TNFR1拮抗剂不抑制TNFα与TNFR1的结合，但却抑制通过TNFR1介导的信号转导。例如，TNFR1拮抗剂可抑制TNFα诱导的TNFR1成簇，而这是通过TNFR1的信号转导之前的步骤。这类拮抗剂具有若干优势。例如，在这样的拮抗剂存在下，TNFα能结合在细胞表面表达的并离开细胞环境的TNFR1，但是不会激活TNFR1介导的信号转导。因此，TNFR1信号诱导的额外TNF和其它炎症介质的产生将会被抑制。同样，能结合TNFR1并抑制通过TNFR1介导的信号转导、但却不抑制TNFα与TNFR1结合的TNFR1拮抗剂，将不抑制内源产生的可溶性TNFR1的TNFα的结合及抑制活性。因此，将这样的拮抗剂给予有需要的哺乳动物，可补充内源调节途径，所述途径在体内抑制TNFα的活性和TNFR1的活性。本发明也涉及配体，所述配体(i)能结合TNFR1(例如在域1)，(ii)不拮抗TNFR1介导的信号转导的活化，和(iii)不抑制TNFα与TNFR1的结合。这类配体能结合可溶性TNFR1，但不阻止可溶性受体结合TNFα，因此将这样的拮抗剂给予有需要的哺乳动物，可补充内源调节途径，所述途径在体内通过延长血清中可溶性受体的半衰期而抑制TNFα的活性。这些优势尤其与这样的配体有关：所述配体被精制成为具有较大流体动力学尺寸(hydrodynamic size)，例如通过连接PEG基团、血清白蛋白、转铁蛋白、转铁蛋白受体或者至少其转铁蛋白结合部分、抗体Fc区，或者通过与抗体区缀合。例如，可以把具有以下特性的物质(例如多肽)精制成为具有较大的抗体的抗原结合片段或成为抗体(例如精制成为Fab、Fab′、F(ab)2、F(ab′)2、IgG、scFv)：(i)能结合TNFR1(即在域1)，(ii)不拮抗TNFR1介导的信号转导的活化，和(iii)不抑制TNFα与TNFR1(例如dAb单体)的结合。也可以通过将TNFR1结合剂与能结合本文所述的能延长体内半衰期的抗原或表位的结合域(例如抗体或抗体片段)缀合或连接，从而增加配体的流体动力学尺寸及其血清半衰期(参见附录1)。例如，TNFR1结合剂(例如多肽)可以与抗血清白蛋白或抗新生动物Fc受体抗体或抗体片段(例如抗SA或抗新生动物Fc受体dAb、Fab、Fab′或scFv)或与抗SA亲和体或抗新生动物Fc受体亲和体缀合或连接。Some TNFR1 antagonists do not inhibit the binding of TNFα to TNFR1, but inhibit the signal transduction mediated by TNFR1. For example, TNFRl antagonists can inhibit TNFα-induced clustering of TNFRl, which is a step preceding signaling through TNFRl. Such antagonists have several advantages. For example, in the presence of such an antagonist, TNF[alpha] is able to bind TNFRl expressed on the cell surface and out of the cellular environment, but does not activate TNFRl-mediated signal transduction. Therefore, the production of additional TNF and other inflammatory mediators induced by TNFR1 signaling will be suppressed. Likewise, an antagonist of TNFR1 that binds TNFR1 and inhibits signaling mediated through TNFR1, but does not inhibit the binding of TNFα to TNFR1, will not inhibit the TNFα-binding and inhibitory activity of endogenously produced soluble TNFR1. Thus, administration of such antagonists to a mammal in need thereof complements the endogenous regulatory pathways that inhibit the activity of TNF[alpha] and the activity of TNFRl in vivo. The present invention also relates to ligands that (i) bind TNFR1 (eg, at domain 1), (ii) do not antagonize TNFR1-mediated activation of signal transduction, and (iii) do not inhibit the binding of TNFα to TNFR1 . Such ligands bind soluble TNFR1 but do not prevent soluble receptors from binding TNFα, so administration of such antagonists to mammals in need complements endogenous regulatory pathways that act in vivo by prolonging the expression of soluble receptors in serum. The half-life of TNFα inhibits the activity of TNFα. These advantages are especially relevant for ligands that have been refined to have a larger hydrodynamic size, for example by attachment of PEG groups, serum albumin, transferrin, transferrin receptor or At least its transferrin binding portion, antibody Fc region, or by conjugation to an antibody region. For example, substances (such as polypeptides) that have the following properties can be refined into larger antigen-binding fragments of antibodies or into antibodies (such as Fab, Fab', F(ab)2 , F(ab')2 , IgG , scFv): (i) binds TNFR1 (ie, in domain 1), (ii) does not antagonize TNFR1-mediated activation of signal transduction, and (iii) does not inhibit the binding of TNFα to TNFR1 (eg, a dAb monomer). The hydrodynamic size of the ligand and its serum can also be increased by conjugating or linking a TNFR1-binding agent to a binding domain (e.g., an antibody or antibody fragment) that binds to an antigen or epitope described herein that can extend half-life in vivo Half-life (see Appendix 1). For example, a TNFR1-binding agent (e.g., a polypeptide) can be combined with an anti-serum albumin or an anti-neonatal Fc receptor antibody or antibody fragment (e.g., an anti-SA or anti-neonatal Fc receptor dAb, Fab, Fab' or scFv) or with an anti-SA Affibody or Anti-Neozoan Fc Receptor Affibody Conjugated or Linked.
用于本发明的TNFR1结合配体的合适白蛋白、白蛋白片段或白蛋白变异体的实例参见WO 2005/077042A2，所述文献通过引用全部结合到本文中。具体地讲，下面的白蛋白、白蛋白片段或白蛋白变异体可用于本发明：Examples of suitable albumin, albumin fragments or albumin variants for use in the TNFRl binding ligands of the invention are found in WO 2005/077042A2, which is hereby incorporated by reference in its entirety. Specifically, the following albumins, albumin fragments or albumin variants are useful in the present invention:
●SEQ ID NO：1(公开于WO 2005/077042A2，该序列通过引用明确地结合到本说明书中)；●SEQ ID NO: 1 (disclosed in WO 2005/077042A2, this sequence is explicitly incorporated in this specification by reference);
●白蛋白片段或变异体，其包含WO 2005/077042A2中SEQID NO：1的氨基酸1-387或由其组成；an albumin fragment or variant comprising or consisting of amino acids 1-387 of SEQ ID NO: 1 in WO 2005/077042A2;
●白蛋白或其片段或变异体，其包含选自以下的氨基酸序列：(a)WO 2005/077042A2中SEQ ID NO：1的氨基酸54-61；(b)WO 2005/077042A2中SEQ ID NO：1的氨基酸76-89；(c)WO 2005/077042A2中SEQ ID NO：1的氨基酸92-100；(d)WO 2005/077042A2中SEQ ID NO：1的氨基酸170-176；(e)WO 2005/077042A2中SEQ ID NO：1的氨基酸247-252；(f)WO 2005/077042A2中SEQ ID NO：1的氨基酸266-277；(g)WO 2005/077042A2中SEQ ID NO：1的氨基酸280-288；(h)WO 2005/077042A2中SEQ ID NO：1的氨基酸362-368；(i)WO 2005/077042A2中SEQ ID NO：1的氨基酸439-447；(j)WO 2005/077042A2中SEQ ID NO：1的氨基酸462-475；(k)WO 2005/077042A2中SEQ ID NO：1的氨基酸478-486；和(l)WO 2005/077042A2中SEQ ID NO：1的氨基酸560-566。Albumin or a fragment or variant thereof comprising an amino acid sequence selected from: (a) amino acids 54-61 of SEQ ID NO: 1 in WO 2005/077042A2; (b) SEQ ID NO in WO 2005/077042A2: Amino acids 76-89 of 1; (c) amino acids 92-100 of SEQ ID NO: 1 in WO 2005/077042A2; (d) amino acids 170-176 of SEQ ID NO: 1 in WO 2005/077042A2; (e) WO 2005 Amino acids 247-252 of SEQ ID NO: 1 in /077042A2; (f) amino acids 266-277 of SEQ ID NO: 1 in WO 2005/077042A2; (g) amino acids 280-280- of SEQ ID NO: 1 in WO 2005/077042A2 288; (h) amino acids 362-368 of SEQ ID NO: 1 in WO 2005/077042A2; (i) amino acids 439-447 of SEQ ID NO: 1 in WO 2005/077042A2; (j) SEQ ID in WO 2005/077042A2 Amino acids 462-475 of NO: 1; (k) amino acids 478-486 of SEQ ID NO: 1 in WO 2005/077042A2; and (l) amino acids 560-566 of SEQ ID NO: 1 in WO 2005/077042A2.
用于本发明的TNFR1结合配体的合适白蛋白、其片段和变异体的其它实例参见WO 03/076567A2，所述文献通过引用全部结合到本文中。具体地讲，下面的白蛋白、片段或变异体可用于本发明：Further examples of suitable albumin, fragments and variants thereof for use in the TNFRl binding ligands of the invention are found in WO 03/076567A2, which is hereby incorporated by reference in its entirety. Specifically, the following albumins, fragments or variants can be used in the present invention:
●人血清白蛋白，参见WO 03/076567A2，例如在图3中(该序列信息通过引用明确地结合到本说明书中)；human serum albumin, see WO 03/076567A2, for example in Figure 3 (this sequence information is expressly incorporated in this specification by reference);
●人血清白蛋白(HA)，它由585个氨基酸的单一非糖基化多肽链组成，分子量为66，500(参见Meloun等，FEBS Letters58：136(1975)；Behrens等，Fed.Proc.34：591(1975)；Lawn等，Nucleic Acids Research 9：6102-6114(1981)；Minghetti等，J.Biol.Chem.261：6747(1986))；Human serum albumin (HA), which consists of a single non-glycosylated polypeptide chain of 585 amino acids with a molecular weight of 66,500 (see Meloun et al., FEBS Letters 58:136 (1975); Behrens et al., Fed.Proc.34 : 591 (1975); Lawn et al., Nucleic Acids Research 9: 6102-6114 (1981); Minghetti et al., J. Biol. Chem. 261: 6747 (1986));
●白蛋白多态变异体或其类似物或片段，参见Weitkamp等，Ann.Hum.Genet.37：219(1973)；Polymorphic variants of albumin or analogs or fragments thereof, see Weitkamp et al., Ann. Hum. Genet. 37: 219 (1973);
●白蛋白片段或变异体，参见EP 322094，例如HA(1-373)、HA(1-388)、HA(1-389)、HA(1-369)和HA(1-419)以及1-369和1-419间的片段；Albumin fragments or variants, see EP 322094, such as HA(1-373), HA(1-388), HA(1-389), HA(1-369) and HA(1-419) and 1- 369 and fragments between 1-419;
●白蛋白片段或变异体，参见EP 399666，例如HA(1-177)和HA(1-200)和HA(1-X)之间的片段，其中X为178-199间的任意数字。Albumin fragments or variants, see EP 399666, eg HA(1-177) and fragments between HA(1-200) and HA(1-X), where X is any number between 178-199.
当一个或多个半衰期延长部分(例如白蛋白、转铁蛋白及其片段和类似物)用于本发明的TNFR1结合配体时，可以用任何合适方法将其缀合，例如通过与TNFR1结合部分(例如抗TNFR1 dAb或抗体片段)直接融合，例如通过使用编码融合蛋白的单个核苷酸构建体，其中融合蛋白编码成为单一多肽链和半衰期延长部分(位于TNFR1结合部分的N端或C端)。或者，可以通过使用各部分间的肽接头(例如WO 03/076567 A2或WO 2004/003019所述的肽接头(这些接头的全部公开内容通过引用结合到本说明书中，以提供用于本发明的实例))，来完成缀合反应。When one or more half-life extending moieties (e.g., albumin, transferrin, and fragments and analogs thereof) are used in the TNFR1-binding ligands of the invention, they may be conjugated by any suitable method, for example by binding to the TNFR1-binding moiety (e.g. anti-TNFR1 dAb or antibody fragment) direct fusion, e.g. by using a single nucleotide construct encoding a fusion protein encoded as a single polypeptide chain and a half-life extending moiety (either N- or C-terminal to the TNFR1-binding moiety) . Alternatively, it can be achieved by using peptide linkers between the parts, such as those described in WO 03/076567 A2 or WO 2004/003019 (the entire disclosures of these linkers are incorporated herein by reference to provide Example)), to complete the conjugation reaction.
在更具体的实施方案中，能结合TNFR1并抑制通过TNFR1介导的信号转导、但却不抑制TNFα与TNFR1结合的TNFR1拮抗剂，能结合TNFR1的域1或TNFR1的域4。在某些实施方案中，这种TNFR1拮抗剂是能结合TNFR1的域1或TNFR1的域4的dAb单体或配体。In a more specific embodiment, an antagonist of TNFR1 that binds TNFR1 and inhibits signal transduction mediated through TNFR1, but does not inhibit the binding of TNFα to TNFR1, binds
在一个具体实施方案中，TNFR1拮抗剂(例如dAb单体或配体)能结合TNFR1的域1，并且一旦结合TNFα后能抑制通过TNFR1介导的信号转导。这样的拮抗剂可抑制通过TNFR1的信号转导，但不抑制TNFα与TNFR1的结合和/或TNFR1的脱落，以产生可溶性TNFR1。因此，将这样的拮抗剂给予有需要的哺乳动物，可补充内源调节途径，所述途径在体内抑制TNFα的活性和TNFR1的活性。In a specific embodiment, an antagonist of TNFRl (eg, a dAb monomer or a ligand) binds
其它TNFR1拮抗剂能结合TNFR1，但不结合域4。这类拮抗剂可抑制TNFR1的功能，但不抑制可溶性TNFR1的脱落。因此，将这样的拮抗剂给予有需要的哺乳动物，可补充内源调节途径，所述途径在体内抑制TNFα的活性和TNFR1的活性。Other TNFRl antagonists bind TNFRl but not
在某些实施方案中，所述拮抗剂(例如化合物，新型化学实体、dAb单体、配体)能结合TNFR1的域1并与TAR2m-21-23竞争性结合小鼠TNFR1或与TAR2h-205竞争性结合人TNFR1。在其它实施方案中，所述拮抗剂(例如化合物，新型化学实体、dAb单体、配体)能结合TNFR1的域2或域4。在其它实施方案中，所述拮抗剂(例如化合物、新型化学实体、dAb单体、配体)能结合TNFR1的域3并与TAR2h-131-8、TAR2h-15-8、TAR2h-35-4、TAR2h-154-7、TAR2h-154-10、TAR2h-185-25或TAR2h-27-10竞争性结合TNFR1(例如人和/或小鼠TNFR1)。In certain embodiments, the antagonist (e.g., compound, novel chemical entity, dAb monomer, ligand) binds
一些配体(在优选的实施方案中为TNFR1拮抗剂)能结合人TNFR1和小鼠TNFR1。这类配体(例如拮抗剂、dAb单体)提供了这样的优势：可用相同配体进行临床前和临床研究，而不需要用合适替代配体来进行临床前研究。Some ligands (in preferred embodiments, TNFRl antagonists) bind both human TNFRl and mouse TNFRl. Such ligands (eg antagonists, dAb monomers) offer the advantage that the same ligand can be used for preclinical and clinical studies without the need for a suitable surrogate ligand for preclinical studies.
在其它实施方案中，所述拮抗剂或配体是对TNFR1或其抗原结合片段(例如Fab片段、Fab′片段、F(ab′)2片段或Fv片段(例如scFV))具有结合特异性的抗体。在其它实施方案中，所述拮抗剂或配体是单价的，例如dAb或抗体的单价抗原结合片段，例如Fab片段、Fab′片段或Fv片段。In other embodiments, the antagonist or ligand has binding specificity for TNFR1 or an antigen-binding fragmentthereof (e.g., a Fab fragment, a Fab' fragment, an F(ab') fragment, or an Fv fragment (e.g., scFv)). Antibody. In other embodiments, the antagonist or ligand is monovalent, such as a dAb or a monovalent antigen-binding fragment of an antibody, such as a Fab fragment, Fab' fragment, or Fv fragment.
在本说明书中所述的本发明的其它实施方案中，在本发明的拮抗剂或配体中不使用“dAb”，是考虑技术人员可使用包含能结合TNFR1的dAb的CDR的结构域(例如移植到合适蛋白质支架或骨架上的CDR，例如亲和体、SpA支架、LDL受体A类区或EGF区)或者可以是包含针对TNFR1的结合位点的蛋白质结构域，例如其中所述结构域选自亲和体、SpA区、LDL受体A类区或EGF区。因此，总的来说，本发明的公开内容应视为提供了拮抗剂、配体以及使用所述结构域替代dAb的方法。In other embodiments of the invention described in this specification, "dAbs" are not used in the antagonists or ligands of the invention, considering that the skilled artisan can use domains comprising the CDRs of dAbs that bind TNFR1 (e.g. CDRs grafted onto a suitable protein scaffold or backbone, such as Affibodies, SpA scaffolds, LDL receptor class A regions, or EGF regions) or may be protein domains comprising a binding site for TNFR1, such as where the domain selected from Affibody, SpA region, LDL receptor class A region or EGF region. In general, therefore, the present disclosure should be viewed as providing antagonists, ligands, and methods of using such domains in place of dAbs.
优选的TNFR1拮抗剂是本文所述的配体。配体包含对TNFR1具有结合特异性的免疫球蛋白单可变区或域抗体(dAb)或所述dAb的合适形式的互补决定区。配体可以是由这样的dAb组成或基本上由这样的dAb组成的多肽。配体也可以是包含合适形式(例如抗体形式(例如IgG样形式、scFv、Fab、Fab′、F(ab′)2))的dAb(或dAb的CDR)的多肽，包含能结合TNFR1的dAb以及能结合另一靶蛋白、抗原或表位(例如血清白蛋白)的第二dAb的双特异性配体，或者本文所述的多特异性配体。Preferred antagonists of TNFRl are the ligands described herein. The ligand comprises an immunoglobulin single variable region or domain antibody (dAb) with binding specificity for TNFRl or a complementarity determining region of a suitable form of said dAb. A ligand may be a polypeptide consisting or consisting essentially of such a dAb. The ligand may also be a polypeptide comprising a dAb (or a CDR of a dAb) in a suitable format, such as an antibody format (eg, IgG-like format, scFv, Fab, Fab', F(ab')2 ), comprising a dAb capable of binding TNFR1 and a dual specific ligand of a second dAb capable of binding another target protein, antigen or epitope (eg, serum albumin), or a multispecific ligand as described herein.
TNFR1拮抗剂，包括本发明任何方面的配体、以及用于构建所述配体的dAb单体，都可有利地从它们的关联靶上解离下来，根据表面等离子共振的测定，Kd为300nM～5pM(即3×10-7M～5×10-12M)、优选50nM～20pM、或5nM～200pM或1nM～100pM，1×10-7M以下、1×10-8M以下、1×10-9M以下、1×10-10M以下、1×10-11M以下；和/或K解离速率常数为5×10-1s-1～1×10-7s-1、优选1×10-2s-1～1×10-6s-1、或5×10-3s-1～1×10-5s-1、或5×10-1s-1以下、或1×10-2s-1以下、或1×10-3s-1以下、或1×104s-1以下、或1×10-5s-1以下、或1×10-6s-1以下。Kd速率常数定义为K解离/K缔合。Antagonists of TNFR1, including ligands of any aspect of the invention, and dAb monomers used to construct said ligands, advantageously dissociate from their cognate targets with a Kof 300nM～5pM (that is, 3×10-7 M～5×10-12 M), preferably 50nM～20pM, or 5nM～200pM or 1nM～100pM, 1×10-7 M or less, 1×10-8 M or less, Below 1×10-9 M, below 1×10-10 M, below 1×10-11 M; and/or Kdissociation rate constant is 5×10-1 s-1 ~1×10-7 s-1 , preferably 1×10-2 s-1 to 1×10-6 s-1 , or 5×10-3 s-1 to 1×10-5 s-1 , or less than 5×10-1 s-1 , or 1×10-2 s-1 or less, or 1×10-3 s-1 or less, or 1×104 s-1 or less, or 1×10-5 s-1 or less, or 1×10-6 s-1 or less. The Kd rate constant is defined as Kdissociation /Kassociation .
在其它实施方案中，所述拮抗剂结合TNFR1并抑制TNFR1的一个或多个功能(例如受体成簇、受体信号传导或TNFα与TNFR1的结合)，而且也结合TNF受体超家族的另一成员。优选该类拮抗剂也抑制TNF受体超家族其它成员的功能(例如成员簇集、信号转导或该成员与其关联配体的结合)。TNF受体超家族是本领域公知的一组蛋白质，它们包括TNFR1(p55、CD120a、p60、TNF受体超家族成员1A、TNFRSF1A)、TNFR2(p75、p80、CD120b、TNF受体超家族成员1B、TNFRSF1B)、In other embodiments, the antagonist binds TNFR1 and inhibits one or more functions of TNFR1 (e.g., receptor clustering, receptor signaling, or binding of TNFα to TNFR1), and also binds another member of the TNF receptor superfamily. a member. Preferably such antagonists also inhibit the function of other members of the TNF receptor superfamily (eg, member clustering, signal transduction, or binding of that member to its cognate ligand). TNF receptor superfamily is a group of proteins well known in the art, they include TNFR1 (p55, CD120a, p60, TNF receptor superfamily member 1A, TNFRSF1A), TNFR2 (p75, p80, CD120b, TNF receptor superfamily member 1B , TNFRSF1B),
CD18(TNFRSF3，LTBR，TNFR2-RP，TNFR-RP，TNFCR，TNF-R-III)，OX40(TNFRSF4，ACT35，TXGP1L)，CD40(TNFRSF5，p50，Bp50)，Fas(CD95，TNFRSF6，APO-1，APTI)，DcR3(TNFRSF6B)，CD27(TN-FRSF7，Tp55，S152)，CD30(TNFRSF8，Ki-1，D1S166E)，CD137(TNFRSF9，4-1BB，ILA)，TRAILR-1(TNFRSF10A，DR4，Apo2)，TRAIL-R2(TNFRSF10B，DR5，KILLER，TRICK2A，TRICKB)，TRAILR3(TNFRSF10C，DcR1，LIT，TRID)，TRAILR4(TNFRSF10D，DcR2，TRUNDD)，RANK(TNFRSF11A)，OPG(TNFRSF11B，OCIF，TR1)，DR3(TNFRSF12，TRAMP，WSL-1，LARD，WSL-LR，DDR3，TR3，APO-3)，DR3L(TNFRSF12L)，TAC1(TNFRSF13B)，BAFFR(TNFRSF13C)，HVEM(TNFRSF14，ATAR，TR2，LIGHTR，HVEA)，NGFR(TNFRSF16)，BCMA(TNFRSF17，BCM)，AITR(TNFRSF18，GITR)，TNFRSF19，FLJ14993(TNFRSF19L，RELT)，DR6(TNFRSF21)，SOBa(TNFRSF22，Tnfrh2，2810028K06Rik)，mSOB(THFRSF23，Tnfrh1)CD18 (TNFRSF3, LTBR, TNFR2-RP, TNFR-RP, TNFCR, TNF-R-III), OX40 (TNFRSF4, ACT35, TXGP1L), CD40 (TNFRSF5, p50, Bp50), Fas (CD95, TNFRSF6, APO-1 , APTI), DcR3 (TNFRSF6B), CD27 (TN-FRSF7, Tp55, S152), CD30 (TNFRSF8, Ki-1, D1S166E), CD137 (TNFRSF9, 4-1BB, ILA), TRAILR-1 (TNFRSF10A, DR4, Apo2), TRAIL-R2 (TNFRSF10B, DR5, KILLER, TRICK2A, TRICKB), TRAILR3 (TNFRSF10C, DcR1, LIT, TRID), TRAILR4 (TNFRSF10D, DcR2, TRUNDD), RANK (TNFRSF11A), OPG (TNFRSF11B, OCIF, TR1 ), DR3 (TNFRSF12, TRAMP, WSL-1, LARD, WSL-LR, DDR3, TR3, APO-3), DR3L (TNFRSF12L), TAC1 (TNFRSF13B), BAFFR (TNFRSF13C), HVEM (TNFRSF14, ATAR, TR2, LIGHTR, HVEA), NGFR (TNFRSF16), BCMA (TNFRSF17, BCM), AITR (TNFRSF18, GITR), TNFRSF19, FLJ14993 (TNFRSF19L, RELT), DR6 (TNFRSF21), SOBa (TNFRSF22, Tnfrh2, 2810028K06Rik), m2SOB3 (THFRSF , Tnfrh1)
在某些实施方案中，所述拮抗剂包含能结合TNFR1并抑制TNFR1功能的第一dAb以及能结合TNF受体超家族另一成员的第二dAb，所述另一成员例如以上列举的TNFR2(CD120b)、OX40、CD40、Fas(CD95)、TRAILR-1、TRAILR-2、TAC1、BCMA等。在另一个实施方案中，所述拮抗剂包含能结合TNFR1并抑制TNFR1功能、并且也能结合TNF受体超家族另一成员的dAb单体(所述功能例如受体成簇、受体信号传导或TNFα与TNFR1的结合)，所述另一成员例如以上列举的TNFR2(CD120b)、OX40、CD40、Fas(CD95)、TRAILR-1、TRAILR-2、TAC1、BCMA等。In certain embodiments, the antagonist comprises a first dAb that binds TNFR1 and inhibits TNFR1 function and a second dAb that binds another member of the TNF receptor superfamily, such as TNFR2 ( CD120b), OX40, CD40, Fas (CD95), TRAILR-1, TRAILR-2, TAC1, BCMA, etc. In another embodiment, the antagonist comprises a dAb monomer that binds TNFR1 and inhibits the function of TNFR1 and also binds another member of the TNF receptor superfamily (such functions as receptor clustering, receptor signaling or the combination of TNFα and TNFR1), the other members are, for example, TNFR2 (CD120b), OX40, CD40, Fas (CD95), TRAILR-1, TRAILR-2, TAC1, BCMA, etc. listed above.
与TNFR1结合的配体和dAb单体Ligands and dAb monomers that bind to TNFR1
本发明提供包含抗TNFR1 dAb单体的配体(例如包含这种dAb的双特异性配体)，根据表面等离子共振的测定，所述单体与TNF受体I结合的Kd为300nM～5pM(即3×10-7M～5×10-12M)、优选50nM～20pM、更优选5nM～200pM、最优选1nM～100pM，例如1×10-7M以下、优选1×10-8M以下、更优选1×10-9M以下、有利的是1×10-10M以下、最优选1×10-11M以下；和/或K解离速率常数为5×10-1s-1～1×10-7s-1、优选1×10-2s-1～1×10-6s-1、更优选5×10-3s-1～1×10-5s-1例如5×10-1s-1以下、优选1×10-2s-1以下、有利的是1×10-3s-1以下、更优选1×10-4s-1以下、还更优选1×10-5s-1以下、最优选1×10-6s-1以下。The present invention provides a ligand comprising an anti-TNFR1 dAb monomer (such as a dual-specific ligand comprising such a dAb), and according to the determination of surface plasmon resonance, the Kd of the monomer binding to TNF receptor I is 300nM～5pM (that is, 3×10-7 M to 5×10-12 M), preferably 50nM to 20pM, more preferably 5nM to 200pM, most preferably 1nM to 100pM, for example, 1×10-7 M or less, preferably 1×10-8 M Below, more preferably below 1×10-9 M, advantageously below 1×10-10 M, most preferably below 1×10-11 M; and/or the Kdissociation rate constant is 5×10-1 s-1 ~1×10-7 s-1 , preferably 1×10-2 s-1 ~1×10-6 s-1 , more preferably 5×10-3 s-1 ~1×10-5 s-1 for example 5 ×10-1 s-1 or less, preferably 1×10-2 s-1 or less, advantageously 1×10-3 s-1 or less, more preferably 1×10-4 s-1 or less, still more preferably 1× 10-5 s-1 or less, most preferably 1×10-6 s-1 or less.
优选配体或dAb单体抑制TNFα与TNFα受体1(p55受体)的结合，其抑制浓度50(IC50)为500nM～50pM、优选100nM～50pM、更优选10nM～100pM、有利的是1nM～100pM；例如50nM以下、优选5nM以下、更优选500pM以下、有利的是200pM以下、最优选100pM以下。优选TNF受体1的靶是人TNFα。Preferably, the ligand or dAb monomer inhibits the binding of TNFα to TNFα receptor 1 (p55 receptor), and its inhibitory concentration 50 (IC50 ) is 500nM to 50pM, preferably 100nM to 50pM, more preferably 10nM to 100pM, and advantageously 1nM ~ 100 pM; eg below 50 nM, preferably below 5 nM, more preferably below 500 pM, advantageously below 200 pM, most preferably below 100 pM. A preferred target of
优选配体或dAb结合人TNFR1并抑制人TNFα与人TNFR1的结合，或者抑制因响应TNFα结合而通过TNFR1的信号传导。例如，在某些实施方案中，所述配体或dAb单体能结合TNFR1并抑制TNFR1介导的信号传导，但基本上不抑制TNFα与TNFR1的结合。在某些实施方案中，所述配体或dAb单体抑制由TNFα诱导的细胞表面上TNFR1的交联或成簇。这类配体或dAb(例如本文所述的TAR2m-21-23)是有利的，因为它们可拮抗细胞表面的TNFR1，但基本上不降低内源可溶性TNFR1的抑制活性。例如，在受体结合测定中，所述配体或dAb能结合TNFR1，但抑制TNFα与TNFR1的结合不超过约10％、不超过约5％、不超过约4％、不超过约3％、不超过约2％或不超过约1％。并且，在这些实施方案中，在标准细胞测定中，所述配体或dAb抑制TNFα诱导的TNFR1交联和/或TNFR1介导的信号转导至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％、至少约90％、至少约95％或至少约99％。Preferably the ligand or dAb binds to human TNFRl and inhibits the binding of human TNF[alpha] to human TNFRl, or inhibits signaling through TNFRl in response to TNF[alpha] binding. For example, in certain embodiments, the ligand or dAb monomer binds TNFR1 and inhibits TNFR1-mediated signaling, but does not substantially inhibit the binding of TNFα to TNFR1. In certain embodiments, the ligand or dAb monomer inhibits TNF[alpha]-induced crosslinking or clustering of TNFRl on the cell surface. Such ligands or dAbs (such as TAR2m-21-23 described herein) are advantageous because they antagonize cell surface TNFRl without substantially reducing the inhibitory activity of endogenous soluble TNFRl. For example, in a receptor binding assay, the ligand or dAb is capable of binding TNFR1 but inhibits the binding of TNFα to TNFR1 by no more than about 10%, by no more than about 5%, by no more than about 4%, by no more than about 3%, Not more than about 2% or not more than about 1%. And, in these embodiments, the ligand or dAb inhibits TNFα-induced TNFR1 cross-linking and/or TNFR1-mediated signal transduction by at least about 10%, at least about 20%, at least about 30% in a standard cellular assay. %, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.
如本文所述，配体可以是单价的(例如dAb单体)或多价的(例如双特异性、多特异性)。在具体的实施方案中，所述配体是能结合TNFR1的域1的dAb单体。相对于其它结合形式(例如单克隆抗体)来说，能结合TNFR1的域1的域抗体单体具有小足迹(smallfootprint)。因此，这样的dAb单体可选择性阻断域1，但不干扰TNFR1其它结构域的功能。例如，能结合TNFR1的域1的dAb单体可拮抗TNFR1，但不抑制TNFα与TNPR1的结合或TNFR1的脱落。As described herein, a ligand can be monovalent (eg, a dAb monomer) or multivalent (eg, bispecific, multispecific). In specific embodiments, the ligand is a dAb monomer that binds
在更具体的实施方案中，所述配体是能结合TNFR1的域1并与TAR2m-21-23竞争性结合小鼠TNFR1或与TAR2h-205竞争性结合人TNFR1的dAb单体。In a more specific embodiment, the ligand is a dAb monomer that binds
在其它实施方案中，所述配体是多价的，包含两个或更多个能结合TNFR1的dAb单体。多价配体可含有两个或更多个拷贝的能结合TNFR1的特定dAb，或者含有两个或更多个能结合TNFR1的dAb。例如，本文所述的配体可以是二聚体、三聚体或多聚体，其包含两个或更多个拷贝的能结合TNFR1的特定dAb，或者两个或更多个能结合TNFR1的不同dAb。在一些实例种，配体是同型二聚体或同型三聚体，它们分别包含两个或三个拷贝的能结合TNFR1的特定dAb。在标准细胞测定中，优选的多价配体基本上不激动TNFR1(作为TNFR1激动剂)(即在测定中，当浓度为1nM、10nM、100nM、1μM、10μM、100μM、1000μM或5,000μM时，导致不超过约5％的由TNFα(100pg/ml)诱导的TNFR1介导的活性)。In other embodiments, the ligand is multivalent comprising two or more dAb monomers that bind TNFRl. A multivalent ligand may contain two or more copies of a particular dAb that binds TNFRl, or two or more dAbs that bind TNFRl. For example, a ligand described herein may be a dimer, trimer, or multimer comprising two or more copies of a specific dAb that binds TNFR1, or two or more copies of a specific dAb that binds TNFR1. Different dAbs. In some examples, the ligand is a homodimer or homotrimer comprising two or three copies, respectively, of a specific dAb that binds TNFRl. Preferred multivalent ligands do not substantially agonize TNFR1 (as TNFR1 agonists) in standard cellular assays (i.e., at concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1000 μM, or 5,000 μM in the assay, results in no more than about 5% of the TNFRl-mediated activity induced by TNF[alpha] (100 pg/ml)).
在某些实施方案中，多价配体含有两个或更多个能结合TNFR1所需表位或结构域的dAb。例如，多价配体可包含两个或更多个拷贝的能结合TNFR1的域1所需表位的dAb。In certain embodiments, the multivalent ligand contains two or more dAbs that bind a desired epitope or domain of TNFRl. For example, a multivalent ligand may comprise two or more copies of a dAb that binds the desired epitope of
在其它实施方案中，多价配体含有两个或更多个能结合TNFR1不同表位或结构域的dAb。在一个实例中，多价配体包含第一dAb和第二dAb，其中第一dAb能结合TNFR1的域1中的第一表位，第二dAb能结合TNFR1的域1中不同的第二表位。在其它实例中，多价配体包含能结合TNFR1的两个或更多个所需表位或结构域的dAb。例如，多价配体可包含结合以下TNFR1的结构域的dAb：域1和域2、域1和域3、域1和域4、域2和域3、域2和域4、或域3和域4。In other embodiments, the multivalent ligand contains two or more dAbs that bind different epitopes or domains of TNFRl. In one example, the multivalent ligand comprises a first dAb and a second dAb, wherein the first dAb binds a first epitope in
在某些实施方案中，多价配体是双特异性配体，所述配体包含能结合TNFR1的域1的dAb，以及能结合TNFR1的域3的dAb。该类配体可以高亲和力结合TNFR1，并且比起其它配体形式(例如dAb单体)来说，对于在其表面高密度过量表达TNFR1或表达TNFR1的细胞的结合选择性更高。In certain embodiments, the multivalent ligand is a dual specific ligand comprising a dAb that binds
在其它具体的实施方案中，多价配体包含两个或更多个能结合TNFR1的域1的dAb或者两个或更多个拷贝的特定dAb。该类多价配体可以低亲和力结合TNFR1单体，但却以高亲合力结合受体多聚体(例如在受体配体复合物中所观察到的三聚体)。因此，可以将该形式的配体给予有效靶向受体，所述受体已彼此成簇或缔合和/或与TNFR1介导的信号转导所需的配体(例如TNFα)簇集或缔合。In other specific embodiments, the multivalent ligand comprises two or more dAbs or two or more copies of a particular dAb that binds
一些配体或dAb单体结合TNFR1并抑制TNFα与TNFR1的结合。在某些实施方案中，这样的配体或dAb单体能结合TNFR1的域2和/或域3。在具体的实施方案中，所述配体或dAb单体能结合TNFR1的域3。在更具体的实施方案中，所述配体或dAb单体能结合TNFR1的域3并与TAR2h-10-27、TAR2h-131-8、TAR2h-15-8、TAR2h-35-4、TAR2h-154-7、TAR2h-154-10或TAR2h-185-25竞争性结合TNFR1。Some ligands or dAb monomers bind TNFR1 and inhibit the binding of TNFα to TNFR1. In certain embodiments, such ligands or dAb monomers are capable of binding
其它配体或dAb单体不抑制TNFα与TNFR1的结合。这类拮抗剂具有作为诊断试剂的优势，因为它们可用于结合和检测、量化或测定样品中的TNFR1，但是不与样品中的TNF竞争性结合TNFR1。因此，可以准确测定样品中是否存在TNFR1以及有多少TNFR1。Other ligands or dAb monomers did not inhibit the binding of TNFα to TNFR1. Such antagonists have advantages as diagnostic reagents because they can be used to bind and detect, quantify or measure TNFRl in a sample, but do not compete with TNF in the sample for binding to TNFRl. Thus, it is possible to accurately determine whether and how much TNFR1 is present in a sample.
一些配体和dAb单体不抑制TNFα与TNFR1的结合，但却抑制通过TNFR1介导的信号转导。例如，配体或dAb单体可抑制TNFα诱导的TNFR1成簇，而这是在通过TNFR1的信号转导之前的步骤。这类配体或dAb单体具有若干优势，正如本文所讨论的有关具有这些特性的拮抗剂。在具体的实施方案中，该类配体或dAb单体能结合TNFR1的域1或TNFR1的域4。在某些实施方案中，所述配体是能结合TNFR1的域1或TNFR1的域4的dAb单体。Some ligands and dAb monomers do not inhibit the binding of TNFα to TNFR1, but inhibit the signal transduction mediated by TNFR1. For example, ligands or dAb monomers can inhibit TNF[alpha]-induced clustering of TNFR1, a step preceding signaling through TNFR1. Such ligands or dAb monomers have several advantages, as discussed herein with respect to antagonists having these properties. In specific embodiments, such ligands or dAb monomers are capable of binding
在一个具体的实施方案中，所述配体或dAb单体能结合TNFR1的域1，并且一旦结合TNFα后就能抑制通过TNFR1介导的信号转导。这类配体或dAb单体可抑制通过TNFR1的信号转导，但不抑制TNFα与TNFR1的结合和/或TNFR1的脱落产生可溶性TNFR1。因此，将这样的配体或dAb单体给予有需要的哺乳动物，可补充内源调节途径，所述途径在体内抑制TNFα的活性和TNFR1的活性。In a specific embodiment, the ligand or dAb monomer is capable of binding
其它配体或dAb单体能结合TNFR1，但不结合域4。这类配体或dAb单体可抑制TNFR1的功能，但不抑制可溶性TNFR1的脱落。因此，将这样的拮抗剂给予有需要的哺乳动物，可补充内源调节途径，所述途径在体内抑制TNFα的活性和TNFR1的活性。Other ligands or dAb monomers can bind TNFR1 but not
在标准测定(例如本文所述的标准L929或标准HeLa IL-8测定)中，优选的配体或dAb单体中和TNFα或TNFR1(抑制其活性)，其中和剂量50(ND50)为500nM～50pM、优选100nM～50pM、更优选10nM～100pM、有利的是1nM～100pM；例如50nM以下、优选5nM以下、更优选500pM以下、有利的是200pM以下、最优选100pM以下。Preferred ligands or dAb monomers neutralize TNFα or TNFR1 (inhibit their activity) in standard assays (such as the standard L929 or standard HeLa IL-8 assays described herein) with a neutralizing dose 50 (ND50 ) of 500 nM ~50pM, preferably 100nM~50pM, more preferably 10nM~100pM, advantageously 1nM~100pM; for example below 50nM, preferably below 5nM, more preferably below 500pM, advantageously below 200pM, most preferably below 100pM.
在某些实施方案中，所述配体或dAb单体特异性结合人肿瘤坏死因子受体1(TNFR1；p55)，根据表面等离子共振的测定，所述配体或dAb单体从人TNFR1上解离下来的解离常数(Kd)为50nM～20pM，K解离速率常数为5×10-1s-1～1×10-7s-1。In some embodiments, the ligand or the dAb monomer specifically binds to human tumor necrosis factor receptor 1 (TNFR1; p55), and according to the determination of surface plasmon resonance, the ligand or the dAb monomer is separated from human TNFR1 The dissociated dissociation constant (Kd ) is 50nM～20pM, and the Kdissociation rate constant is 5×10-1 s-1 ～1×10-7 s-1 .
在其它实施方案中，所述配体或dAb单体特异性结合TNFR1(其Kd如本文所述)并且在标准小鼠LPS/D-半乳糖胺诱发的脓毒性休克模型中抑制致死率(即与合适对照相比，其预防致死或减少致死率至少约10％)。在标准小鼠LPS/D-半乳糖胺诱发的脓毒性休克模型中，与合适对照相比，当给予约5mg/kg或更优选约1mg/kg的dAb单体时，优选的dAb单体抑制致死率至少约25％或至少约50％。In other embodiments, the ligand or dAb monomer specifically binds TNFR1 (with aKd as described herein) and inhibits lethality in a standard mouse LPS/D-galactosamine-induced septic shock model ( That is, it prevents or reduces mortality by at least about 10% compared to a suitable control). In a standard mouse LPS/D-galactosamine-induced septic shock model, when a dAb monomer is administered at about 5 mg/kg or more preferably at about 1 mg/kg, the preferred dAb monomer inhibits The lethality is at least about 25% or at least about 50%.
在其它实施方案中，在标准细胞测定中，所述配体或dAb单体结合TNFR1并拮抗TNFR1活性的ND50≤100nM，在浓度≤10μM时，dAb激动TNFR1的活性≤5％。In other embodiments, the ligand or dAb monomer binds to TNFR1 and antagonizes TNFR1 activity with anND50 of < 100 nM, and the dAb agonizes TNFR1 by < 5% of its activity at a concentration of < 10 μM in a standard cellular assay.
在具体的实施方案中，在标准细胞测定中，所述配体或dAb单体基本上不激动TNFR1(起到TNFR1激动剂的作用)(即在测定中，当浓度为1nM、10nM、100nM、1μM、10μM、100μM、1000μM或5,000μM时，导致不超过约5％的由TNFα(100pg/ml)诱导的TNPR1介导的活性)。In specific embodiments, the ligand or dAb monomer does not substantially agonize TNFR1 (acts as a TNFR1 agonist) in a standard cellular assay (i.e., in an assay, at a concentration of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1000 μM or 5,000 μM resulted in no more than about 5% of TNPR1-mediated activity induced by TNFα (100 pg/ml)).
在某些实施方案中，所述配体或dAb单体基本上能抵抗聚集。例如，在某些实施方案中，当将1-5mg/ml、5-10mg/ml、10-20mg/ml、20-50mg/ml、50-100mg/ml、100-200mg/ml或200-500mg/ml配体或dAb溶液溶于药物配制常用溶剂(例如盐水、缓冲盐水、柠檬酸缓冲盐水、水、乳化剂和任何这些含有可接受的赋形剂的溶剂，所述赋形剂例如由FDA批准的赋形剂)时，将小于约10％、小于约9％、小于约8％、小于约7％、小于约6％、小于约5％、小于约4％、小于约3％、小于约2％或小于约1％的配体或dAb单体聚集物，在约22℃、22-25℃、25-30℃、30-37℃、37-40℃、40-50℃、50-60℃、60-70℃、70-80℃、15-20℃、10-15℃、5-10℃、2-5℃、0-2℃、-10℃至0℃、-20℃至-10℃、-40℃至-20℃、-60℃至-40℃或-80℃至-60℃的温度下，保持一段时间，例如10分钟、1小时、8小时、24小时、2天、3天、4天、1周、2周、3周、1月、2月、3月、4月、6月、1年或2年。In certain embodiments, the ligand or dAb monomer is substantially resistant to aggregation. For example, in certain embodiments, when 1-5 mg/ml, 5-10 mg/ml, 10-20 mg/ml, 20-50 mg/ml, 50-100 mg/ml, 100-200 mg/ml or 200-500 mg /ml ligand or dAb solution dissolved in common solvents for pharmaceutical formulation (e.g. saline, buffered saline, citrate buffered saline, water, emulsifiers and any of these solvents containing acceptable excipients such as those approved by FDA approved excipients), less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than Ligand or dAb monomer aggregates of about 2% or less than about 1%, at about 22°C, 22-25°C, 25-30°C, 30-37°C, 37-40°C, 40-50°C, 50- 60°C, 60-70°C, 70-80°C, 15-20°C, 10-15°C, 5-10°C, 2-5°C, 0-2°C, -10°C to 0°C, -20°C to - At a temperature of 10°C, -40°C to -20°C, -60°C to -40°C or -80°C to -60°C, keep it for a period of time, such as 10 minutes, 1 hour, 8 hours, 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year or 2 years.
可采用显微镜检、通过目测或分光镜检测定溶液浊度等任何合适方法，对聚集进行评价。优选通过动态光散射来评价聚集。抗聚集的配体或dAb单体具有若干优势。例如，通过采用合适的生物生产系统(例如大肠杆菌)进行表达，容易以可溶性蛋白质的形式产生这类配体或dAb单体并且收率高，然后可以比常规多肽更高的浓度来配制和/或贮存，同时聚集和活性的损失较低。Aggregation can be assessed by any suitable method such as microscopic examination, determination of solution turbidity by visual inspection or spectroscopic examination. Aggregation is preferably assessed by dynamic light scattering. Anti-aggregation ligands or dAb monomers have several advantages. Such ligands or dAb monomers are readily produced in high yields as soluble proteins, for example, by expression using a suitable biological production system (e.g., E. coli), which can then be formulated at higher concentrations than conventional polypeptides and/or Or storage with lower loss of aggregation and activity.
另外，生产抗聚集的配体或dAb单体，可以比生产其它抗原或表位结合多肽(例如常规抗体)更经济。例如，用于体内应用的抗原或表位结合多肽的制备通常包括除去聚集多肽的过程(例如凝胶过滤)。如果不能除去这样的聚集物，会使制剂不适用于体内应用，例如，因为希望起到拮抗剂作用的抗原结合多肽如果聚集的话，可通过诱导靶抗原的交联或成簇而起到激动剂的作用。聚集蛋白也可通过在接受它们的患者体内诱导免疫应答而降低治疗性多肽的功效。In addition, the production of anti-aggregation ligands or dAb monomers can be more economical than production of other antigen or epitope binding polypeptides such as conventional antibodies. For example, preparation of antigen- or epitope-binding polypeptides for in vivo use typically includes processes (eg, gel filtration) to remove aggregated polypeptides. Failure to remove such aggregates would render the formulation unsuitable for in vivo use, for example, because an antigen-binding polypeptide desired to act as an antagonist, if aggregated, could act as an agonist by inducing cross-linking or clustering of the target antigen role. Aggrins can also reduce the efficacy of therapeutic polypeptides by inducing an immune response in patients receiving them.
相比之下，可以制备本发明的抗聚集配体或dAb单体，用于体内应用，而无需包括除去聚集物的过程，并且可用于体内应用，而没有聚集多肽所引起的上述缺点。In contrast, anti-aggregation ligands or dAb monomers of the invention can be prepared for in vivo use without involving a process to remove aggregates, and can be used in vivo without the above-mentioned disadvantages caused by aggregated polypeptides.
在某些实施方案中，所述配体或dAb单体当加热到温度(Ts)和冷却到温度(Tc)时能可逆地解折叠，其中Ts高于dAb的解链温度(Tm)，而Tc低于dAb的解链温度。例如，dAb单体当加热到80℃和冷却到约室温时能可逆地解折叠。可逆地解折叠的多肽在解折叠时会丧失功能，而一旦重折叠后又会恢复功能。这类多肽不同于在解折叠时聚集或不适当重折叠(即不能恢复功能)的多肽(错折叠的多肽)。In certain embodiments, the ligand or dAb monomer is capable of reversibly unfolding when heated to a temperature (Ts) and cooled to a temperature (Tc), wherein Ts is greater than the melting temperature (Tm) of the dAb and The Tc is lower than the melting temperature of the dAb. For example, dAb monomers can reversibly unfold when heated to 80°C and cooled to about room temperature. Polypeptides that unfold reversibly lose function when unfolded and regain function once refolded. Such polypeptides are distinguished from polypeptides that aggregate when unfolded or that refold improperly (ie, fail to restore function) (misfolded polypeptides).
可以采用任何合适方法，通过直接或间接测定多肽结构来评价多肽解折叠和重折叠。例如，可以通过圆二色性(CD)(例如远UV CD、近UV CD)、荧光(例如色氨酸侧链的荧光)、对蛋白酶解的敏感性、核磁共振(NMR)，或者通过检测或测定依赖于合适折叠的多肽功能(例如与靶配体的结合、与通用配体的结合)，来测定多肽结构。在一个实例中，采用功能测定，评价多肽解折叠，其中结合功能(例如结合通用配体和/或靶配体，结合底物)的丧失，表明该多肽是解折叠的。Polypeptide unfolding and refolding can be assessed by direct or indirect determination of polypeptide structure using any suitable method. For example, it can be measured by circular dichroism (CD) (e.g., far UV CD, near UV CD), fluorescence (e.g., of the side chain of tryptophan), susceptibility to proteolysis, nuclear magnetic resonance (NMR), or by detecting Or determine polypeptide structure by determining polypeptide function that depends on proper folding (eg, binding to a target ligand, binding to a universal ligand). In one example, polypeptide unfolding is assessed using a functional assay, wherein loss of binding function (eg, binding of a generic ligand and/or target ligand, binding of a substrate) indicates that the polypeptide is unfolded.
可以采用解折叠或变性曲线，测定配体或dAb单体的解折叠和重折叠的程度。可通过纵坐标为温度与横坐标为折叠多肽相对浓度作图，绘制解折叠曲线。可以采用任何合适的方法(例如CD、荧光、结合测定)，直接或间接测定折叠配体或dAb单体的相对浓度。例如，可以制备配体或dAb单体溶液，然后通过CD测定溶液的椭圆率。所得椭圆率值表示折叠配体或dAb单体的相对浓度为100％。再通过逐渐升高溶液温度，使溶液中的配体或dAb单体解折叠，然后以合适增量(例如温度每升高一度后)测定椭圆率。然后通过逐渐降低溶液温度，使溶液中配体或dAb单体重折叠，再以合适增量测定椭圆率。可以对数据作图，绘制解折叠曲线和重折叠曲线。解折叠曲线和重折叠曲线具有特征性的S形，它包括配体或dAb单体分子折叠部分，配体或dAb单体分子解折叠到不同程度时的解折叠/重折叠转换部分，以及配体或dAb单体分子解折叠部分。重折叠曲线的Y轴截距是恢复的重折叠配体或dAb单体的相对数量。至少约50％、或至少约60％、或至少约70％、或至少约75％、或至少约80％、或至少约85％、或至少约90％、或至少约95％的恢复，表明配体或dAb单体可逆地解折叠。The extent of unfolding and refolding of the ligand or dAb monomer can be determined using an unfolding or denaturation curve. An unfolding curve can be drawn by plotting temperature on the ordinate versus relative concentration of folded polypeptide on the abscissa. The relative concentration of folded ligand or dAb monomer can be determined directly or indirectly by any suitable method (eg, CD, fluorescence, binding assays). For example, a ligand or dAb monomer solution can be prepared and the ellipticity of the solution determined by CD. The resulting ellipticity values represent 100% relative concentration of folded ligand or dAb monomer. Then, the ligand or dAb monomer in the solution is unfolded by gradually increasing the temperature of the solution, and then the ellipticity is measured at an appropriate increment (for example, after each degree of temperature increase). Then by gradually reducing the solution temperature, the ligand or dAb monomer in the solution is refolded, and then the ellipticity is measured in an appropriate increment. The data can be plotted, drawing unfolding curves and refolding curves. The unfolding curve and refolding curve have a characteristic S-shape, which includes the folding part of the ligand or dAb monomer molecule, the unfolding/refolding transition part when the ligand or dAb monomer molecule is unfolded to different degrees, and the ligand or dAb monomer molecule. The unfolded portion of the body or dAb monomer molecule. The Y-axis intercept of the refolding curve is the relative amount of refolded ligand or dAb monomer recovered. A recovery of at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, indicating The ligand or dAb monomer unfolds reversibly.
在一个优选的实施方案中，通过制备配体或dAb单体溶液并绘制加热解折叠和重折叠曲线，测定配体或dAb单体解折叠的可逆性。可以在任何合适溶剂中制备配体或dAb单体溶液，所述溶剂例如含水缓冲液，其pH适于让配体或dAb单体溶解(例如pH比等电点(pI)约高或低3个单位)。配体或dAb单体溶液足够浓，以便可检测出解折叠/折叠。例如，配体或dAb单体溶液可以是约0.1μM至约100μM、或优选约1μM至约10μM。In a preferred embodiment, the reversibility of ligand or dAb monomer unfolding is determined by preparing a solution of the ligand or dAb monomer and plotting heating unfolding and refolding curves. Ligand or dAb monomer solutions can be prepared in any suitable solvent, such as an aqueous buffer, at a pH suitable for dissolution of the ligand or dAb monomer (e.g., pH about 3 higher or lower than the isoelectric point (pi). units). Ligand or dAb monomer solutions are sufficiently concentrated so that unfolding/folding is detectable. For example, the ligand or dAb monomer solution may be from about 0.1 μM to about 100 μM, or preferably from about 1 μM to about 10 μM.
如果配体或dAb单体的解链温度(Tm)是已知的，则可将溶液加热至比Tm低约10度(Tm-10)，然后通过椭圆率或荧光(例如远-UV CD可从200nm至250nm，固定波长CD为235nm或225nm；色氨酸荧光发射光谱为300-450nm，激发为298nm)进行评价折叠，以提供100％的相对折叠配体或dAb单体。再按预定增量(例如约0.1度至约1度的增加)，将溶液加热至比Tm高至少10度(Tm+10)，在每次增量时测定椭圆率或荧光。然后，按预定增量，通过冷却到至少Tm-10，使配体或dAb单体重折叠，在每次增量时测定椭圆率或荧光。如果不知道配体或dAb单体的解链温度，可以通过从约25℃至约100℃逐渐增量加热，使溶液解折叠，再逐渐增量冷却到至少约25℃使其重折叠，在每次加热和冷却增量时测定椭圆率或荧光。可以对所得数据作图，绘制解折叠曲线和重折叠曲线，其中重折叠曲线的Y轴截距是恢复的重折叠蛋白质的相对数量。If the melting temperature (Tm) of the ligand or dAb monomer is known, the solution can be heated to about 10 degrees below the Tm (Tm-10) and then analyzed by ellipticity or fluorescence (e.g. far-UV CD can Folding was evaluated from 200nm to 250nm, fixed wavelength CD at 235nm or 225nm; tryptophan fluorescence emission spectrum at 300-450nm, excitation at 298nm) to provide 100% relative folded ligand or dAb monomer. The solution is then heated to at least 10 degrees above Tm (Tm+10) in predetermined increments (eg, about 0.1 degree to about 1 degree increments), and the ellipticity or fluorescence is measured at each increment. The ligand or dAb monomer is then refolded by cooling to at least Tm-10 in predetermined increments, with ellipticity or fluorescence measured at each increment. If the melting temperature of the ligand or dAb monomer is unknown, the solution can be unfolded by incremental heating from about 25°C to about 100°C, followed by incremental cooling to at least about 25°C to allow refolding, at Ellipticity or fluorescence was measured at each heating and cooling increment. The resulting data can be plotted to generate an unfolding curve and a refolding curve, where the Y-axis intercept of the refolding curve is the relative amount of refolded protein recovered.
在某些实施方案中，本发明的配体或dAb单体当以有效量给予时，在慢性炎性疾病模型中有效。通常有效量为约1mg/kg至约10mg/kg(例如约1mg/kg、约2mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg或约10mg/kg)。本文所述的慢性炎性疾病模型是本领域技术人员公知的，用于预测在人体内的疗效。现有技术没有给出在这些模型中使用TNFR1拮抗剂(例如本文所述的单价拮抗剂、配体)的启示，也没有给出它们是否有效的启示。In certain embodiments, a ligand or dAb monomer of the invention is effective in a model of chronic inflammatory disease when administered in an effective amount. Usually the effective amount is about 1 mg/kg to about 10 mg/kg (for example about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg). The chronic inflammatory disease models described herein are well known to those skilled in the art and are used to predict therapeutic effects in humans. The prior art does not suggest the use of TNFRl antagonists (eg, the monovalent antagonists, ligands described herein) in these models, nor does it suggest whether they are effective.
在具体的实施方案中，所述配体或dAb单体在标准小鼠胶原诱发的关节炎模型(实施例15A)中是有效的。例如与合适对照相比，在标准小鼠胶原诱发的关节炎模型中，给予有效量的配体可降低四肢平均关节炎评分总和例如约1至约16、约3至约16、约6至约16、约9至约16或约12至约16。在另一实例中，与合适对照相比，在标准小鼠胶原诱发的关节炎模型中，给予有效量的配体可延迟关节炎症状发作例如达约1天、约2天、约3天、约4天、约5天、约6天、约7天、约10天、约14天、约21天或约28天。在另一实例中，在标准小鼠胶原诱发的关节炎模型中，给予有效量的配体可导致四肢平均关节炎评分总和为0至约3、约3至约5、约5至约7、约7至约15、约9至约15、约10至约15、约12至约15或约14至约15。In specific embodiments, the ligand or dAb monomer is effective in a standard mouse collagen-induced arthritis model (Example 15A). For example, in a standard mouse collagen-induced arthritis model, administration of an effective amount of the ligand can reduce the sum of the four limb mean arthritis scores, e.g., from about 1 to about 16, from about 3 to about 16, from about 6 to about 16. From about 9 to about 16 or from about 12 to about 16. In another example, administration of an effective amount of a ligand delays the onset of arthritis symptoms, e.g., by about 1 day, about 2 days, about 3 days, compared to a suitable control, in a standard mouse collagen-induced arthritis model. About 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, or about 28 days. In another example, in a standard mouse collagen-induced arthritis model, administration of an effective amount of a ligand results in a sum of four limb mean arthritis scores of 0 to about 3, about 3 to about 5, about 5 to about 7, From about 7 to about 15, from about 9 to about 15, from about 10 to about 15, from about 12 to about 15, or from about 14 to about 15.
在其它实施方案中，所述配体或dAb单体在小鼠关节炎ΔARE模型中是有效的(实施例15B)。例如，与合适对照相比，在小鼠关节炎ΔARE模型中，给予有效量的配体可降低平均关节炎评分例如达约0.1至约2.5、约0.5至约2.5、约1至约2.5、约1.5至约2.5、或约2至约2.5。在另一实例中，与合适对照相比，在小鼠关节炎ΔARE模型中，给予有效量的配体可延迟关节炎症状发作例如达约1天、约2天、约3天、约4天、约5天、约6天、约7天、约10天、约14天、约21天或约28天。在另一实例中，在小鼠关节炎ΔARE模型中，给予有效量的配体可导致平均关节炎评分为0至约0.5、约0.5至约1、约1至约1.5、约1.5至约2、或约2至约2.5。In other embodiments, the ligand or dAb monomer is effective in the mouse arthritis ΔARE model (Example 15B). For example, in a mouse arthritis ΔARE model, administration of an effective amount of a ligand can reduce the mean arthritis score, e.g., by about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5, or about 2 to about 2.5. In another example, administration of an effective amount of a ligand delays the onset of arthritis symptoms, e.g., by about 1 day, about 2 days, about 3 days, about 4 days in a mouse arthritis ΔARE model, compared to a suitable control , about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, or about 28 days. In another example, administration of an effective amount of a ligand results in a mean arthritis score of 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2 in the mouse arthritis ΔARE model , or from about 2 to about 2.5.
在其它实施方案中，所述配体或dAb单体在小鼠炎性肠病(IBD)ΔARE模型(实施例15B)中是有效的。例如，与合适对照相比，在小鼠IBDΔARE模型中，给予有效量的配体可降低平均急性和/或慢性炎症评分例如达约0.1至约2.5、约0.5至约2.5、约1至约2.5、约1.5至约2.5、或约2至约2.5。在另一实例中，与合适对照相比，在小鼠IBD ΔARE模型中，给予有效量的配体可延迟IBD症状发作例如达约1天、约2天、约3天、约4天、约5天、约6天、约7天、约10天、约14天、约21天或约28天。在另一实例中，在小鼠IBDΔARE模型中，给予有效量的配体可导致平均急性和/或慢性炎症评分为0至约0.5、约0.5至约1、约1至约1.5、约1.5至约2或约2至约2.5。In other embodiments, the ligand or dAb monomer is effective in the mouse inflammatory bowel disease (IBD) ΔARE model (Example 15B). For example, administration of an effective amount of a ligand can reduce the mean acute and/or chronic inflammation score, e.g., by about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5 in the mouse IBD ΔARE model compared to a suitable control , about 1.5 to about 2.5, or about 2 to about 2.5. In another example, in a mouse IBD ΔARE model, administration of an effective amount of a ligand delays the onset of IBD symptoms, e.g., by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, or about 28 days. In another example, in the mouse IBDΔARE model, administration of an effective amount of ligand results in a mean acute and/or chronic inflammation score of 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2 or about 2 to about 2.5.
在其它实施方案中，所述配体或dAb单体在小鼠葡聚糖硫酸钠(DSS)诱发的IBD模型中是有效的(实施例15C)。例如，与合适对照相比，在小鼠DSS IBD模型中，给予有效量的配体可降低平均严重性评分例如达约0.1至约2.5、约0.5至约2.5、约1至约2.5、约1.5至约2.5、或约2至约2.5。在另一实例中，与合适对照相比，在小鼠DSS IBD模型中，给予有效量的配体可延迟IBD症状发作例如达约1天、约2天、约3天、约4天、约5天、约6天、约7天、约10天、约14天、约21天或约28天。在另一实例中，在小鼠DSS IBD模型中，给予有效量的配体可导致平均严重性评分为0至约0.5、约0.5至约1、约1至约1.5、约1.5至约2或约2至约2.5。In other embodiments, the ligand or dAb monomer is effective in a mouse dextran sodium sulfate (DSS) induced IBD model (Example 15C). For example, in a mouse DSS IBD model, administration of an effective amount of a ligand can reduce the mean severity score, e.g., by about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 compared to a suitable control to about 2.5, or about 2 to about 2.5. In another example, in a mouse DSS IBD model, administration of an effective amount of a ligand delays the onset of IBD symptoms, e.g., by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, or about 28 days. In another example, administration of an effective amount of a ligand results in a mean severity score of 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2, or about 2 to about 2.5.
在具体实施方案中，所述配体或dAb单体在小鼠吸烟所致慢性阻塞性肺病(COPD)模型中是有效的(实施例15D)。例如，与合适对照相比，给予有效量的配体可降低或延迟COPD症状发作。In specific embodiments, the ligand or dAb monomer is effective in a mouse model of smoking-induced chronic obstructive pulmonary disease (COPD) (Example 15D). For example, administration of an effective amount of a ligand reduces or delays the onset of symptoms of COPD compared to a suitable control.
在具体实施方案中，所述配体或dAb单体特异性结合TNFR1并且包含TAR2-10的氨基酸序列(SEQ ID NO：31)或与其具有至少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％或至少99％同源性的序列。In particular embodiments, the ligand or dAb monomer specifically binds to TNFR1 and comprises the amino acid sequence of TAR2-10 (SEQ ID NO: 31) or has at least 80%, at least 85%, at least 90%, at least 91% thereof %, 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% homologous.
在具体的实施方案中，所述配体或dAb单体特异性结合TNFR1并包含TAR2-5的氨基酸序列(SEQ ID NO：195)或与其具有至少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％或至少99％同源性的序列。In specific embodiments, the ligand or dAb monomer specifically binds to TNFR1 and comprises the amino acid sequence of TAR2-5 (SEQ ID NO: 195) or has at least 80%, at least 85%, at least 90%, at least A sequence that is 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% homologous.
在其它实施方案中，所述配体包含域抗体(dAb)单体，所述dAb单体与肿瘤坏死因子受体1(TNFR1，p55，CD120a)特异性结合的Kd为300nM～5pM，并且所述dAb单体的氨基酸序列与选自以下的氨基酸序列或dAb具有至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2h-12(SEQ ID NO：32)、TAR2h-13(SEQ ID NO：33)、TAR2h-14(SEQ ID NO：34)、TAR2h-16(SEQ ID NO：35)、TAR2h-17(SEQ ID NO：36)、TAR2h-18(SEQ ID NO：37)、TAR2h-19(SEQ ID NO：38)、TAR2h-20(SEQ ID NO：39)、TAR2h-21(SEQ ID NO：40)、TAR2h-22(SEQ ID NO：41)、TAR2h-23(SEQ ID NO：42)、TAR2h-24(SEQ ID NO：43)、TAR2h-25(SEQ ID NO：44)、TAR2h-26(SEQ ID NO：45)、TAR2h-27(SEQ ID NO：46)、TAR2h-29(SEQ ID NO：47)、TAR2h-30(SEQ ID NO：48)、TAR2h-32(SEQ ID NO：49)、TAR2h-33(SEQ ID NO：50)、TAR2h-10-1(SEQ ID NO：51)、TAR2h-10-2(SEQ ID NO：52)、TAR2h-10-3(SEQ ID NO：53)、TAR2h-10-4(SEQ ID NO：54)、TAR2h-10-5(SEQ ID NO：55)、TAR2h-10-6(SEQ ID NO：56)、TAR2h-10-7(SEQ ID NO：57)、TAR2h-10-8(SEQ ID NO：58)、TAR2h-10-9(SEQ ID NO：59)、TAR2h-10-10(SEQ ID NO：60)、TAR2h-10-11(SEQ ID NO：61)、TAR2h-10-12(SEQ ID NO：62)、TAR2h-10-13(SEQ ID NO：63)、TAR2h-10-14(SEQ ID NO：64)、TAR2h-10-15(SEQ ID NO：65)、TAR2h-10-16(SEQ ID NO：66)、TAR2h-10-17(SEQ ID NO：67)、TAR2h-10-18(SEQ ID NO：68)、TAR2h-10-19(SEQ ID NO：69)、TAR2h-10-20(SEQ ID NO：70)、TAR2h-10-21(SEQ ID NO：71)、TAR2h-10-22(SEQ ID NO：72)、TAR2h-10-27(SEQ ID NO：73)、TAR2h-10-29(SEQ ID NO：74)、TAR2h-10-31(SEQ ID NO：75)、TAR2h-10-35(SEQ ID NO：76)、TAR2h-10-36(SEQ ID NO：77)、TAR2h-10-37(SEQ ID NO：78)、TAR2h-10-38(SEQ ID NO：79)、TAR2h-10-45(SEQ ID NO：80)、TAR2h-10-47(SEQ ID NO：81)、TAR2h-10-48(SEQ ID NO：82)、TAR2h-10-57(SEQ ID NO：83)、TAR2h-10-56(SEQ ID NO：84)、TAR2h-10-58(SEQ ID NO：85)、TAR2h-10-66(SEQ ID NO：86)、TAR2h-10-64(SEQ ID NO：87)、TAR2h-10-65(SEQ ID NO：88)、TAR2h-10-68(SEQ ID NO：89)、TAR2h-10-69(SEQ ID NO：90)、TAR2h-10-67(SEQ ID NO：91)、TAR2h-10-61(SEQ ID NO：92)、TAR2h-10-62(SEQ ID NO：93)、TAR2h-10-63(SEQ ID NO：94)、TAR2h-10-60(SEQ ID NO：95)、TAR2h-10-55(SEQ ID NO：96)、TAR2h-10-59(SEQ ID NO：97)、TAR2h-10-70(SEQ ID NO：98)、TAR2h-34(SEQ ID NO：373)、TAR2h-35(SEQ ID NO：374)、TAR2h-36(SEQ ID NO：375)、TAR2h-37(SEQ ID NO：376)、TAR2h-38(SEQ ID NO：377)、TAR2h-39(SEQ ID NO：378)、TAR2h-40(SEQID NO：379)、TAR2h-41(SEQ ID NO：380)、TAR2h-42(SEQ ID NO：381)、TAR2h-43(SEQ ID NO：382)、TAR2h-44(SEQ ID NO：383)、TAR2h-45(SEQ ID NO：384)、TAR2h-47(SEQ ID NO：385)、TAR2h-48(SEQ ID NO：386)、TAR2h-50(SEQ ID NO：387)、TAR2h-51(SEQID NO：388)、TAR2h-66(SEQ ID NO：389)、TAR2h-67(SEQ ID NO：390)、TAR2h-68(SEQ ID NO：391)、TAR2h-70(SEQ ID NO：392)、TAR2h-71(SEQ ID NO：393)、TAR2h-72(SEQ ID NO：394)、TAR2h-73(SEQ ID NO：395)、TAR2h-74(SEQ ID NO：396)、TAR2h-75(SEQID NO：397)、TAR2h-76(SEQ ID NO：398)、TAR2h-77(SEQ ID NO：399)、TAR2h-78(SEQ ID NO：400)、TAR2h-79(SEQ ID NO：401)和TAR2h-15(SEQ ID NO：431)。In other embodiments, the ligand comprises a domain antibody (dAb) monomer, and the Kd of the specific binding of the dAb monomer to tumor necrosis factor receptor 1 (TNFR1, p55, CD120a) is 300 nM to 5 pM, and The amino acid sequence of the dAb monomer has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-12 (SEQ ID NO: 32), TAR2h-13 (SEQ ID NO : 33), TAR2h-14 (SEQ ID NO: 34), TAR2h-16 (SEQ ID NO: 35), TAR2h-17 (SEQ ID NO: 36), TAR2h-18 (SEQ ID NO: 37), TAR2h- 19 (SEQ ID NO: 38), TAR2h-20 (SEQ ID NO: 39), TAR2h-21 (SEQ ID NO: 40), TAR2h-22 (SEQ ID NO: 41), TAR2h-23 (SEQ ID NO: 42), TAR2h-24 (SEQ ID NO: 43), TAR2h-25 (SEQ ID NO: 44), TAR2h-26 (SEQ ID NO: 45), TAR2h-27 (SEQ ID NO: 46), TAR2h-29 (SEQ ID NO: 47), TAR2h-30 (SEQ ID NO: 48), TAR2h-32 (SEQ ID NO: 49), TAR2h-33 (SEQ ID NO: 50), TAR2h-10-1 (SEQ ID NO : 51), TAR2h-10-2 (SEQ ID NO: 52), TAR2h-10-3 (SEQ ID NO: 53), TAR2h-10-4 (SEQ ID NO: 54), TAR2h-10-5 (SEQ ID NO: 55), TAR2h-10-6 (SEQ ID NO: 56), TAR2h-10-7 (SEQ ID NO: 57), TAR2h-10-8 (SEQ ID NO: 58), TAR2h-10-9 (SEQ ID NO: 59), TAR2h-10-10 (SEQ ID NO: 60), TAR2h-10-11 (SEQ ID NO: 61), TAR2h-10-12 (SEQ ID NO: 62), TAR2h-10 -13 (SEQ ID NO: 63), TAR2h-10-14 (SEQ ID NO: 64), TAR2h-10-15 (SEQ ID NO: 65), TAR2h-10-16 (SEQ ID NO: 66), TAR2h -10-17 (SEQ ID NO: 67), TAR2h-10-18 (SEQ ID NO: 68), TAR2h-10-19 (SEQ ID NO: 69), TAR2h-10-20 (SEQ ID NO: 70) , TAR2h-10-21 (SEQ ID NO: 71), TAR2h-10-22 (SEQ ID NO: 72), TAR2h-10-27 (SEQ ID NO: 73), TAR2h-10-29 (SEQ ID NO: 74), TAR2h-10-31 (SEQ ID NO: 75), TAR2h-10-35 (SEQ ID NO: 76), TAR2h-10-36 (SEQ ID NO: 77), TAR2h-10-37 (SEQ ID NO: 78), TAR2h-10-38 (SEQ ID NO: 79), TAR2h-10-45 (SEQ ID NO: 80), TAR2h-10-47 (SEQ ID NO: 81), TAR2h-10-48 ( SEQ ID NO: 82), TAR2h-10-57 (SEQ ID NO: 83), TAR2h-10-56 (SEQ ID NO: 84), TAR2h-10-58 (SEQ ID NO: 85), TAR2h-10- 66 (SEQ ID NO: 86), TAR2h-10-64 (SEQ ID NO: 87), TAR2h-10-65 (SEQ ID NO: 88), TAR2h-10-68 (SEQ ID NO: 89), TAR2h- 10-69 (SEQ ID NO: 90), TAR2h-10-67 (SEQ ID NO: 91), TAR2h-10-61 (SEQ ID NO: 92), TAR2h-10-62 (SEQ ID NO: 93), TAR2h-10-63 (SEQ ID NO: 94), TAR2h-10-60 (SEQ ID NO: 95), TAR2h-10-55 (SEQ ID NO: 96), TAR2h-10-59 (SEQ ID NO: 97 ), TAR2h-10-70 (SEQ ID NO: 98), TAR2h-34 (SEQ ID NO: 373), TAR2h-35 (SEQ ID NO: 374), TAR2h-36 (SEQ ID NO: 375), TAR2h- 37 (SEQ ID NO: 376), TAR2h-38 (SEQ ID NO: 377), TAR2h-39 (SEQ ID NO: 378), TAR2h-40 (SEQ ID NO: 379), TAR2h-41 (SEQ ID NO: 380 ), TAR2h-42 (SEQ ID NO: 381), TAR2h-43 (SEQ ID NO: 382), TAR2h-44 (SEQ ID NO: 383), TAR2h-45 (SEQ ID NO: 384), TAR2h-47 ( SEQ ID NO: 385), TAR2h-48 (SEQ ID NO: 386), TAR2h-50 (SEQ ID NO: 387), TAR2h-51 (SEQ ID NO: 388), TAR2h-66 (SEQ ID NO: 389), TAR2h-67 (SEQ ID NO: 390), TAR2h-68 (SEQ ID NO: 391), TAR2h-70 (SEQ ID NO: 392), TAR2h-71 (SEQ ID NO: 393), TAR2h-72 (SEQ ID NO: 394), TAR2h-73 (SEQ ID NO: 395), TAR2h-74 (SEQ ID NO: 396), TAR2h-75 (SEQ ID NO: 397), TAR2h-76 (SEQ ID NO: 398), TAR2h- 77 (SEQ ID NO: 399), TAR2h-78 (SEQ ID NO: 400), TAR2h-79 (SEQ ID NO: 401) and TAR2h-15 (SEQ ID NO: 431).
在其它实施方案中，所述配体包含域抗体(dAb)单体，所述dAb单体与肿瘤坏死因子受体1(TNFR1，p55，CD120a)特异性结合的Kd为300nM～5pM，并且所述dAb单体的氨基酸序列与选自以下的氨基酸序列或dAb具有至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2h-131-8(SEQ ID NO：433)、TAR2h-131-24(SEQ ID NO：434)、TAR2h-15-8(SEQ ID NO：435)、TAR2h-15-8-1 SEQ ID NO：436)、TAR2h-15-8-2(SEQ ID NO：437)、TAR2h-185-23(SEQ ID NO：438)、TAR2h-154-10-5(SEQ ID NO：439)、TAR2h-14-2(SEQ ID NO：440)、TAR2h-151-8(SEQ ID NO：441)、TAR2h-152-7(SEQ ID NO：442)、TAR2h-35-4(SEQ ID NO：443)、TAR2h-154-7(SEQ ID NO：444)、TAR2h-80(SEQ ID NO：445)、TAR2h-81(SEQ ID NO：446)、TAR2h-82(SEQ ID NO：447)、TAR2h-83(SEQ ID NO：448)、TAR2h-84(SEQID NO：449)、TAR2h-85(SEQ ID NO：450)、TAR2h-86(SEQ ID NO：451)、TAR2h-87(SEQ ID NO：452)、TAR2h-88(SEQ ID NO：453)、TAR2h-89(SEQ ID NO：454)、TAR2h-90(SEQ ID NO：455)、TAR2h-91(SEQ ID NO：456)、TAR2h-92(SEQ ID NO：457)、TAR2h-93(SEQID NO：458)、TAR2h-94(SEQ ID NO：459)、TAR2h-95(SEQ ID NO：460)、TAR2h-96(SEQ ID NO：461)、TAR2h-97(SEQ ID NO：462)、TAR2h-99(SEQ ID NO：463)、TAR2h-100(SEQ ID NO：464)、TAR2h-101(SEQ ID NO：465)、TAR2h-102(SEQ ID NO：466)、TAR2h-103(SEQ ID NO：467)、TAR2h-104(SEQ ID NO：468)、TAR2h-105(SEQ ID NO：469)、TAR2h-106(SEQ ID NO：470)、TAR2h-107(SEQ ID NO：471)、TAR2h-108(SEQ ID NO：472)、TAR2h-109(SEQ ID NO：473)、TAR2h-110(SEQ ID NO：474)、TAR2h-111(SEQ ID NO：475)、TAR2h-112(SEQ ID NO：476)、TAR2h-113(SEQ ID NO：477)、TAR2h-114(SEQ ID NO：478)、TAR2h-115(SEQ ID NO：479)、TAR2h-116(SEQ ID NO：480)、TAR2h-117(SEQ ID NO：481)、TAR2h-118(SEQ ID NO：482)、TAR2h-119(SEQ ID NO：483)、TAR2h-120(SEQ ID NO：484)、TAR2h-121(SEQ ID NO：485)、TAR2h-122(SEQ ID NO：486)、TAR2h-123(SEQ ID NO：487)、TAR2h-124(SEQ ID NO：488)、TAR2h-125(SEQ ID NO：489)、TAR2h-126(SEQ ID NO：490)、TAR2h-127(SEQ ID NO：490)、TAR2h-128(SEQ ID NO：492)、TAR2h-129(SEQ ID NO：493)、TAR2h-130(SEQ ID NO：494)、TAR2h-131(SEQ ID NO：495)、TAR2h-132(SEQ ID NO：496)、TAR2h-133(SEQ ID NO：497)、TAR2h-151(SEQ ID NO：498)、TAR2h-152(SEQ ID NO：499)、TAR2h-153(SEQ ID NO：500)、TAR2h-154(SEQ ID NO：501)、TAR2h-159(SEQ ID NO：502)、TAR2h-165(SEQ ID NO：503)、TAR2h-166(SEQ ID NO：504)、TAR2h-168(SEQ ID NO：505)、TAR2h-171(SEQ ID NO：506)、TAR2h-172(SEQ ID NO：507)、TAR2h-173(SEQ ID NO：508)、TAR2h-174(SEQ ID NO：509)、TAR2h-176(SEQ ID NO：510)、TAR2h-178(SEQ ID NO：511)、TAR2h-201(SEQ ID NO：512)、TAR2h-202(SEQ ID NO：513)、TAR2h-203(SEQ ID NO：514)、TAR2h-204(SEQ ID NO：515)、TAR2h-185-25(SEQ ID NO：516)、TAR2h-154-10(SEQ ID NO：517)和TAR2h-205(SEQ ID NO：627)。In other embodiments, the ligand comprises a domain antibody (dAb) monomer, and the Kd of the specific binding of the dAb monomer to tumor necrosis factor receptor 1 (TNFR1, p55, CD120a) is 300 nM to 5 pM, and The amino acid sequence of the dAb monomer has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-131-8 (SEQ ID NO: 433), TAR2h-131-24 (SEQ ID NO: 434), TAR2h-15-8 (SEQ ID NO: 435), TAR2h-15-8-1 (SEQ ID NO: 436), TAR2h-15-8-2 (SEQ ID NO: 437), TAR2h-185-23 (SEQ ID NO: 438), TAR2h-154-10-5 (SEQ ID NO: 439), TAR2h-14-2 (SEQ ID NO: 440), TAR2h-151-8 (SEQ ID NO : 441), TAR2h-152-7 (SEQ ID NO: 442), TAR2h-35-4 (SEQ ID NO: 443), TAR2h-154-7 (SEQ ID NO: 444), TAR2h-80 (SEQ ID NO : 445), TAR2h-81 (SEQ ID NO: 446), TAR2h-82 (SEQ ID NO: 447), TAR2h-83 (SEQ ID NO: 448), TAR2h-84 (SEQ ID NO: 449), TAR2h-85 (SEQ ID NO: 450), TAR2h-86 (SEQ ID NO: 451), TAR2h-87 (SEQ ID NO: 452), TAR2h-88 (SEQ ID NO: 453), TAR2h-89 (SEQ ID NO: 454 ), TAR2h-90 (SEQ ID NO: 455), TAR2h-91 (SEQ ID NO: 456), TAR2h-92 (SEQ ID NO: 457), TAR2h-93 (SEQ ID NO: 458), TAR2h-94 (SEQ ID NO: 459), TAR2h-95 (SEQ ID NO: 460), TAR2h-96 (SEQ ID NO: 461), TAR2h-97 (SEQ ID NO: 462), TAR2h-99 (SEQ ID NO: 463), TAR2h-100 (SEQ ID NO: 464), TAR2h-101 (SEQ ID NO: 465), TAR2h-102 (SEQ ID NO: 466), TAR2h-103 (SEQ ID NO: 467), TAR2h-104 (SEQ ID NO: 468), TAR2h-105 (SEQ ID NO: 469), TAR2h-106 (SEQ ID NO: 470), TAR2h-107 (SEQ ID NO: 471), TAR2h-108 (SEQ ID NO: 472), TAR2h -109 (SEQ ID NO: 473), TAR2h-110 (SEQ ID NO: 474), TAR2h-111 (SEQ ID NO: 475), TAR2h-112 (SEQ ID NO: 476), TAR2h-113 (SEQ ID NO : 477), TAR2h-114 (SEQ ID NO: 478), TAR2h-115 (SEQ ID NO: 479), TAR2h-116 (SEQ ID NO: 480), TAR2h-117 (SEQ ID NO: 481), TAR2h- 118 (SEQ ID NO: 482), TAR2h-119 (SEQ ID NO: 483), TAR2h-120 (SEQ ID NO: 484), TAR2h-121 (SEQ ID NO: 485), TAR2h-122 (SEQ ID NO: 486), TAR2h-123 (SEQ ID NO: 487), TAR2h-124 (SEQ ID NO: 488), TAR2h-125 (SEQ ID NO: 489), TAR2h-126 (SEQ ID NO: 490), TAR2h-127 (SEQ ID NO: 490), TAR2h-128 (SEQ ID NO: 492), TAR2h-129 (SEQ ID NO: 493), TAR2h-130 (SEQ ID NO: 494), TAR2h-131 (SEQ ID NO: 495 ), TAR2h-132 (SEQ ID NO: 496), TAR2h-133 (SEQ ID NO: 497), TAR2h-151 (SEQ ID NO: 498), TAR2h-152 (SEQ ID NO: 499), TAR2h-153 ( SEQ ID NO:500), TAR2h-154 (SEQ ID NO:501), TAR2h-159 (SEQ ID NO:502), TAR2h-165 (SEQ ID NO:503), TAR2h-166 (SEQ ID NO:504) , TAR2h-168 (SEQ ID NO: 505), TAR2h-171 (SEQ ID NO: 506), TAR2h-172 (SEQ ID NO: 507), TAR2h-173 (SEQ ID NO: 508), TAR2h-174 (SEQ ID NO: 509), TAR2h-176 (SEQ ID NO: 510), TAR2h-178 (SEQ ID NO: 511), TAR2h-201 (SEQ ID NO: 512), TAR2h-202 (SEQ ID NO: 513), TAR2h-203 (SEQ ID NO:514), TAR2h-204 (SEQ ID NO:515), TAR2h-185-25 (SEQ ID NO:516), TAR2h-154-10 (SEQ ID NO:517) and TAR2h- 205 (SEQ ID NO: 627).
在优选的实施方案中，所述配体或dAb的氨基酸序列与选自以下dAb的氨基酸序列具有至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％同源性：TAR2h-10-27(SEQ ID NO：73)、TAR2h-10-57(SEQ ID NO：83)、TAR2h-10-56(SEQ ID NO：84)、TAR2h-10-58(SEQ ID NO：85)、TAR2h-10-66(SEQ ID NO：86)、TAR2h-10-64(SEQ ID NO：87)、TAR2h-10-65(SEQ ID NO：88)、TAR2h-10-68(SEQ ID NO：89)、TAR2h-10-69(SEQ ID NO：90)、TAR2h-10-67(SEQ ID NO：91)、TAR2h-10-61(SEQ ID NO：92)、TAR2h-10-62(SEQ ID NO：93)、TAR2h-10-63(SEQ ID NO：94)、TAR2h-10-60(SEQ ID NO：95)、TAR2h-10-55(SEQ ID NO：96)、TAR2h-10-59(SEQ ID NO：97)和TAR2h-10-70(SEQ ID NO：98)。In preferred embodiments, the ligand or dAb has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% identical to the amino acid sequence of a dAb selected from the group consisting of: At least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-10-27 (SEQ ID NO: 73), TAR2h-10-57 (SEQ ID NO : 83), TAR2h-10-56 (SEQ ID NO: 84), TAR2h-10-58 (SEQ ID NO: 85), TAR2h-10-66 (SEQ ID NO: 86), TAR2h-10-64 (SEQ ID NO: 87), TAR2h-10-65 (SEQ ID NO: 88), TAR2h-10-68 (SEQ ID NO: 89), TAR2h-10-69 (SEQ ID NO: 90), TAR2h-10-67 (SEQ ID NO: 91), TAR2h-10-61 (SEQ ID NO: 92), TAR2h-10-62 (SEQ ID NO: 93), TAR2h-10-63 (SEQ ID NO: 94), TAR2h-10 -60 (SEQ ID NO: 95), TAR2h-10-55 (SEQ ID NO: 96), TAR2h-10-59 (SEQ ID NO: 97) and TAR2h-10-70 (SEQ ID NO: 98).
特别优选的配体或dAb的氨基酸序列与SEQ ID NO：73的氨基酸序列具有至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％同源性。Particularly preferred ligands or dAbs have an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95% identical to the amino acid sequence of SEQ ID NO:73. At least about 96%, at least about 97%, at least about 98%, or at least about 99% homology.
在其它实施方案中，所述配体包含域抗体(dAb)单体，所述dAb单体与肿瘤坏死因子受体1(TNFR1，p55，CD120a)特异性结合的Kd为300nM～5pM，并且所述dAb单体的氨基酸序列与选自以下的氨基酸序列或dAb具有至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2m-14(SEQ ID NO：167)、TAR2m-15(SEQ ID NO：168)、TAR2m-19(SEQ IDNO：169)、TAR2m-20(SEQ ID NO：170)、TAR2m-21(SEQ ID NO：171)、TAR2m-24(SEQ ID NO：172)、TAR2m-21-23(SEQ ID NO：173)、TAR2m-21-07(SEQ ID NO：174)、TAR2m-21-43(SEQ ID NO：175)、TAR2m-21-48(SEQ ID NO：176)、TAR2m-21-10(SEQ ID NO：177)、TAR2m-21-06(SEQ ID NO：178)、TAR2m-21-17(SEQ ID NO：179)。In other embodiments, the ligand comprises a domain antibody (dAb) monomer, and the Kd of the specific binding of the dAb monomer to tumor necrosis factor receptor 1 (TNFR1, p55, CD120a) is 300 nM to 5 pM, and The amino acid sequence of the dAb monomer has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2m-14 (SEQ ID NO: 167), TAR2m-15 (SEQ ID NO : 168), TAR2m-19 (SEQ ID NO: 169), TAR2m-20 (SEQ ID NO: 170), TAR2m-21 (SEQ ID NO: 171), TAR2m-24 (SEQ ID NO: 172), TAR2m-21 -23 (SEQ ID NO: 173), TAR2m-21-07 (SEQ ID NO: 174), TAR2m-21-43 (SEQ ID NO: 175), TAR2m-21-48 (SEQ ID NO: 176), TAR2m -21-10 (SEQ ID NO: 177), TAR2m-21-06 (SEQ ID NO: 178), TAR2m-21-17 (SEQ ID NO: 179).
在某些实施方案中，所述配体包含dAb单体，所述dAb单体能结合TNFR1并与本文所公开的任何dAb竞争性结合TNFR1(例如小鼠TNFR1和/或人TNFR1)。In certain embodiments, the ligand comprises a dAb monomer that binds TNFR1 and competes with any of the dAbs disclosed herein for binding to TNFR1 (eg, mouse TNFR1 and/or human TNFR1 ).
优选的配体或dAb单体当在大肠杆菌或毕赤酵母(例如巴斯德毕赤酵母)中表达时，其分泌量至少约为0.5mg/L。在其它优选的实施方案中，当在大肠杆菌或毕赤酵母(例如巴斯德毕赤酵母)中表达时，dAb单体的分泌量至少约0.75mg/L、至少约1mg/L、至少约4mg/L、至少约5mg/L、至少约10mg/L、至少约15mg/L、至少约20mg/L、至少约25mg/L、至少约30mg/L、至少约35mg/L、至少约40mg/L、至少约45mg/L、或至少约50mg/L、或至少约100mg/L、或至少约200mg/L、或至少约300mg/L、或至少约400mg/L、或至少约500mg/L、或至少约600mg/L、或至少约700mg/L、或至少约800mg/L、至少约900mg/L、或至少约1g/L。在其它优选的实施方案中，当在大肠杆菌或毕赤酵母(例如巴斯德毕赤酵母)中表达时，dAb单体的分泌量至少约1mg/L到至少约1g/L、至少约1mg/L到至少约750mg/L、至少约100mg/L到至少约1g/L、至少约200mg/L到至少约1g/L、至少约300mg/L到至少约1g/L、至少约400mg/L到至少约1g/L、至少约500mg/L到至少约1g/L、至少约600mg/L到至少约1g/L、至少约700mg/L到至少约1g/L、至少约800mg/L到至少约1g/L、或至少约900mg/L到至少约1g/L。尽管在大肠杆菌或毕赤酵母(例如巴斯德毕赤酵母)中表达时，本文所述的配体和dAb单体可以分泌，但是它们也可以采用任何其它合适方法来制备，例如不需要用大肠杆菌或毕赤酵母的合成化学方法或生物学制备方法。Preferred ligands or dAb monomers are secreted in amounts of at least about 0.5 mg/L when expressed in E. coli or Pichia pastoris (eg, Pichia pastoris). In other preferred embodiments, the dAb monomer is secreted in an amount of at least about 0.75 mg/L, at least about 1 mg/L, at least about 4mg/L, at least about 5mg/L, at least about 10mg/L, at least about 15mg/L, at least about 20mg/L, at least about 25mg/L, at least about 30mg/L, at least about 35mg/L, at least about 40mg/L L, at least about 45mg/L, or at least about 50mg/L, or at least about 100mg/L, or at least about 200mg/L, or at least about 300mg/L, or at least about 400mg/L, or at least about 500mg/L, Or at least about 600 mg/L, or at least about 700 mg/L, or at least about 800 mg/L, at least about 900 mg/L, or at least about 1 g/L. In other preferred embodiments, the dAb monomer is secreted in an amount of at least about 1 mg/L to at least about 1 g/L, at least about 1 mg when expressed in E. coli or Pichia pastoris (e.g., Pichia pastoris) /L to at least about 750 mg/L, at least about 100 mg/L to at least about 1 g/L, at least about 200 mg/L to at least about 1 g/L, at least about 300 mg/L to at least about 1 g/L, at least about 400 mg/L to at least about 1 g/L, at least about 500 mg/L to at least about 1 g/L, at least about 600 mg/L to at least about 1 g/L, at least about 700 mg/L to at least about 1 g/L, at least about 800 mg/L to at least About 1 g/L, or at least about 900 mg/L to at least about 1 g/L. Although the ligands and dAb monomers described herein can be secreted when expressed in E. coli or Pichia pastoris (e.g., Pichia pastoris), they can also be prepared by any other suitable method, e.g., without using Synthetic chemical methods or biological preparation methods of Escherichia coli or Pichia pastoris.
dAb单体可包含任何合适的免疫球蛋白可变区、优选包含人可变区或包含人构架区的可变区。在某些实施方案中，dAb单体包含本文所述的通用构架。The dAb monomer may comprise any suitable immunoglobulin variable region, preferably a human variable region or a variable region comprising a human framework region. In certain embodiments, dAb monomers comprise the general frameworks described herein.
通用构架可以是VL构架(Vλ或Vκ)，例如包含由人种系DPK1、DPK2、DPK3、DPK4、DPK5、DPK6、DPK7、DPK8、DPK9、DPK10、DPK12、DPK13、DPK15、DPK16、DPK18、DPK19、DPK20、DPK21、DPK22、DPK23、DPK24、DPK25、DPK26或DPK28免疫球蛋白基因区段所编码的构架氨基酸序列的构架。如有必要，VL构架还可包含由人种系Jκ1、Jκ2、Jκ3、Jκ4、或Jκ5免疫球蛋白基因区段所编码的构架氨基酸序列。The general framework may be aVL framework (Vλ or Vκ), for example comprising a human germline DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19 , DPK20, DPK21 , DPK22, DPK23, DPK24, DPK25, DPK26 or DPK28 immunoglobulin gene segment encoded framework amino acid sequence framework. If desired, theVL framework may also comprise framework amino acid sequences encoded by human germline JK1, JK2, JK3, JK4, or JK5 immunoglobulin gene segments.
在其它实施方案中，通用构架可以是VH构架，例如包含由人种系DP4、DP7、DP8、DP9、DP10、DP31、DP33、DP38、DP45、DP46、DP47、DP49、DP50、DP51、DP53、DP54、DP65、DP66、DP67、DP68或DP69免疫球蛋白基因区段所编码的构架氨基酸序列的构架。如有必要，VH构架还可包含由人种系JH1、JH2、JH3、JH4、JH4b、JH5和JH6免疫球蛋白基因区段所编码的构架氨基酸序列。In other embodiments, the universal framework may be aVH framework, for example comprising a human germline DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP38, DP45, DP46, DP47, DP49, DP50, DP51, DP53, A framework of framework amino acid sequences encoded by DP54, DP65, DP66, DP67, DP68 or DP69 immunoglobulin gene segments. TheVH framework may also contain, if necessary, human germlineJH1 ,JH2 ,JH3 ,JH4 ,JH4b ,JH5 , andJH6 immunoglobulin gene segments. Framework amino acid sequence.
在具体的实施方案中，dAb单体配体包含DPK9 VL构架或选自DP47、DP45和DP38的VH构架。In specific embodiments, the dAb monomer ligand comprises a DPK9VL framework or aVH framework selected from DP47, DP45 and DP38.
dAb单体可包含通用配体的结合位点，所述通用配体例如蛋白A、蛋白L和蛋白G。A dAb monomer may comprise a binding site for a generic ligand, such as protein A, protein L, and protein G.
在某些实施方案中，dAb单体包含一个或多个构架区，所述架构区的氨基酸序列与人种系抗体基因区段所编码的相应构架区的氨基酸序列相同，或者一个或多个所述构架区的氨基酸序列共同包含最多5个与人种系抗体基因区段所编码的所述相应构架区的氨基酸序列不同的氨基酸。In certain embodiments, the dAb monomer comprises one or more framework regions having the same amino acid sequence as the corresponding framework region encoded by a human germline antibody gene segment, or one or more of the framework regions The amino acid sequences of the framework regions collectively comprise up to 5 amino acids that differ from the amino acid sequences of the corresponding framework regions encoded by human germline antibody gene segments.
在其它实施方案中，dAb单体的FW1、FW2、FW3和FW4的氨基酸序列与人种系抗体基因区段所编码的相应构架区的氨基酸序列相同，或者FW1、FW2、FW3和FW4的氨基酸序列共同包含最多10个与所述人种系抗体基因区段所编码的相应构架区的氨基酸序列不同的氨基酸。In other embodiments, the amino acid sequences of FW1, FW2, FW3, and FW4 of the dAb monomer are identical to the amino acid sequences of the corresponding framework regions encoded by human germline antibody gene segments, or the amino acid sequences of FW1, FW2, FW3, and FW4 collectively comprise up to 10 amino acids that differ from the amino acid sequences of the corresponding framework regions encoded by said human germline antibody gene segments.
在其它实施方案中，dAb单体包含FW1、FW2和FW3区，而且所述FW1、FW2和FW3区的氨基酸序列与人种系抗体基因区段所编码的相应构架区的氨基酸序列相同。In other embodiments, the dAb monomer comprises FW1, FW2, and FW3 regions, and the amino acid sequences of the FW1, FW2, and FW3 regions are identical to the amino acid sequences of the corresponding framework regions encoded by human germline antibody gene segments.
在某些实施方案中，dAb单体不含骆驼科动物免疫球蛋白可变区，或者不含骆驼科动物种系抗体基因区段所编码的免疫球蛋白可变区所特有的一个或多个构架氨基酸。In certain embodiments, the dAb monomer does not contain a camelid immunoglobulin variable region, or one or more of the immunoglobulin variable regions that are characteristic of an immunoglobulin variable region encoded by a camelid germline antibody gene segment. Structural amino acids.
与血清白蛋白结合的配体和dAb单体Ligands and dAb monomers that bind serum albumin
本发明提供配体或dAb单体(例如包含这样的dAb的双特异性配体)，其与血清白蛋白(SA)结合的Kd为1nM～500μM(即1×10-9～5×10-4)、优选100nM～10μM。优选对于含有第一抗SA dAb和针对另一靶标的第二dAb的双特异性配体来说，第二dAb对其靶标的亲和力(例如经采用BiaCore进行表面等离子共振测定的Kd和/或K解离)为第一dAb对SA的亲和力的1-100000倍(优选100-100000倍、更优选1000-100000倍、或10000-100000倍)。例如，第一dAb结合SA的亲和力约为10μM，第二dAb结合其靶标的亲和力为100pM。优选的血清白蛋白是人血清白蛋白(HSA)。在一个实施方案中，第一dAb(或dAb单体)与SA(例如HSA)结合的Kd约为50nM、优选70nM、更优选100nM、150nM或200nM。The present invention provides ligands or dAb monomers (such as dual-specific ligands comprising such dAbs) that bind serum albumin (SA) with a Kd of 1 nM to 500 μM (ie, 1×10−9 to 5×10-4 ), preferably 100 nM to 10 μM. Preferably for a dual specific ligand comprising a first anti-SA dAb and a second dAb directed against another target, the affinity of the second dAb to its target (e.g.Kd and/or as determined by surface plasmon resonance using BiaCore) Kdissociation ) is 1-100000 times (preferably 100-100000 times, more preferably 1000-100000 times, or 10000-100000 times) the affinity of the first dAb for SA. For example, the first dAb binds SA with an affinity of approximately 10 μM and the second dAb binds its target with an affinity of 100 pM. A preferred serum albumin is human serum albumin (HSA). In one embodiment, the first dAb (or dAb monomer) binds SA (eg, HSA) with aKd of about 50 nM, preferably 70 nM, more preferably 100 nM, 150 nM or 200 nM.
在某些实施方案中，对SA具有特异性的dAb单体包含MSA-16的氨基酸序列(SEQ ID NO：28)或与其具有至少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％、或至少99％同源性的序列。In certain embodiments, the dAb monomer specific for SA comprises or has at least 80%, at least 85%, at least 90%, at least 91%, at least A sequence that is 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.
在其它实施方案中，对SA具有特异性的dAb单体包含MSA-26的氨基酸序列(SEQ ID NO：30)或与其具有至少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％、或至少99％同源性的序列。In other embodiments, the dAb monomer specific for SA comprises or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92% of the amino acid sequence of MSA-26 (SEQ ID NO: 30) %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the sequence.
在某些实施方案中，与SA结合的dAb单体能抵抗聚集、可逆地解折叠和/或包含如上所述的构架区，该架构区用于能结合TNFR1的dAb单体。In certain embodiments, dAb monomers that bind SA resist aggregation, unfold reversibly, and/or comprise framework regions as described above for dAb monomers that bind TNFRl.
核酸分子、载体和宿主细胞Nucleic acid molecules, vectors and host cells
本发明也提供分离和/或重组的核酸分子，所述分子编码本文所述的配体和dAb单体。在某些实施方案中，分离和/或重组的核酸包含的核苷酸序列编码能特异性结合肿瘤坏死因子受体1(TNFR)的域抗体(dAb)单体，其中所述核苷酸序列与选自以下的核苷酸序列具有至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2h-12(SEQ ID NO：32)、TAR2h-13(SEQ ID NO：33)、TAR2h-14(SEQ ID NO：34)、TAR2h-16(SEQ ID NO：35)、TAR2h-17(SEQ ID NO：36)、TAR2h-18(SEQ ID NO：37)、TAR2h-19(SEQ ID NO：38)、TAR2h-20(SEQ ID NO：39)、TAR2h-21(SEQ ID NO：40)、TAR2h-22(SEQ ID NO：41)、TAR2h-23(SEQ ID NO：42)、TAR2h-24(SEQ ID NO：43)、TAR2h-25(SEQ ID NO：44)、TAR2h-26(SEQ ID NO：45)、TAR2h-27(SEQ ID NO：46)、TAR2h-29(SEQ ID NO：47)、TAR2h-30(SEQ ID NO：48)、TAR2h-32(SEQ ID NO：49)、TAR2h-33(SEQ ID NO：50)、TAR2h-10-1(SEQ IDNO：51)、TAR2h-10-2(SEQ ID NO：52)、TAR2h-10-3(SEQ ID NO：53)、TAR2h-10-4(SEQ ID NO：54)、TAR2h-10-5(SEQ ID NO：55)、TAR2h-10-6(SEQ ID NO：56)、TAR2h-10-7(SEQ ID NO：57)、TAR2h-10-8(SEQ ID NO：58)、TAR2h-10-9(SEQ ID NO：59)、TAR2h-10-10(SEQ ID NO：60)、TAR2h-10-11(SEQ ID NO：61)、TAR2h-10-12(SEQ ID NO：62)、TAR2h-10-13(SEQ ID NO：63)、TAR2h-10-14(SEQ ID NO：64)、TAR2h-10-15(SEQ ID NO：65)、TAR2h-10-16(SEQ ID NO：66)、TAR2h-10-17(SEQ ID NO：67)、TAR2h-10-18(SEQ ID NO：68)、TAR2h-10-19(SEQ ID NO：69)、TAR2h-10-20(SEQ ID NO：70)、TAR2h-10-21(SEQ ID NO：71)、TAR2h-10-22(SEQ ID NO：72)、TAR2h-10-27(SEQ ID NO：73)、TAR2h-10-29(SEQ ID NO：74)、TAR2h-10-31(SEQ ID NO：75)、TAR2h-10-35(SEQ ID NO：76)、TAR2h-10-36(SEQ ID NO：77)、TAR2h-10-37(SEQ ID NO：78)、TAR2h-10-38(SEQ ID NO：79)、TAR2h-10-45(SEQ ID NO：80)、TAR2h-10-47(SEQ ID NO：81)、TAR2h-10-48(SEQ ID NO：82)、TAR2h-10-57(SEQ ID NO：83)、TAR2h-10-56(SEQ ID NO：84)、TAR2h-10-58(SEQ ID NO：85)、TAR2h-10-66(SEQ ID NO：86)、TAR2h-10-64(SEQ ID NO：87)、TAR2h-10-65(SEQ ID NO：88)、TAR2h-10-68(SEQ ID NO：89)、TAR2h-10-69(SEQ ID NO：90)、TAR2h-10-67(SEQ ID NO：91)、TAR2h-10-61(SEQ ID NO：92)、TAR2h-10-62(SEQ ID NO：93)、TAR2h-10-63(SEQ ID NO：94)、TAR2h-10-60(SEQ ID NO：95)、TAR2h-10-55(SEQ ID NO：96)、TAR2h-10-59(SEQ ID NO：97)、TAR2h-10-70(SEQ ID NO：98)、TAR2h-34(SEQ ID NO：402)、TAR2h-35(SEQ ID NO：403)、TAR2h-36(SEQ ID NO：404)、TAR2h-37(SEQ ID NO：405)、TAR2h-38(SEQ ID NO：406)、TAR2h-39(SEQID NO：407)、TAR2h-40(SEQ ID NO：408)、TAR2h-41(SEQ ID NO：409)、TAR2h-42(SEQ ID NO：410)、TAR2h-43(SEQ ID NO：411)、TAR2h-44(SEQ ID NO：412)、TAR2h-45(SEQ ID NO：413)、TAR2h-47(SEQ ID NO：414)、TAR2h-48(SEQ ID NO：415)、TAR2h-50(SEQID NO：416)、TAR2h-51(SEQ ID NO：417)、TAR2h-66(SEQ ID NO：418)、TAR2h-67(SEQ ID NO：419)、TAR2h-68(SEQ ID NO：420)、TAR2h-70(SEQ ID NO：421)、TAR2h-71(SEQ ID NO：422)、TAR2h-72(SEQ ID NO：423)、TAR2h-73(SEQ ID NO：424)、TAR2h-74(SEQID NO：425)、TAR2h-75(SEQ ID NO：426)、TAR2h-76(SEQ ID NO：427)、TAR2h-77(SEQ ID NO：428)、TAR2h-78(SEQ ID NO：429)、TAR2h-79(SEQ ID NO：430)和TAR2h-15(SEQ ID NO：432)。The invention also provides isolated and/or recombinant nucleic acid molecules encoding the ligands and dAb monomers described herein. In some embodiments, the nucleotide sequence contained in the isolated and/or recombinant nucleic acid encodes a domain antibody (dAb) monomer that can specifically bind tumor necrosis factor receptor 1 (TNFR), wherein the nucleotide sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, At least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-12 (SEQ ID NO: 32), TAR2h-13 (SEQ ID NO: 33), TAR2h-14 ( SEQ ID NO: 34), TAR2h-16 (SEQ ID NO: 35), TAR2h-17 (SEQ ID NO: 36), TAR2h-18 (SEQ ID NO: 37), TAR2h-19 (SEQ ID NO: 38) , TAR2h-20 (SEQ ID NO: 39), TAR2h-21 (SEQ ID NO: 40), TAR2h-22 (SEQ ID NO: 41), TAR2h-23 (SEQ ID NO: 42), TAR2h-24 (SEQ ID NO: 43), TAR2h-25 (SEQ ID NO: 44), TAR2h-26 (SEQ ID NO: 45), TAR2h-27 (SEQ ID NO: 46), TAR2h-29 (SEQ ID NO: 47), TAR2h-30 (SEQ ID NO: 48), TAR2h-32 (SEQ ID NO: 49), TAR2h-33 (SEQ ID NO: 50), TAR2h-10-1 (SEQ ID NO: 51), TAR2h-10-2 (SEQ ID NO: 52), TAR2h-10-3 (SEQ ID NO: 53), TAR2h-10-4 (SEQ ID NO: 54), TAR2h-10-5 (SEQ ID NO: 55), TAR2h-10 -6 (SEQ ID NO: 56), TAR2h-10-7 (SEQ ID NO: 57), TAR2h-10-8 (SEQ ID NO: 58), TAR2h-10-9 (SEQ ID NO: 59), TAR2h -10-10 (SEQ ID NO: 60), TAR2h-10-11 (SEQ ID NO: 61), TAR2h-10-12 (SEQ ID NO: 62), TAR2h-10-13 (SEQ ID NO: 63) , TAR2h-10-14 (SEQ ID NO: 64), TAR2h-10-15 (SEQ ID NO: 65), TAR2h-10-16 (SEQ ID NO: 66), TAR2h-10-17 (SEQ ID NO: 67), TAR2h-10-18 (SEQ ID NO: 68), TAR2h-10-19 (SEQ ID NO: 69), TAR2h-10-20 (SEQ ID NO: 70), TAR2h-10-21 (SEQ ID NO: 71), TAR2h-10-22 (SEQ ID NO: 72), TAR2h-10-27 (SEQ ID NO: 73), TAR2h-10-29 (SEQ ID NO: 74), TAR2h-10-31 ( SEQ ID NO: 75), TAR2h-10-35 (SEQ ID NO: 76), TAR2h-10-36 (SEQ ID NO: 77), TAR2h-10-37 (SEQ ID NO: 78), TAR2h-10- 38 (SEQ ID NO: 79), TAR2h-10-45 (SEQ ID NO: 80), TAR2h-10-47 (SEQ ID NO: 81), TAR2h-10-48 (SEQ ID NO: 82), TAR2h- 10-57 (SEQ ID NO: 83), TAR2h-10-56 (SEQ ID NO: 84), TAR2h-10-58 (SEQ ID NO: 85), TAR2h-10-66 (SEQ ID NO: 86), TAR2h-10-64 (SEQ ID NO: 87), TAR2h-10-65 (SEQ ID NO: 88), TAR2h-10-68 (SEQ ID NO: 89), TAR2h-10-69 (SEQ ID NO: 90 ), TAR2h-10-67 (SEQ ID NO: 91), TAR2h-10-61 (SEQ ID NO: 92), TAR2h-10-62 (SEQ ID NO: 93), TAR2h-10-63 (SEQ ID NO : 94), TAR2h-10-60 (SEQ ID NO: 95), TAR2h-10-55 (SEQ ID NO: 96), TAR2h-10-59 (SEQ ID NO: 97), TAR2h-10-70 (SEQ ID NO: 98), TAR2h-34 (SEQ ID NO: 402), TAR2h-35 (SEQ ID NO: 403), TAR2h-36 (SEQ ID NO: 404), TAR2h-37 (SEQ ID NO: 405), TAR2h-38 (SEQ ID NO: 406), TAR2h-39 (SEQ ID NO: 407), TAR2h-40 (SEQ ID NO: 408), TAR2h-41 (SEQ ID NO: 409), TAR2h-42 (SEQ ID NO : 410), TAR2h-43 (SEQ ID NO: 411), TAR2h-44 (SEQ ID NO: 412), TAR2h-45 (SEQ ID NO: 413), TAR2h-47 (SEQ ID NO: 414), TAR2h- 48 (SEQ ID NO: 415), TAR2h-50 (SEQ ID NO: 416), TAR2h-51 (SEQ ID NO: 417), TAR2h-66 (SEQ ID NO: 418), TAR2h-67 (SEQ ID NO: 419 ), TAR2h-68 (SEQ ID NO: 420), TAR2h-70 (SEQ ID NO: 421), TAR2h-71 (SEQ ID NO: 422), TAR2h-72 (SEQ ID NO: 423), TAR2h-73 ( SEQ ID NO: 424), TAR2h-74 (SEQ ID NO: 425), TAR2h-75 (SEQ ID NO: 426), TAR2h-76 (SEQ ID NO: 427), TAR2h-77 (SEQ ID NO: 428), TAR2h-78 (SEQ ID NO: 429), TAR2h-79 (SEQ ID NO: 430) and TAR2h-15 (SEQ ID NO: 432).
在其它实施方案中，分离和/或重组的核酸包含的核苷酸序列编码能特异性结合肿瘤坏死因子受体1(TNFR1)的域抗体(dAb)单体，其中所述核苷酸序列与选自以下的核苷酸序列具有至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2h-131-8(SEQ ID NO：518)、TAR2h-131-24(SEQ ID NO：519)、TAR2h-15-8(SEQ ID NO：520)、TAR2h-15-8-1SEQ ID NO：521)、TAR2h-15-8-2(SEQ ID NO：522)、TAR2h-185-23(SEQ ID NO：523)、TAR2h-154-10-5(SEQ ID NO：524)、TAR2h-14-2(SEQ ID NO：525)、TAR2h-151-8(SEQ ID NO：526)、TAR2h-152-7(SEQ ID NO：527)、TAR2h-35-4(SEQ ID NO：528)、TAR2h-154-7(SEQID NO：529)、TAR2h-80(SEQ ID NO：530)、TAR2h-81(SEQ ID NO：531)、TAR2h-82(SEQ ID NO：532)、TAR2h-83(SEQ ID NO：533)、TAR2h-84(SEQ ID NO：534)、TAR2h-85(SEQ ID NO：535)、TAR2h-86(SEQ ID NO：536)、TAR2h-87(SEQ ID NO：537)、TAR2h-88(SEQID NO：538)、TAR2h-89(SEQ ID NO：539)、TAR2h-90(SEQ ID NO：540)、TAR2h-91(SEQ ID NO：541)、TAR2h-92(SEQ ID NO：542)、TAR2h-93(SEQ ID NO：543)、TAR2h-94(SEQ ID NO：544)、TAR2h-95(SEQ ID NO：545)、TAR2h-96(SEQ ID NO：546)、TAR2h-97(SEQID NO：547)、TAR2h-99(SEQ ID NO：548)、TAR2h-100(SEQ ID NO：549)、TAR2h-101(SEQ ID NO：550)、TAR2h-102(SEQ ID NO：551)、TAR2h-103(SEQ ID NO：552)、TAR2h-104(SEQ ID NO：553)、TAR2h-105(SEQ ID NO：554)、TAR2h-106(SEQ ID NO：555)、TAR2h-107(SEQ ID NO：556)、TAR2h-108(SEQ ID NO：557)、TAR2h-109(SEQ ID NO：558)、TAR2h-110(SEQ ID NO：559)、TAR2h-111(SEQ ID NO：560)、TAR2h-112(SEQ ID NO：561)、TAR2h-113(SEQ ID NO：562)、TAR2h-114(SEQ ID NO：563)、TAR2h-115(SEQ ID NO：564)、TAR2h-116(SEQ ID NO：565)、TAR2h-117(SEQ ID NO：566)、TAR2h-118(SEQ ID NO：567)、TAR2h-119(SEQ ID NO：568)、TAR2h-120(SEQ ID NO：569)、TAR2h-121(SEQ ID NO：570)、TAR2h-122(SEQ ID NO：571)、TAR2h-123(SEQ ID NO：572)、TAR2h-124(SEQ ID NO：573)、TAR2h-125(SEQ ID NO：574)、TAR2h-126(SEQ ID NO：575)、TAR2h-127(SEQ ID NO：576)、TAR2h-128(SEQ ID NO：577)、TAR2h-129(SEQ ID NO：578)、TAR2h-130(SEQ ID NO：579)、TAR2h-131(SEQ ID NO：580)、TAR2h-132(SEQ ID NO：581)、TAR2h-133(SEQ ID NO：582)、TAR2h-151(SEQ ID NO：583)、TAR2h-152(SEQ ID NO：584)、TAR2h-153(SEQ ID NO：585)、TAR2h-154(SEQ ID NO：586)、TAR2h-159(SEQ ID NO：587)、TAR2h-165(SEQ ID NO：588)、TAR2h-166(SEQ ID NO：589)、TAR2h-168(SEQ ID NO：590)、TAR2h-171(SEQ ID NO：591)、TAR2h-172(SEQ ID NO：592)、TAR2h-173(SEQ ID NO：593)、TAR2h-174(SEQ ID NO：594)、TAR2h-176(SEQ ID NO：595)、TAR2h-178(SEQ ID NO：596)、TAR2h-201(SEQ ID NO：597)、TAR2h-202(SEQ ID NO：598)、TAR2h-203(SEQ ID NO：599)、TAR2h-204(SEQ ID NO：600)、TAR2h-185-25(SEQ ID NO：601)、TAR2h-154-10(SEQ ID NO：602)和TAR2h-205(SEQ ID NO：628)。In other embodiments, the isolated and/or recombinant nucleic acid comprises a nucleotide sequence encoding a domain antibody (dAb) monomer that specifically binds tumor necrosis factor receptor 1 (TNFR1), wherein the nucleotide sequence is identical to A nucleotide sequence selected from the group consisting of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least About 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-131-8 (SEQ ID NO:518), TAR2h-131-24 (SEQ ID NO:519), TAR2h -15-8 (SEQ ID NO: 520), TAR2h-15-8-1 (SEQ ID NO: 521), TAR2h-15-8-2 (SEQ ID NO: 522), TAR2h-185-23 (SEQ ID NO: 523), TAR2h-154-10-5 (SEQ ID NO: 524), TAR2h-14-2 (SEQ ID NO: 525), TAR2h-151-8 (SEQ ID NO: 526), TAR2h-152-7 ( SEQ ID NO: 527), TAR2h-35-4 (SEQ ID NO: 528), TAR2h-154-7 (SEQ ID NO: 529), TAR2h-80 (SEQ ID NO: 530), TAR2h-81 (SEQ ID NO : 531), TAR2h-82 (SEQ ID NO: 532), TAR2h-83 (SEQ ID NO: 533), TAR2h-84 (SEQ ID NO: 534), TAR2h-85 (SEQ ID NO: 535), TAR2h- 86 (SEQ ID NO:536), TAR2h-87 (SEQ ID NO:537), TAR2h-88 (SEQ ID NO:538), TAR2h-89 (SEQ ID NO:539), TAR2h-90 (SEQ ID NO:540 ), TAR2h-91 (SEQ ID NO: 541), TAR2h-92 (SEQ ID NO: 542), TAR2h-93 (SEQ ID NO: 543), TAR2h-94 (SEQ ID NO: 544), TAR2h-95 ( SEQ ID NO: 545), TAR2h-96 (SEQ ID NO: 546), TAR2h-97 (SEQ ID NO: 547), TAR2h-99 (SEQ ID NO: 548), TAR2h-100 (SEQ ID NO: 549), TAR2h-101 (SEQ ID NO: 550), TAR2h-102 (SEQ ID NO: 551), TAR2h-103 (SEQ ID NO: 552), TAR2h-104 (SEQ ID NO: 553), TAR2h-105 (SEQ ID NO: 554), TAR2h-106 (SEQ ID NO: 555), TAR2h-107 (SEQ ID NO: 556), TAR2h-108 (SEQ ID NO: 557), TAR2h-109 (SEQ ID NO: 558), TAR2h -110 (SEQ ID NO: 559), TAR2h-111 (SEQ ID NO: 560), TAR2h-112 (SEQ ID NO: 561), TAR2h-113 (SEQ ID NO: 562), TAR2h-114 (SEQ ID NO : 563), TAR2h-115 (SEQ ID NO: 564), TAR2h-116 (SEQ ID NO: 565), TAR2h-117 (SEQ ID NO: 566), TAR2h-118 (SEQ ID NO: 567), TAR2h- 119 (SEQ ID NO: 568), TAR2h-120 (SEQ ID NO: 569), TAR2h-121 (SEQ ID NO: 570), TAR2h-122 (SEQ ID NO: 571), TAR2h-123 (SEQ ID NO: 572), TAR2h-124 (SEQ ID NO:573), TAR2h-125 (SEQ ID NO:574), TAR2h-126 (SEQ ID NO:575), TAR2h-127 (SEQ ID NO:576), TAR2h-128 (SEQ ID NO: 577), TAR2h-129 (SEQ ID NO: 578), TAR2h-130 (SEQ ID NO: 579), TAR2h-131 (SEQ ID NO: 580), TAR2h-132 (SEQ ID NO: 581 ), TAR2h-133 (SEQ ID NO: 582), TAR2h-151 (SEQ ID NO: 583), TAR2h-152 (SEQ ID NO: 584), TAR2h-153 (SEQ ID NO: 585), TAR2h-154 ( SEQ ID NO: 586), TAR2h-159 (SEQ ID NO: 587), TAR2h-165 (SEQ ID NO: 588), TAR2h-166 (SEQ ID NO: 589), TAR2h-168 (SEQ ID NO: 590) , TAR2h-171 (SEQ ID NO: 591), TAR2h-172 (SEQ ID NO: 592), TAR2h-173 (SEQ ID NO: 593), TAR2h-174 (SEQ ID NO: 594), TAR2h-176 (SEQ ID NO: 595), TAR2h-178 (SEQ ID NO: 596), TAR2h-201 (SEQ ID NO: 597), TAR2h-202 (SEQ ID NO: 598), TAR2h-203 (SEQ ID NO: 599), TAR2h-204 (SEQ ID NO: 600), TAR2h-185-25 (SEQ ID NO: 601), TAR2h-154-10 (SEQ ID NO: 602) and TAR2h-205 (SEQ ID NO: 628).
在一个优选的实施方案中，分离和/或重组的核酸包含的核苷酸序列与选自以下的核苷酸具有至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2m-10-27(SEQ ID NO：141)、TAR2m-10-57(SEQ ID NO：151)、TAR2m-10-56(SEQ ID NO：152)、TAR2m-10-58(SEQ ID NO：153)、TAR2m-10-66(SEQ ID NO：154)、TAR2m-10-64(SEQ ID NO：155)、TAR2m-10-65(SEQ ID NO：156)、TAR2m-10-68(SEQ ID NO：157)、TAR2m-10-69(SEQ ID NO：158)、TAR2m-10-67(SEQ ID NO：159)、TAR2m-10-61(SEQ ID NO：160)、TAR2m-10-62(SEQ ID NO：161)、TAR2m-10-63(SEQ ID NO：162)、TAR2m-10-60(SEQ ID NO：163)、TAR2m-10-55(SEQ ID NO：164)、TAR2m-10-59(SEQ ID NO：165)和TAR2m-10-70(SEQ ID NO：166)。In a preferred embodiment, the isolated and/or recombinant nucleic acid comprises a nucleotide sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, At least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2m-10-27 (SEQ ID NO: 141), TAR2m-10 -57 (SEQ ID NO: 151), TAR2m-10-56 (SEQ ID NO: 152), TAR2m-10-58 (SEQ ID NO: 153), TAR2m-10-66 (SEQ ID NO: 154), TAR2m -10-64 (SEQ ID NO: 155), TAR2m-10-65 (SEQ ID NO: 156), TAR2m-10-68 (SEQ ID NO: 157), TAR2m-10-69 (SEQ ID NO: 158) , TAR2m-10-67 (SEQ ID NO: 159), TAR2m-10-61 (SEQ ID NO: 160), TAR2m-10-62 (SEQ ID NO: 161), TAR2m-10-63 (SEQ ID NO: 162), TAR2m-10-60 (SEQ ID NO: 163), TAR2m-10-55 (SEQ ID NO: 164), TAR2m-10-59 (SEQ ID NO: 165) and TAR2m-10-70 (SEQ ID NO: 165) NO: 166).
在其它实施方案中，分离和/或重组的核酸包含的核苷酸序列编码能特异性结合肿瘤坏死因子受体1(TNFR1)的域抗体(dAb)单体，其中所述核苷酸序列与选自以下的核苷酸序列具有至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％同源性：TAR2m-14(SEQ ID NO：180)、TAR2m-15(SEQID NO：181)、TAR2m-19(SEQ ID NO：182)、TAR2m-20(SEQ ID NO：183)、TAR2m-21(SEQ ID NO：184)、TAR2m-24(SEQ ID NO：185)、TAR2m-21-23(SEQ ID NO：186)、TAR2m-21-07(SEQ ID NO：187)、TAR2m-21-43(SEQ ID NO：188)、TAR2m-21-48(SEQ ID NO：189)、TAR2m-21-10(SEQ ID NO：190)、TAR2m-21-06(SEQ ID NO：191)和TAR2m-21-17(SEQ ID NO：192)和TAR2m-21-23a(SEQ ID NO：626)。In other embodiments, the isolated and/or recombinant nucleic acid comprises a nucleotide sequence encoding a domain antibody (dAb) monomer that specifically binds tumor necrosis factor receptor 1 (TNFR1), wherein the nucleotide sequence is identical to A nucleotide sequence selected from the group consisting of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least About 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2m-14 (SEQ ID NO: 180), TAR2m-15 (SEQ ID NO: 181), TAR2m-19 (SEQ ID NO: 182), TAR2m-20 (SEQ ID NO: 183), TAR2m-21 (SEQ ID NO: 184), TAR2m-24 (SEQ ID NO: 185), TAR2m-21-23 (SEQ ID NO: 186) , TAR2m-21-07 (SEQ ID NO: 187), TAR2m-21-43 (SEQ ID NO: 188), TAR2m-21-48 (SEQ ID NO: 189), TAR2m-21-10 (SEQ ID NO: 190), TAR2m-21-06 (SEQ ID NO: 191) and TAR2m-21-17 (SEQ ID NO: 192) and TAR2m-21-23a (SEQ ID NO: 626).
本发明也提供包含本发明重组核酸分子的载体。在某些实施方案中，载体是包含一个或多个与本发明重组核酸操作性连接的表达控制元件或序列的表达载体。合适的载体(例如质粒、噬菌粒)和表达控制元件进一步描述如下。The invention also provides vectors comprising recombinant nucleic acid molecules of the invention. In certain embodiments, the vector is an expression vector comprising one or more expression control elements or sequences operably linked to a recombinant nucleic acid of the invention. Suitable vectors (eg, plasmids, phagemids) and expression control elements are further described below.
本发明也提供重组宿主细胞，所述细胞包含本发明的重组核酸分子或载体。合适的宿主细胞以及用于制备本发明重组宿主细胞的方法进一步描述如下。The invention also provides recombinant host cells comprising a recombinant nucleic acid molecule or vector of the invention. Suitable host cells and methods for preparing recombinant host cells of the invention are further described below.
本发明也提供制备本发明的配体(例如dAb单体、双特异性配体、多特异性配体)的方法，该方法包括在适于所述重组核酸表达的条件下维持包含本发明重组核酸的重组宿主细胞，由此表达重组核酸并产生配体。在某些实施方案中，该方法还包括分离出配体。The invention also provides a method for preparing a ligand (eg, dAb monomer, dual-specific ligand, multispecific ligand) of the invention, the method comprising maintaining a recombinant nucleic acid comprising the recombinant nucleic acid of the invention under conditions suitable for expression of said recombinant nucleic acid. Recombinant host cells for nucleic acids whereby recombinant nucleic acids are expressed and ligands are produced. In certain embodiments, the method further includes isolating the ligand.
配体形式Ligand form
本发明的配体可以精制成为单特异性或多特异性抗体或抗体片段或者单特异性或多特异性非免疫球蛋白结构。合适的形式包括任何合适的多肽结构，其中抗体可变区或其CDR可以结合起来，以便在结构上赋予对抗原的结合特异性。各种合适的抗体形式是本领域已知的，例如IgG样形式、嵌合抗体、人源化抗体、人抗体、单链抗体、双特异性抗体、抗体重链、抗体轻链、抗体重链和/或轻链的同型二聚体和异型二聚体、任何前述抗原结合片段(例如Fv片段(例如单链Fv(scFv)、二硫键连接的Fv)、Fab片段、Fab′片段、F(ab′)2片段)、单可变区(例如VH、VL)、dAb和任何前述的修饰形式(例如通过共价连接聚烷二醇(例如聚乙二醇、聚丙二醇、聚丁二醇)或其它合适的聚合物)而修饰)。参见PCT/GB03/002804(2003年6月30日申请，指定国为美国，公布号为WO 2004/081026)有关单可变区和dAb的PEG化、其合适的制备方法、PEG化单可变区和dAb单体和多聚体的延长的体内半衰期、合适的PEG、优选的流体动力学尺寸的PEG和优选的流体动力学尺寸的PEG化单可变区和dAb单体和多聚体。PCT/GB03/002804(WO 2004/081026)的全部公开内容，包括上面提及的部分，全都通过引用结合到本文中。The ligands of the invention may be refined as monospecific or multispecific antibodies or antibody fragments or monospecific or multispecific non-immunoglobulin structures. Suitable formats include any suitable polypeptide structure in which antibody variable regions or CDRs thereof may be combined so as to confer binding specificity for the antigen structurally. Various suitable antibody formats are known in the art, such as IgG-like formats, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, antibody heavy chains and/or homodimers and heterodimers of light chains, any of the foregoing antigen-binding fragments (e.g., Fv fragments (e.g., single-chain Fv (scFv), disulfide-linked Fv), Fab fragments, Fab' fragments, Fab fragments, Fab fragments, (ab')2 fragments), single variable domains (e.g.VH ,VL ), dAbs and any of the foregoing modified forms (e.g. by covalently linking polyalkylene glycols (e.g. polyethylene glycol, polypropylene glycol, polybutylene glycol Diol) or other suitable polymer) and modified). See PCT/GB03/002804 (applied on June 30, 2003, the designated country is the United States, publication number is WO 2004/081026) for PEGylation of single variable domains and dAbs, suitable methods for their preparation, PEGylated single variable Prolonged in vivo half-life of domain and dAb monomers and multimers, suitable PEG, preferred hydrodynamic size of PEG and preferred hydrodynamic size of PEGylated single variable domain and dAb monomers and multimers. The entire disclosure of PCT/GB03/002804 (WO 2004/081026), including the above mentioned parts, is hereby incorporated by reference.
在具体的实施方案中，所述配体(例如dAb单体、二聚体或多聚体、双特异性形式、多特异性形式)是PEG化配体，并且能结合TNFR1的域1和任选抑制TNFR1功能。优选的PEG化配体抑制通过TNFR1的信号转导。优选的PEG化配体与TNFR1的域1结合的亲和力与未PEG化的相同配体的亲和力相同。例如，在一个实施方案中，所述配体是能结合TNFR1的域1并抑制通过TNFR1的信号转导的PEG化dAb单体，其中PEG化dAb单体与TNFR1的域1结合的亲和力与非PEG化形式的dAb的亲和力之间的差异不超过约1000倍、优选不超过约100倍，更优选不超过约10倍，或者前者的亲和力相对于未PEG化形式的亲和力来说基本不变。In specific embodiments, the ligand (e.g. dAb monomer, dimer or multimer, bispecific format, multispecific format) is a PEGylated ligand and is capable of binding
结合TNFR1的膜结合(跨膜)和可溶性形式的域1、但不抑制TNFα与受体形式结合的本发明配体，可用于阻断膜结合受体上的域1(例如以抑制受体成簇和/或抑制信号转导)并且也结合可溶性形式，以延长半衰期，特别是当与PEG连接或按照上述其它方法时。在一个优选的实施方案中，所述配体不结合TNFR1的膜结合(跨膜)和可溶性形式的域2。在另一优选的实施方案中，所述配体不结合TNFR1的膜结合(跨膜)和可溶性形式的域3。在另一优选的实施方案中，所述配体不结合TNFR1的膜结合(跨膜)和可溶性形式的域2或域3。Ligands of the invention that bind
在某些实施方案中，所述配体是IgG样形式。这类形式具有常规IgG分子的4条链结构(2条重链和2条轻链)，其中一个或多个可变区(VH和/或VL)已经被具有所需特异性的dAb即单可变区取代。优选各可变区(2个VH区和2个VL区)被dAb即单可变区取代。包括在IgG样形式中的dAb即单可变区可具有相同特异性或不同特异性。在某些实施方案中，IgG样形式是四价的，并且可具有1、2、3或4种特异性。例如，IgG样形式可以是单特异性的，并且包含4个具有相同特异性的dAb；可以是双特异性的，并且包含3个具有相同特异性的dAb和另一具有不同特异性的dAb；可以是双特异性的，并且包含2个具有相同特异性的dAb和两个具有共同、却不同特异性的dAb；可以是三特异性的，并且包含具有相同特异性的第一和第二dAb，具有不同特异性的第三dAb，具有不同于第一、第二和第三dAb的特异性的第四dAb；或者可以是四特异性的，并且包含4个各自具有不同特异性的dAb。可以制备IgG样形式的抗原结合片段(例如Fab、F(ab′)2、Fab′、Fv、scFy)。优选的IgG样形式或其抗原结合片段不交联TNFR1。In certain embodiments, the ligand is in an IgG-like format. Such formats have the 4-chain structure of a conventional IgG molecule (2 heavy and 2 light chains), in which one or more variable domains (VH and/orVL ) have been synthesized by a dAb with the desired specificity That is, a single variable domain substitution. Preferably each variable domain (2VH domains and 2VL domains) is replaced by a dAb, ie a single variable domain. The dAbs, ie, single variable domains, included in an IgG-like format may have the same specificity or different specificities. In certain embodiments, IgG-like forms are tetravalent and may have 1, 2, 3 or 4 specificities. For example, an IgG-like format may be monospecific and comprise 4 dAbs of the same specificity; may be bispecific and comprise 3 dAbs of the same specificity and another dAb of a different specificity; Can be bispecific and contain 2 dAbs with the same specificity and two dAbs with common but different specificities; can be trispecific and contain a first and a second dAb with the same specificity , a third dAb with a different specificity, a fourth dAb with a different specificity than the first, second and third dAbs; or may be tetraspecific and comprise 4 dAbs each with a different specificity. Antigen-binding fragments can be prepared in an IgG-like format (eg, Fab, F(ab')2 , Fab', Fv, scFy). Preferred IgG-like forms or antigen-binding fragments thereof do not cross-link TNFR1.
本发明的配体，包括dAb单体、二聚体和三聚体，可以连接抗体Fc区，该区包含CH2和CH3区的一个或两个，以及任选的铰链区。例如，编码将单核苷酸序列连接Fc区的配体的载体，可用于制备所述多肽。Ligands of the invention, including dAb monomers, dimers and trimers, can be linked to an antibody Fc region comprising one or both of the CH2 and CH3 regions, and optionally a hinge region. For example, a vector encoding a ligand linking a single nucleotide sequence to an Fc region can be used to prepare the polypeptide.
按照本发明的以下方面，本发明还提供前述dAb单体的二聚体、三聚体和多聚体。According to the following aspects of the present invention, the present invention also provides dimers, trimers and multimers of the aforementioned dAb monomers.
本发明的配体可以结合和/或制成非免疫球蛋白多配体结构的形式，以形成多价复合物，其结合具有相同抗原的靶分子，因而提供较高亲合力。在某些实施方案中，至少一个可变区结合抗原，以延长多聚体的半衰期。例如天然细菌受体(例如SpA)已经用作移植CDR的支架，以产生能特异性结合一个或多个表位的配体。该方法的细节参见US5,831,012。其它合适的支架包括基于纤连蛋白和亲和体的支架。合适方法的细节参见WO 98/58965。其它合适的支架包括脂质运载蛋白和CTLA4(参见van den Beuken等，J,Mol.Biol.(2001)310，591-601)以及例如描述于WO00/69907(Medical Research Council)的支架，所述支架是基于例如细菌GroEL的环状结构或其它陪伴分子多肽。Ligands of the invention can bind and/or form non-immunoglobulin multi-ligand structures to form multivalent complexes that bind target molecules with the same antigen, thus providing higher avidity. In certain embodiments, at least one variable region binds antigen to prolong the half-life of the multimer. For example natural bacterial receptors such as SpA have been used as scaffolds for grafting CDRs to generate ligands that specifically bind one or more epitopes. Details of this method are found in US 5,831,012. Other suitable scaffolds include fibronectin and affibody based scaffolds. Details of suitable methods are found in WO 98/58965. Other suitable scaffolds include lipocalin and CTLA4 (see van den Beuken et al., J, Mol. Biol. (2001) 310, 591-601 ) and scaffolds such as described in WO00/69907 (Medical Research Council), which Scaffolds are based on eg the loop structure of bacterial GroEL or other chaperone polypeptides.
蛋白质支架可以进行组合；例如，CDR可以移植到CTLA4支架上并且与免疫球蛋白VH区或VL区一起使用，以构成配体。同样，纤连蛋白、脂质运载蛋白和其它支架也可以进行组合。Protein scaffolds can be combined; for example, CDRs can be grafted onto CTLA4 scaffolds and used with immunoglobulinVH orVL regions to form ligands. Likewise, fibronectin, lipocalin, and other scaffolds can also be combined.
双特异性配体和多特异性配体Dual specific ligands and multispecific ligands
本发明人在其同时待审的国际专利申请WO 03/002609以及同时待审的未公布的UK专利申请0230203.2中，已经描述了双特异性免疫球蛋白配体，所述配体包含各自具有不同特异性的免疫球蛋白单可变区。这些结构域可以彼此竞争性或独立地结合靶分子上的抗原或表位。The present inventors have described dual specific immunoglobulin ligands comprising each of a different Specific immunoglobulin single variable domain. These domains can bind antigens or epitopes on the target molecule competitively with each other or independently.
本发明的双特异性配体优选包含重链区和轻链区的组合。例如，双特异性配体可包含VH区和VL区，这两区可以连接在一起呈scFv的形式。另外，配体可包含一个或多个CH区或CL区。例如，配体可包含CH1区、CH2区或CH3区和/或CL区、Cμ1、Cμ2、Cμ3或Cμ4区、或其任何组合。铰链区也可包含在其中。这样的各区组合可以是例如模拟天然抗体，例如IgG或IgM或其片段，例如Fv、scFv、Fab或F(ab′)2分子。也考虑了其它结构，例如包含VH、VL、CH1和CL区的IgG分子的单臂。The dual specific ligands of the invention preferably comprise a combination of heavy and light chain regions. For example, a dual specific ligand may comprise aVH domain and aVL domain, which may be joined together in the form of a scFv. Additionally, the ligand may comprise one or moreCH orCL regions. For example, the ligand can comprise aCH1 domain, aCH2 domain or aCH3 domain and/or aCL domain, a Cμ1, Cμ2, Cμ3 or Cμ4 domain, or any combination thereof. A hinge region may also be included. Such combinations of domains may for example mimic natural antibodies, eg IgG or IgM or fragments thereof eg Fv, scFv, Fab or F(ab')2 molecules. Other structures are also contemplated, such as a single arm of an IgG molecule comprisingVH ,VL ,CHI andCL regions.
在本发明的一个优选的实施方案中，可变区选自单域V基因库。通常，单域抗体库都展示在丝状噬菌体表面上。在一个优选的实施方案中，通过噬菌体库与抗原的结合，来选择各单域抗体。In a preferred embodiment of the invention, the variable region is selected from a repertoire of single domain V genes. Typically, single domain antibody repertoires are displayed on the surface of filamentous phage. In a preferred embodiment, individual single domain antibodies are selected by binding the phage library to the antigen.
在本发明的一个优选的实施方案中，在互补可变区不存在时，可以通过与其靶抗原或表位的结合来选择各单可变区。在一个替代实施方案中，在互补可变区存在时，可以通过与其靶抗原或表位的结合来选择单可变区。因此，在第三互补可变区存在时，可以选择第一单可变区，在第四互补可变区存在时，可以选择第二可变区。互补的第三或第四可变区可以是天然关联可变区(它们具有与待测单域相同的特异性)或非关联互补区-例如“dummy”可变区。In a preferred embodiment of the invention, in the absence of complementary variable domains, each single variable domain can be selected by binding to its target antigen or epitope. In an alternative embodiment, single variable domains may be selected by binding to their target antigen or epitope when complementary variable domains are present. Thus, the first single variable domain can be selected when the third complementary variable domain is present, and the second variable domain can be selected when the fourth complementary variable domain is present. The complementary third or fourth variable domain may be a naturally associated variable domain (which has the same specificity as the single domain being tested) or a non-cognate complementary domain - eg a "dummy" variable domain.
优选本发明的双特异性配体仅包含两个可变区，尽管几个这样的配体也能结合在一起成为同一蛋白质，例如两个这样的配体可以结合成IgG或多聚体形式的免疫球蛋白，例如IgM。或者，在另一实施方案中，多个双特异性配体结合形成多聚体。例如，两个不同双特异性配体结合在一起，以产生四特异性分子。Preferably the dual specific ligands of the invention comprise only two variable domains, although several such ligands can also be combined together into the same protein, e.g. two such ligands can be combined in IgG or multimeric form Immunoglobulins, such as IgM. Alternatively, in another embodiment, multiple dual specific ligands associate to form multimers. For example, two different dual specific ligands are combined to create a tetraspecific molecule.
本领域技术人员可以理解，本发明方法所产生的双特异性配体的轻链可变区和重链可变区，可以在同一多肽链上或者在不同多肽链上。就可变区在不同多肽链上而言，它们可以通过接头、通常是柔性接头(例如多肽链)、化学连接基团而连接，或者通过本领域已知的任何其它方法而连接。Those skilled in the art can understand that the light chain variable region and the heavy chain variable region of the dual-specific ligand produced by the method of the present invention can be on the same polypeptide chain or on different polypeptide chains. To the extent the variable regions are on different polypeptide chains, they may be linked by a linker, usually a flexible linker (eg, a polypeptide chain), a chemical linking group, or by any other method known in the art.
在第一格局中，本发明提供双特异性配体的进一步改进，该改进是由本发明人开发的，其中配体的一个特异性针对生物体内存在的蛋白质或多肽，它们通过与其结合而起到延长配体半衰期的作用。In a first configuration, the present invention provides a further improvement of dual-specific ligands developed by the inventors, wherein one specificity of the ligand is directed against a protein or polypeptide present in an organism, and they act by binding to it. The effect of prolonging the half-life of the ligand.
因此，第一方面，提供双特异性配体，所述配体包含对第一抗原或表位具有结合特异性的第一免疫球蛋白单可变区以及对第二抗原或表位具有结合活性的第二互补免疫球蛋白单可变区，其中所述抗原或表位的一个或两个起到延长配体体内半衰期的作用，而且其中所述第一和第二区缺乏共享相同特异性的互补区，前提条件是所述双特异性配体不由抗HSA VH区和抗β半乳糖苷酶Vκ区组成。优选的第一或第二可变区都不结合人血清白蛋白(HSA)。Thus, in a first aspect, there is provided a dual specific ligand comprising a first immunoglobulin single variable domain having binding specificity for a first antigen or epitope and binding activity for a second antigen or epitope A second complementary immunoglobulin single variable domain, wherein one or both of said antigens or epitopes act to prolong the half-life of the ligand in vivo, and wherein said first and second domains lack a domain that shares the same specificity Complementary regions, with the proviso that the dual specific ligand does not consist of an anti-HSAVH region and an anti-β-galactosidase Vκ region. Preferably neither the first nor the second variable domain binds human serum albumin (HSA).
延长本文所述配体的半衰期的抗原或表位最好位于生物体内存在的蛋白质或多肽上。实例包括胞外基质蛋白、血蛋白和生物体不同组织中存在的蛋白质。蛋白质例如通过作为膨胀剂(bulking agent)，或者通过将配体锚定在所需作用位点上，而起到降低配体从血液中清除的速率。延长体内半衰期的抗原/表位的实例在以下附录1中给出。Antigens or epitopes that extend the half-life of the ligands described herein are preferably located on proteins or polypeptides present in the organism. Examples include extracellular matrix proteins, blood proteins and proteins present in different tissues of an organism. The protein acts to reduce the rate at which the ligand is cleared from the blood, for example by acting as a bulking agent, or by anchoring the ligand at the desired site of action. Examples of antigens/epitopes that extend half-life in vivo are given in
延长的半衰期可用于免疫球蛋白、尤其是抗体、最尤其是小体积的抗体片段的体内应用。这类片段(Fv、二硫键连接的Fv、Fab、scFv、dAb)会从体内快速清除掉；因此，尽管它们能快速到达身体的大部分部位，并且也能够快速生产和容易操作，但是它们在体内的短持续性限制了它们在体内的应用。本发明解决了这一问题，即通过使配体在体内半衰期延长，继而延长配体功能活性在体内的持续时间。The extended half-life is useful for in vivo applications of immunoglobulins, especially antibodies, and most especially antibody fragments of small size. Such fragments (Fv, disulfide-linked Fv, Fab, scFv, dAb) are rapidly cleared from the body; thus, although they reach most parts of the body rapidly and are also quick to produce and easy to manipulate, they Short persistence in vivo limits their in vivo applications. The present invention solves this problem by prolonging the half-life of the ligand in vivo, which in turn prolongs the duration of the functional activity of the ligand in vivo.
药代动力学分析方法和配体半衰期的测定方法是本领域技术人员熟知的。有关细节可参见Kenneth，A等，Chemical Stability ofPharmaceuticals：A Handbook for Pharmacists和Peters等，Pharmacokinetc analysis：A Practical Approach(1996)。参考文献也可参见“Pharmacokinetics”，M Gibaldi&D Perron，Marcel Dekker出版，2ndRev.ex edition(1982)，该文献介绍了药代动力学参数，例如tα和tβ半衰期和曲线下面积(AUC)。Methods for pharmacokinetic analysis and determination of ligand half-life are well known to those skilled in the art. For details see Kenneth, A et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetc analysis: A Practical Approach (1996). References are also found in "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker,2nd Rev. ex edition (1982), which presents pharmacokinetic parameters such as tα and tβ half-lives and the area under the curve (AUC).
半衰期(tα和tβ)和AUC可以根据配体血清浓度与时间的曲线而确定。WinNonlin分析包(可得自Pharsight Corp.，Mountain View，CA94040，USA)可用于例如曲线建模。在第一相(α相)，配体主要是在患者体内分布，同时有一些消除。第二相(β相)是终末相，当配体已经分布且血清浓度随配体从患者体内消除而下降。tα半衰期是第一相的半衰期，tβ半衰期则是第二相的半衰期。因此，最好本发明提供本发明配体或包含本发明配体的组合物，所述配体的tα半衰期范围为15分钟或更长时间。在一个实施方案中，范围的下限是30分钟、45分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、10小时、11小时或12小时。另外，或者，本发明的配体或组合物的tα半衰期范围为最多12小时且包括12小时。在一个实施方案中，范围的上限是11小时、10小时、9小时、8小时、7小时、6小时或5小时。合适范围的实例为1-6小时、2-5小时或3-4小时。Half-lives (tα and tβ) and AUC can be determined from the curve of ligand serum concentration versus time. The WinNonlin analysis package (available from Pharsight Corp., Mountain View, CA 94040, USA) can be used, for example, for curve modeling. In the first phase (alpha phase), the ligand is mainly distributed in the patient with some elimination. The second phase (beta phase) is the terminal phase, when the ligand has been distributed and the serum concentration drops as the ligand is eliminated from the patient. The tα half-life is the half-life of the first phase, and the tβ half-life is the half-life of the second phase. Accordingly, it is preferred that the present invention provide a ligand of the invention, or a composition comprising a ligand of the invention, that has a tα half-life in the range of 15 minutes or more. In one embodiment, the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. Additionally, or alternatively, the ligands or compositions of the invention have a t[alpha] half-life in the range of up to and including 12 hours. In one embodiment, the upper end of the range is 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours or 5 hours. Examples of suitable ranges are 1-6 hours, 2-5 hours or 3-4 hours.
最好本发明提供本发明配体或包含本发明配体的组合物，所述配体的tβ半衰期范围为2.5小时或更长时间。在一个实施方案中，范围的下限是3小时、4小时、5小时、6小时、7小时、10小时、11小时或12小时。另外，或者，本发明的配体或组合物的tβ半衰期范围为最多21天且包括21天。在一个实施方案中，范围的上限是12小时、24小时、2天、3天、5天、10天、15天或20天。最好本发明的配体或组合物的tβ半衰期范围为12-60小时。在另一实施方案中，范围为12-48小时。在再一实施方案中，范围为12-26小时。Preferably the invention provides a ligand of the invention or a composition comprising a ligand of the invention, said ligand having a t[beta] half-life in the range of 2.5 hours or more. In one embodiment, the lower end of the range is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. Additionally, or alternatively, the ligands or compositions of the invention have a t[beta] half-life ranging up to and including 21 days. In one embodiment, the upper end of the range is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days. Preferably, the ligands or compositions of the invention have a t[beta] half-life in the range of 12-60 hours. In another embodiment, the range is 12-48 hours. In yet another embodiment, the range is 12-26 hours.
另外，除了以上标准之外，本发明提供配体或包含本发明配体的组合物，其AUC值(曲线下面积)范围为1mg.min/ml以上。在一个实施方案中，范围的下限是5、10、15、20、30、100、200或300，单位为mg.min/ml。另外，或者，本发明配体或组合物的AUC范围高达600mg.min/ml。在一个实施方案中，范围的上限是500、400、300、200、150、100、75或50，单位为mg.min/ml。最好本发明配体的AUC范围选自以下：15-150mg.min/ml、15-100mg.min/ml、15-75mg.min/ml和15-50mg.min/ml。Additionally, in addition to the above criteria, the present invention provides a ligand or a composition comprising a ligand of the present invention having an AUC value (area under the curve) in the range of 1 mg.min/ml or more. In one embodiment, the lower end of the range is 5, 10, 15, 20, 30, 100, 200 or 300 in mg.min/ml. Additionally, alternatively, the ligands or compositions of the invention have an AUC in the range up to 600 mg.min/ml. In one embodiment, the upper end of the range is 500, 400, 300, 200, 150, 100, 75 or 50 in mg.min/ml. Preferably, the AUC range of the ligand of the present invention is selected from the following: 15-150 mg.min/ml, 15-100 mg.min/ml, 15-75 mg.min/ml and 15-50 mg.min/ml.
在第一个实施方案中，双特异性配体包含两个互补可变区，即两个可变区，它们在其天然环境下能结合在一起成为关联对或关联组，甚至在本发明的情况下，它们分别结合其关联表位。例如，互补可变区可以是免疫球蛋白重链和轻链可变区(VH和VL)。VH区和VL区最好由scFv或Fab抗体片段提供。可变区可连接在一起形成多价配体，例如通过在各V区的C-端提供铰链区并在铰链区的半胱氨酸间提供二硫键；或提供在结构域C-端带有半胱氨酸的dAb，这些半胱氨酸是通过二硫键连接在一起；或者通过产生V-CH和V-CL，以产生Fab形式；或者使用肽接头(例如下文所述的Gly4Ser接头)，以产生二聚体、三聚体和多聚体。In a first embodiment, the dual specific ligand comprises two complementary variable domains, i.e. two variable domains, which in their natural environment can be brought together as a cognate pair or cognate group, even in the context of the present invention , they respectively bind their cognate epitopes. For example, the complementary variable regions may be immunoglobulin heavy and light chain variable regions (VH andVL ). TheVH andVL regions are preferably provided by scFv or Fab antibody fragments. The variable domains can be linked together to form multivalent ligands, for example by providing a hinge region at the C-terminus of each V region and disulfide bonds between cysteines in the hinge regions; or by providing a band at the C-terminus of the domains. dAbs with cysteines linked together by disulfide bonds; either by generating V-CH and V-CL to generate Fab form; or using a peptide linker (such as Gly4 as described below Ser linker) to generate dimers, trimers and multimers.
本发明人已经知道，使用互补可变区可以让两个结构域表面包装在一起并从溶剂中隐退(sequestered)。而且，互补区可彼此稳定。另外，它允许产生双特异性IgG抗体，但没有现有技术所用的杂种杂交瘤的缺点，或者不需要在亚单位界面改造重链或轻链。本发明第一方面的双特异性配体具有至少一个VH/VL对。因此，本发明的双特异性IgG包含两个这样的配对，在Y形分子每个臂上各有一对。因此，不同于常规双特异性抗体或双链抗体(其中所用链的比率决定其制剂是否成功并导致实践的困难)，本发明的双特异性配体没有链平衡的问题。常规双特异性抗体中的链不平衡是由两个不同VL链与两个不同VH链连接所致，其中VL链1与VH链1一起能结合抗原或表位1，VL链2与VH链2一起能结合抗原或表位2，两个正确的配对以某种方式彼此连接。因此，在一个分子中仅当VL链1与VH链1配对、VL链2与VH链2配对时，才产生双特异性。这样的双特异性分子可以两种不同方式产生。第一种，它们可以通过两个各自连接不同抗原或表位的现有VH/VL配对连接而产生(例如在双特异性IgG中)。在这种情况下，VH/VL配对必需以1∶1的比例混合在一起，以便产生所有分子都为双特异性分子群体。这决不会(甚至当互补CH区通过“杵臼结构(knobs into holes)”工程而强化时)导致双特异性分子和仅能结合一个抗原或表位、但不结合另一个抗原或表位的分子的混合物。第二种产生双特异性抗体的方式是通过将两个不同VH链与两个不同VL链同时结合(例如在双特异性双链抗体)。在这种情况下，尽管倾向于优先让VL链1与VH链1配对、VL链2与VH链2配对(这可以通过VL和VH区的“杵臼结构”改造而强化)，但是不会在所有分子中都得到该配对，导致混合制剂，其中发生不正确的配对，使其不能结合抗原或表位。The inventors have known that the use of complementary variable regions allows the two domains to be surface-packed together and sequestered from solvent. Furthermore, the complementary regions can be stabilized with each other. In addition, it allows the production of bispecific IgG antibodies without the disadvantages of hybrid hybridomas used in the prior art, or without the need for heavy or light chain engineering at the subunit interface. The dual specific ligands of the first aspect of the invention have at least oneVH /VL pair. Thus, the bispecific IgG of the invention comprises two such pairs, one on each arm of the Y-shaped molecule. Thus, unlike conventional bispecific antibodies or diabodies, where the ratio of chains used determines the success of their formulation and leads to difficulties in practice, the dual specific ligands of the present invention do not have chain balance problems. The chain imbalance in conventional bispecific antibodies is caused by the linking of two differentVL chains to two differentVH chains, whereVL
按照本发明第一方面的双特异性配体方法构建的双特异性抗体克服了所有这些问题，因为与抗原或表位1的结合存在于VH区或VL区，与抗原或表位2的结合存在于互补VL或VH区。因为VH区和VL区配对以1∶1比例，所有VH/VL配对都是双特异性的，因此采用这些VH/VL配对构建的所有形式(Fv、scFv、Fab、微型抗体、IgG等)都具有100％双特异性活性。The bispecific antibody constructed according to the dual-specific ligand method of the first aspect of the present invention overcomes all these problems, because the binding to the antigen or
在本发明的情况下，第一和第二“表位”被认为是这样的表位：所述表位不同，而且不结合单个单特异性配体。在本发明的第一格局中，它们最好在不同抗原上，其中一个起到延长配体体内半衰期的作用。同样，第一和第二抗原最好是不同的。In the context of the present invention, the first and second "epitopes" are considered to be epitopes which are distinct and which do not bind a single monospecific ligand. In a first aspect of the invention, they are preferably on different antigens, one of which acts to prolong the half-life of the ligand in vivo. Likewise, the first and second antigens are preferably different.
本发明的双特异性配体不包含WO 02/02773所述的配体。因此，本发明的配体不包含互补VH/VL对，其协同结合任何一个或多个抗原或表位。相反，本发明第一方面的配体包含VH/VL互补对，其中V区具有不同特异性。此外，本发明第一方面的配体包含VH/VL互补对，其对非结构相关表位或抗原具有不同特异性。结构相关的表位或抗原是这样的表位或抗原：其具有足够的结构相似性，可以与常规VH/VL互补对结合，其以协同方式结合抗原或表位；就结构相关的表位而言，表位在结构上是足够相似的，使得它们“适合”于VH/VL二聚体的抗原结合位点上形成的同一结合口袋(pocket)。The dual specific ligands of the invention do not comprise the ligands described in WO 02/02773. Accordingly, ligands of the invention do not comprise complementaryVH /VL pairs that bind cooperatively to any one or more antigens or epitopes. In contrast, the ligands of the first aspect of the invention compriseVH /VL complementary pairs, wherein the V regions have different specificities. Furthermore, the ligands of the first aspect of the invention compriseVH /VL complementary pairs having different specificities for non-structurally related epitopes or antigens. A structurally related epitope or antigen is an epitope or antigen that has sufficient structural similarity to bind to a conventionalVH /VL complementary pair that binds the antigen or epitope in a cooperative manner; In terms of VH/VL dimers, the epitopes are sufficiently similar in structure that they "fit" into the same binding pocket formed on the antigen binding site of theVH /VL dimer.
第二方面，本发明提供配体，所述配体包含具有第一抗原或表位结合特异性的第一免疫球蛋白可变区以及具有第二抗原或表位结合特异性的第二免疫球蛋白可变区，其中延长配体体内半衰期的所述第一和第二可变区的一个或两个结合抗原，可变区彼此并不互补。In a second aspect, the invention provides a ligand comprising a first immunoglobulin variable region having a first antigen or epitope binding specificity and a second immunoglobulin having a second antigen or epitope binding specificity Protein variable regions, wherein one or both of said first and second variable regions that extend the half-life of a ligand in vivo binds antigen, the variable regions are not complementary to each other.
在一个实施方案中，通过一个可变区的结合调节了配体通过第二可变区的结合。In one embodiment, binding through one variable domain modulates ligand binding through a second variable domain.
在该实施方案中，可变区可以是例如VH区对或VL区对。在第一位点的抗原结合可调节、例如增强或抑制在第二位点的抗原结合。例如，在第一位点的结合至少部分抑制在第二位点的抗原结合。在这样的实施方案中，配体可以例如通过与延长配体半衰期的蛋白质结合而在患者生物体内得以维持，直到它结合第二靶抗原并从延长半衰期的蛋白质上解离下来。In this embodiment, the variable regions can be, for example, a pair ofVH regions or a pair ofVL regions. Antigen binding at a first site can modulate, eg, enhance or inhibit antigen binding at a second site. For example, binding at a first site at least partially inhibits antigen binding at a second site. In such embodiments, the ligand can be maintained in the patient organism, eg, by binding to the ligand half-life-extending protein, until it binds the second target antigen and dissociates from the half-life-extending protein.
在上述情况下，达到对结合的调节是抗原结合位点彼此结构接近的结果。可以通过连接两个或更多个抗原结合位点的结构组分的特性，达到这样的结构接近，例如通过提供具有相对刚性结构的配体，这样的刚性结构能保持非常接近的抗原结合位点。最好两个或更多个抗原结合位点彼此是物理上非常接近的，通过涉及免疫球蛋白分子内位阻和/或构象变化的过程，使得一个位点能调节另一位点的抗原结合。In the above cases, modulation of binding is achieved as a result of the structural proximity of the antigen binding sites to each other. Such structural proximity can be achieved through the properties of the structural components linking two or more antigen binding sites, for example by providing ligands with relatively rigid structures that hold the antigen binding sites in close proximity . Preferably two or more antigen binding sites are in close physical proximity to each other such that one site can mediate antigen binding by the other through a process involving steric and/or conformational changes within the immunoglobulin molecule .
第一和第二抗原结合域可以共价或非共价结合。在结构域是共价结合的情况下，可以通过例如二硫键或者通过多肽接头例如(Gly4Ser)n而介导结合，其中n＝1-8，例如2、3、4、5或7。The first and second antigen binding domains can be covalently or non-covalently associated. Where the domains are covalently bound, the association may be mediated for example by disulfide bonds or by polypeptide linkers such as (Gly4Ser )n , where n=1-8, such as 2, 3, 4, 5 or 7 .
在采用例如本文所述的噬菌体展示技术从V基因库中选出可变区的情况下，这些可变区可包含通用构架区，使得它们可以被本文所述的特异性通用配体所识别。有关通用构架、通用配体等的使用参见WO 99/20749。在本发明中，有关的噬菌体展示包括噬菌体和/或噬菌粒的使用。Where variable regions are selected from V gene repertoires using, for example, phage display techniques as described herein, these variable regions may comprise common framework regions such that they can be recognized by specific universal ligands as described herein. See WO 99/20749 for use of general frameworks, general ligands, etc. In the present invention, relevant phage display includes the use of phage and/or phagemid.
当使用V基因库时，多肽序列中的变异优选位于可变区结构环内。各可变区的多肽序列可以通过DNA改组或通过突变而改变，以便增强各可变区与其互补对的相互作用。DNA改组是本领域已知的，参见例如Stemmer，Nature 370：389-391(1994)和美国专利第6,297,053号，所述文献都通过引用结合到本文中。其它诱变方法是本领域技术人员众所周知的。When using a repertoire of V genes, variations in the polypeptide sequence are preferably located within the variable region structural loops. The polypeptide sequence of each variable domain can be altered by DNA shuffling or by mutation in order to enhance the interaction of each variable domain with its complementary pair. DNA shuffling is known in the art, see, eg, Stemmer, Nature 370:389-391 (1994) and US Patent No. 6,297,053, both of which are incorporated herein by reference. Other methods of mutagenesis are well known to those skilled in the art.
在本发明的一个优选实施方案中，“双特异性配体”是单链Fv片段。在本发明的一个替代实施方案中，“双特异性配体”由抗体Fab区组成。术语“Fab区”包括Fab样区，其中使用两个VH区或两个VL区。In a preferred embodiment of the invention the "dual specific ligand" is a single chain Fv fragment. In an alternative embodiment of the invention the "dual specific ligand" consists of an antibody Fab region. The term "Fab region" includes Fab-like regions in which twoVH regions or twoVL regions are used.
可变区可来自针对靶抗原或表位的抗体。或者它们可来自单域抗体库，例如在丝状噬菌体表面表达的那些。可以按照下文所述进行选择。The variable regions can be from antibodies raised against the target antigen or epitope. Or they may be derived from single domain antibody repertoires, such as those expressed on the surface of filamentous phage. Selections can be made as described below.
另一方面，本发明提供一种或多种核酸分子，所述核酸至少编码本文所述的双特异性配体。双特异性配体可以由一个核酸分子编码；或者，各结构域可以由各自的核酸分子编码。当配体由一个核酸分子编码时，各结构域可表达为scFv分子形式的融合多肽，或者可以分别表达，再随后连接在一起，例如使用化学连接剂。各自的核酸所表达的配体可通过合适方法连接在一起。In another aspect, the invention provides one or more nucleic acid molecules encoding at least a dual specific ligand described herein. A dual specific ligand can be encoded by one nucleic acid molecule; alternatively, each domain can be encoded by a separate nucleic acid molecule. When the ligand is encoded by one nucleic acid molecule, the domains can be expressed as fusion polypeptides in the form of scFv molecules, or can be expressed separately and subsequently linked together, for example using chemical linkers. The ligands expressed by the respective nucleic acids can be linked together by suitable methods.
核酸还可编码信号序列，用于在表达后将多肽从宿主细胞中输出，而且在表达后还可与丝状噬菌体颗粒的表面组分(或选择展示系统的其它组分)融合。The nucleic acid may also encode a signal sequence for export of the polypeptide from the host cell after expression and may also be fused to a surface component of the filamentous phage particle (or other components of the selection display system) after expression.
另一方面，本发明提供载体，所述载体包含编码本发明的双特异性配体的核酸。In another aspect, the invention provides a vector comprising a nucleic acid encoding a dual specific ligand of the invention.
又一方面，本发明提供用载体转染的宿主细胞，所述载体编码本发明的双特异性配体。In yet another aspect, the invention provides a host cell transfected with a vector encoding a dual specific ligand of the invention.
用这样的载体进行表达，可以例如在噬菌体颗粒表面产生可变区，用于选择。这允许选择所展示的可变区，因此用本发明的方法能选择出“双特异性配体”。Expression with such vectors makes it possible, for example, to generate variable regions on the surface of phage particles for selection. This allows selection of the variable regions displayed and thus "dual specific ligands" can be selected using the methods of the invention.
本发明还提供试剂盒，所述试剂盒至少包括本发明的双特异性配体。The present invention also provides a kit comprising at least the dual specific ligand of the present invention.
第三方面，本发明提供制备配体的方法，所述配体包含具有第一结合特异性的第一免疫球蛋白单可变区以及具有第二(不同)结合特异性的第二单免疫球蛋白单可变区，结合特异性的一个或两个对在体内延长配体半衰期的抗原具有特异性，该方法包括以下步骤：In a third aspect, the invention provides a method of making a ligand comprising a first immunoglobulin single variable domain with a first binding specificity and a second single immunoglobulin with a second (different) binding specificity A single variable domain of a protein, one or both of which are specific for an antigen that extends the half-life of a ligand in vivo, the method comprising the steps of:
a)选择能够结合第一表位的第一可变区，a) selecting a first variable region capable of binding a first epitope,
b)选择能够结合第二表位的第二可变区，b) selecting a second variable region capable of binding a second epitope,
c)连接可变区；和c) linking the variable regions; and
d)选择能够结合所述第一表位和所述第二表位的配体。d) selecting a ligand capable of binding said first epitope and said second epitope.
配体可同时结合第一和第二表位，或者当结合域之间对表位结合具有竞争性时，一个区的结合可阻碍另一区与其关联表位的结合。因此在一个实施方案中，以上步骤(d)需要同时结合第一和第二(可能的话更多)表位；在另一个实施方案中，与第一和第二表位的结合不同时。A ligand may bind both the first and second epitope simultaneously, or when there is competition between the binding domains for epitope binding, binding of one domain may prevent binding of the other domain to its cognate epitope. Thus in one embodiment, step (d) above entails simultaneous binding of the first and second (possibly more) epitopes; in another embodiment, the binding of the first and second epitopes is not simultaneous.
表位优选在各自抗原上。The epitopes are preferably on the respective antigens.
配体最好包含如上所述的免疫球蛋白可变区的VH/VL组合、或VH/VH或VL/VL组合。配体还可包含骆驼科动物VHH区，前提条件是对在体内能延长配体半衰期的抗原具有特异性的VHH区不结合鸡卵清溶菌酶(HEL)、猪胰腺α-淀粉酶或NmC-A；hcg、BSA-连接的RR6偶氮染料或变异链球菌(S.mutans)HG982细胞，参见Conrath等(2001)JBC 276：7346-7350和WO99/23221，这两篇文献都没有介绍针对在体内能延长配体半衰期的抗原特异性用途。The ligand preferably comprises aVH /VL combination, or aVH /VH orVL /VL combination of an immunoglobulin variable region as described above. The ligand may also comprise a camelidVHH region, provided that theVHH region specific for an antigen capable of extending the half-life of the ligand in vivo does not bind chicken egg white lysozyme (HEL), porcine pancreatic alpha-amylase or NmC-A; hcg, BSA-linked RR6 azo dye or S. mutans HG982 cells, see Conrath et al. (2001) JBC 276:7346-7350 and WO99/23221, neither of which are described Antigen-specific use for prolonging ligand half-life in vivo.
在一个实施方案中，在互补可变区不存在的情况下，选择所述第一可变区用于结合所述第一表位。在另一实施方案中，在第三可变区存在的情况下，选择所述第一可变区用于结合所述第一表位/抗原，其中所述第三可变区不同于所述第二可变区并且与第一区互补。In one embodiment, said first variable region is selected for binding said first epitope in the absence of a complementary variable region. In another embodiment, said first variable domain is selected for binding said first epitope/antigen in the presence of a third variable domain, wherein said third variable domain is different from said The second variable region is also complementary to the first region.
同样，可以在互补可变区存在或不存在的情况下，选择第二区。Likewise, the second region can be selected in the presence or absence of a complementary variable region.
除了延长半衰期的蛋白质之外，本发明配体所靶向的抗原或表位可以是任何抗原或表位，但最好是具有治疗益处的抗原或表位。本发明提供配体，包括开放构象、闭合构象和分离的dAb单体配体，它们对任何所述靶标、尤其是本文进一步鉴定的那些靶标具有特异性。所述靶标可以是天然存在或合成的多肽、蛋白质或核酸、或其部分。在该方面，本发明的配体能结合表位或抗原并起到拮抗剂或激动剂(例如EPO受体激动剂)的作用。本领域技术人员将会理解，选择是庞大的而不同的。它们可以是例如人或动物蛋白、细胞因子、细胞因子受体、酶的辅因子或DNA结合蛋白。合适的细胞因子和生长因子包括但不限于：ApoE、Apo-SAA、BDNF、心肌营养蛋白-1、EGF、EGF受体、ENA-78、嗜酸性粒细胞趋化因子(eotaxin)、嗜酸性粒细胞趋化因子-2、Exodus-2、EpoR、FGF(酸性)、FGF(碱性)、成纤维细胞生长因子-10、FLT3配体、CXXXC趋化因子(CX3C)、GDNF、G-CSF、GM-CSF、GF-β1、胰岛素、IFN-γ、IGF-I、IGF-II、IL-1α、IL-1β、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8(72a.a.)、IL-8(77a.a.)、IL-9、IL-10、IL-11、IL-12、IL-13、IL-15、IL-16、IL-17、IL-18(IGIF)、抑制素α、抑制素β、IP-10、角质形成细胞生长因子-2(KGF-2)、KGF、瘦素、LIF、淋巴细胞趋化因子、米勒管(Mullerian)抑制物质、单核细胞集落抑制因子、单核细胞趋化蛋白、M-CSF、MDC(67a.a.)、MDC(69a.a.)、MCP-1(MCAF)、MCP-2、MCP-3、MCP-4、MDC(67a.a.)、MDC(69a.a.)、MIG、MIP-1a、MIP-1β、MIP-3α、MIP-3β、MDP-4、髓性祖细胞抑制因子-1(MPIF-1)、NAP-2、神经营养因子、神经生长因子、β-NGF、NT-3、NT-4、抑瘤素M、PDGF-AA、PDGF-AB、PDGF-BB、PF-4、RANTES、SDF1α、SDF1β、SCF、SCGF、干细胞因子(SCF)、TARC、TGF-α、TGF-β、TGF-β2、TGF-β3、肿瘤坏死因子(TNF)、TNF-α、TNF-β、TNF受体I、TNF受体II、TNIL-1、TPO、VEGF、VEGF受体1、VEGF受体2、VEGF受体3、GCP-2、GRO/MGSA、GRO-β、GRO-γ、HCC1、1-309、HER1、HER2、HER3和HER4。细胞因子受体包含前述细胞因子的受体。可以理解，该名单并非是详尽而无遗漏的。The antigen or epitope targeted by the ligands of the invention may be any antigen or epitope, except for half-life extending proteins, but is preferably an antigen or epitope of therapeutic benefit. The present invention provides ligands, including open conformation, closed conformation and isolated dAb monomer ligands, which are specific for any of the described targets, especially those further identified herein. The target may be a naturally occurring or synthetic polypeptide, protein or nucleic acid, or a portion thereof. In this regard, a ligand of the invention is capable of binding an epitope or antigen and acting as an antagonist or agonist (eg, an EPO receptor agonist). Those skilled in the art will appreciate that the selection is vast and varied. They may be, for example, human or animal proteins, cytokines, cytokine receptors, cofactors for enzymes or DNA binding proteins. Suitable cytokines and growth factors include, but are not limited to: ApoE, Apo-SAA, BDNF, cardiotrophin-1, EGF, EGF receptor, ENA-78, eotaxin, eosinophil Chemokine-2, Exodus-2, EpoR, FGF (acidic), FGF (basic), fibroblast growth factor-10, FLT3 ligand, CXXXC chemokine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8(72a.a.), IL-8(77a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL- 16. IL-17, IL-18 (IGIF), inhibin α, inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, leptin, LIF, lymphocyte chemotactic factor , Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte chemoattractant protein, M-CSF, MDC(67a.a.), MDC(69a.a.), MCP-1(MCAF) , MCP-2, MCP-3, MCP-4, MDC (67a.a.), MDC (69a.a.), MIG, MIP-1a, MIP-1β, MIP-3α, MIP-3β, MDP-4 , myeloid progenitor inhibitory factor-1 (MPIF-1), NAP-2, neurotrophic factor, nerve growth factor, β-NGF, NT-3, NT-4, oncostatin M, PDGF-AA, PDGF- AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, Stem Cell Factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, Tumor Necrosis Factor (TNF) , TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER1, HER2, HER3 and HER4. Cytokine receptors include receptors for the aforementioned cytokines. It is understood that this list is not intended to be exhaustive or exhaustive.
在本发明的一个实施方案中，可变区来自针对抗原或表位的相应抗体。在一个优选的实施方案中，可变区来自抗体单可变区库。In one embodiment of the invention, the variable regions are from corresponding antibodies directed against the antigen or epitope. In a preferred embodiment, the variable domains are from a repertoire of antibody single variable domains.
在第二格局中，本发明提供多特异性配体。根据本发明，术语“多特异性配体”是指具有不止一个本文所定义的表位结合特异性的配体。通常，多特异性配体包含两个或更多个表位结合域。In a second format, the invention provides multispecific ligands. According to the present invention, the term "multispecific ligand" refers to a ligand having more than one epitope binding specificity as defined herein. Typically, multispecific ligands comprise two or more epitope binding domains.
本发明的表位结合域包含蛋白质支架和表位相互作用位点(它们最好在蛋白质支架表面)。根据本发明，最好各表位结合域具有不同表位结合特异性。The epitope binding domains of the invention comprise a protein scaffold and epitope interaction sites (which are preferably on the surface of the protein scaffold). According to the present invention, preferably each epitope binding domain has a different epitope binding specificity.
在本发明的情况下，认为第一和第二“表位”是不同表位且不结合一个单特异性配体的表位。它们可以在不同抗原上，或在同一抗原上、但是有足够距离隔开，使它们不会形成一个这样的实体：所述实体与常规抗体的一个单特异性VH/VL结合对结合。从实验上看，如果单链抗体形式中的单个可变区的两个(域抗体或dAb)各自被针对两个表位的单特异性VH/VL配体竞争的话，则这两个表位间的距离不够远，不能认为是本发明的分离的表位。In the context of the present invention, the first and second "epitopes" are considered to be epitopes that are distinct and do not bind one monospecific ligand. They can be on different antigens, or on the same antigen but separated by a sufficient distance so that they do not form an entity that binds to a monospecificVH /VL binding pair of a conventional antibody. Experimentally, if two of the single variable domains in the scFv format (domain antibody or dAb) are each competed by monospecificVH /VL ligands directed against two epitopes, then the two Epitopes are not far enough apart to be considered isolated epitopes of the invention.
根据本发明，有利的是，每个表位结合域都包含免疫球蛋白可变区。更有利的是，每个免疫球蛋白都可变区可以是轻链可变区(VL)或重链可变区VH。在本发明的第二格局中，免疫球蛋白结构域当存在于本发明配体中时，是非互补的，也就是说，它们不结合形成VH/VL抗原结合位点。因此，本发明第二格局中定义的多特异性配体包含相同亚型的免疫球蛋白结构域，它可以是轻链可变区(VL)或重链可变区(VH)。此外，当本发明的配体呈闭合构象时，免疫球蛋白结构域可以是骆驼科动物VHH型。Advantageously according to the invention, each epitope binding domain comprises an immunoglobulin variable region. Advantageously, the variable region of each immunoglobulin may be a light chain variable region (VL) or a heavy chain variable regionVH . In a second aspect of the invention, the immunoglobulin domains, when present in the ligands of the invention, are non-complementary, that is, they do not associate to form theVH /VL antigen binding site. Thus, the multispecific ligands defined in the second configuration of the invention comprise immunoglobulin domains of the same subtype, which may be the variable region of the light chain (VL ) or the variable region of the heavy chain (VH ). Furthermore, when the ligands of the invention are in a closed conformation, the immunoglobulin domain may be of the camelid VHH type.
在一个替代实施方案中，本发明的配体不包含骆驼科动物VHH区。更具体地讲，与人VH区相比，本发明的配体不含对骆驼科动物VHH区具有特异性的一个或多个氨基酸残基。In an alternative embodiment, the ligands of the invention do not comprise a camelidVHH region. More specifically, the ligands of the invention do not contain one or more amino acid residues specific for a camelidVHH region compared to a humanVH region.
最好单可变区来自抗体，所述抗体是通过针对不同抗原或表位的结合活性而选择出来的。例如，可以通过人免疫，至少部分分离可变区。替代方法是本领域已知的，包括从人类抗体文库中分离和人工抗体基因的合成。Preferably the single variable domains are derived from antibodies selected for their binding activity against different antigens or epitopes. For example, variable regions can be at least partially isolated by human immunization. Alternative methods are known in the art and include isolation from human antibody libraries and synthesis of artificial antibody genes.
可变区最好结合超抗原，例如蛋白A或蛋白L。与超抗原的结合是正确折叠的抗体可变区的特性，允许这样的结构域从例如重组或突变区的文库中分离出来。Preferably, the variable region binds a superantigen, such as protein A or protein L. Binding to superantigens is a property of correctly folded antibody variable regions, allowing the isolation of such domains from, for example, libraries of recombinant or mutated regions.
表位结合域也可以基于并非免疫球蛋白结构域的蛋白质支架或骨架。例如天然细菌受体(例如SpA)已经用作移植CDR的支架，以产生能特异性结合一个或多个表位的配体。该方法的细节参见US5,831,012。其它合适的支架包括基于纤连蛋白和亲和体的支架。合适方法的细节参见WO 98/58965。其它合适的支架包括脂质运载蛋白和CTLA4(参见van den Beuken等，J.Mol.Biol.(2001)310，591-601)以及例如描述于WO0069907(Medical Research Council)的支架，所述支架是基于例如细菌GroEL的环状结构或其它多肽陪伴分子。Epitope binding domains can also be based on protein scaffolds or backbones that are not immunoglobulin domains. For example natural bacterial receptors such as SpA have been used as scaffolds for grafting CDRs to generate ligands that specifically bind one or more epitopes. Details of this method are found in US 5,831,012. Other suitable scaffolds include fibronectin and affibody based scaffolds. Details of suitable methods are found in WO 98/58965. Other suitable scaffolds include lipocalin and CTLA4 (see van den Beuken et al., J.Mol.Biol. (2001) 310, 591-601 ) and the scaffolds such as described in WO0069907 (Medical Research Council), which are Based on loop structures such as bacterial GroEL or other polypeptide chaperones.
蛋白质支架可以进行组合；例如，CDR可以移植到CTLA4支架上并且与免疫球蛋白VH区或VL区一起使用，以构成多价配体。同样，纤连蛋白、脂质运载蛋白和其它支架也可以进行组合。Protein scaffolds can be combined; for example, CDRs can be grafted onto CTLA4 scaffolds and used with immunoglobulinVH orVL regions to form multivalent ligands. Likewise, fibronectin, lipocalin, and other scaffolds can also be combined.
在一个实施方案中，多特异性配体包含表位结合域，它包含结合本文所述的TNFR1的免疫球蛋白单域(dAb)的CDR，所述CDR移植到合适的蛋白质支架或骨架上。In one embodiment, the multispecific ligand comprises an epitope binding domain comprising the CDRs of an immunoglobulin single domain (dAb) that binds TNFRl as described herein, grafted onto a suitable protein scaffold or backbone.
本领域技术人员可以理解，本发明方法所产生的闭合构象多特异性配体的表位结合域，可以在相同多肽链上或者在不同多肽链上。就可变区在不同多肽链上而言，它们可以通过接头、最好是柔性接头(例如多肽链)、化学连接基团而连接，或者通过本领域已知的任何其它方法而连接。Those skilled in the art can understand that the epitope binding domain of the closed conformation multispecific ligand produced by the method of the present invention can be on the same polypeptide chain or on different polypeptide chains. To the extent the variable regions are on different polypeptide chains, they may be linked by a linker, preferably a flexible linker (eg, a polypeptide chain), a chemical linking group, or by any other method known in the art.
第一和第二表位结合域可以共价或非共价结合。在结构域是共价结合的情况下，可以通过例如二硫键介导结合。The first and second epitope binding domains can be covalently or non-covalently associated. Where the domains are covalently associated, the association may be mediated, for example, by disulfide bonds.
在本发明的第二格局的某些实施方案中，表位可以彼此替代而结合。例如，第一表位可以存在于抗原上，在所述抗原与其关联第一结合域结合时，引起第二结合域的位阻或其构象变化，从而替代了与第二结合域结合的表位。In certain embodiments of the second configuration of the invention, the epitopes may bind instead of each other. For example, a first epitope may be present on an antigen which, upon binding of said antigen to its cognate first binding domain, causes steric hindrance or a conformational change in the second binding domain, thereby displacing the epitope bound by the second binding domain .
最好结合下降达25％以上、最好是40％、50％、60％、70％、80％、90％以上、优选高达100％或接近100％，使得结合完全被抑制。可以通过常规抗原结合测定(例如ELISA)、基于荧光的技术(包括FRET)或通过例如测定分子量的表面等离子共振等技术来测定表位的结合。Preferably, the binding is reduced by more than 25%, more preferably 40%, 50%, 60%, 70%, 80%, 90%, preferably up to 100% or close to 100%, so that the binding is completely inhibited. Binding of epitopes can be determined by conventional antigen binding assays (eg, ELISA), fluorescence-based techniques (including FRET), or by techniques such as surface plasmon resonance for molecular weight determination.
此外，本发明提供闭合构象多特异性配体，所述配体包含具有第一表位结合特异性的第一表位结合域以及具有第二表位结合特异性的非互补的第二表位结合域，其中第一和第二结合特异性竞争表位结合，使得闭合构象多特异性配体不同时结合这两个表位。Furthermore, the present invention provides closed conformation multispecific ligands comprising a first epitope binding domain having a first epitope binding specificity and a non-complementary second epitope having a second epitope binding specificity A binding domain wherein the first and second binding specificities compete for epitope binding such that the closed conformation multispecific ligand does not bind both epitopes simultaneously.
本发明的闭合构象多特异性配体不包含WO 02/02773所述的配体。因此，本发明的配体不包含互补VH/VL对，其协同结合任何一个或多个抗原或表位。相反，本发明的配体优选包含非互补的VH-VH或VL-VL对。最好各VH-VH或VL-VL对中的各VH区或VL区具有不同表位结合特异性，而且表位结合位点是这样排列的：使得在一个位点上的表位结合与另一位点上的表位的结合发生竞争。The closed conformation multispecific ligands of the invention do not comprise the ligands described in WO 02/02773. Accordingly, ligands of the invention do not comprise complementaryVH /VL pairs that bind cooperatively to any one or more antigens or epitopes. In contrast, the ligands of the invention preferably comprise non-complementaryVH -VH orVL -VL pairs. Preferably, eachVH region orVL region in eachVH -VH or VL-VLpair has a different epitope binding specificity, and the epitope binding sites are arranged such that at one site The binding of an epitope at one site competes with the binding of an epitope at another site.
最好闭合构象多特异性配体可包含能够结合靶分子的第一区和能够结合延长配体半衰期的分子或基团的第二区。例如，分子或基团可以是膨胀剂(bulky agent)，例如HSA或细胞基质蛋白。本文所用的术语“延长配体半衰期的分子或基团”是指这样的分子或化学基团：当给予动物时，相对于不结合分子或基团的配体来说，当所述分子或化学基团结合本文所述的双特异性配体时，它们延长所述双特异性配体的体内半衰期。延长配体半衰期的分子或基团的实例描述如下。在一个优选的实施方案中，仅在半衰期延长分子或基团被置换时，闭合构象多特异性配体能够结合靶分子。因此，例如，闭合构象多特异性配体可以通过大体积的分子(例如HSA)而在患者血液循环中得以保持。当遇到靶分子时，闭合构象多特异性配体的结合域之间的竞争导致HAS的替代和靶的结合。Preferably, the closed conformation multispecific ligand may comprise a first region capable of binding a target molecule and a second region capable of binding a molecule or group that extends the half-life of the ligand. For example, the molecule or group can be a bulky agent such as HSA or a cell matrix protein. As used herein, the term "molecule or group that extends the half-life of a ligand" refers to a molecule or chemical group that, when administered to an animal, is When the groups bind the dual specific ligands described herein, they increase the half-life of the dual specific ligands in vivo. Examples of molecules or groups that extend the half-life of ligands are described below. In a preferred embodiment, the closed conformation multispecific ligand is capable of binding the target molecule only when the half-life extending molecule or group is displaced. Thus, for example, closed conformation multispecific ligands can be maintained in the blood circulation of a patient by bulky molecules such as HSA. When a target molecule is encountered, competition between the binding domains of the closed conformation multispecific ligand results in displacement of the HAS and binding of the target.
在本发明第二格局的一个优选的实施方案中，表位结合域是免疫球蛋白可变区并选自单域V基因库。通常，单域抗体库都展示在丝状噬菌体表面上。在一个优选的实施方案中，通过噬菌体库与抗原的结合，来选择各单域抗体。在本发明的第二格局的一个优选的实施方案中，表位结合域是免疫球蛋白可变区并选自单域V基因库。通常，单域抗体库都展示在丝状噬菌体表面上。在一个优选的实施方案中，通过噬菌体库与抗原的结合，来选择各单域抗体。In a preferred embodiment of the second configuration of the invention, the epitope binding domain is an immunoglobulin variable region and is selected from a repertoire of single domain V genes. Typically, single domain antibody repertoires are displayed on the surface of filamentous phage. In a preferred embodiment, individual single domain antibodies are selected by binding the phage library to the antigen. In a preferred embodiment of the second configuration of the invention, the epitope binding domain is an immunoglobulin variable region and is selected from a repertoire of single domain V genes. Typically, single domain antibody repertoires are displayed on the surface of filamentous phage. In a preferred embodiment, individual single domain antibodies are selected by binding the phage library to the antigen.
一方面，多特异性配体包含至少两个非互补可变区。例如，配体可包含一对VH区或一对VL区。最好这些结构域并非骆驼科动物来源；优选它们是人结构域或者包含人构架区(FW)和一个或多个异源CDR。CDR和构架区是免疫球蛋白可变区的结构域，正如Kabat的Sequences of Protein of Immunological Interest数据库所定义的。In one aspect, the multispecific ligand comprises at least two non-complementary variable domains. For example, a ligand may comprise a pair ofVH domains or a pair ofVL domains. Preferably these domains are not of camelid origin; preferably they are human domains or comprise human framework regions (FW) and one or more heterologous CDRs. The CDRs and framework regions are the domains of the variable region of an immunoglobulin, as defined by Kabat's Sequences of Protein of Immunological Interest database.
优选的人构架区是种系基因区段DP47和DPK9所编码的结构域。最好VH区或VL区的FW1、FW2和FW3具有来自DP47或DPK9的FW1、FW2或FW3序列。人构架可任选含有突变，例如在本发明配体所用的人构架中共同含有最多约5个氨基酸变化或至多约10个氨基酸变化。Preferred human framework regions are the domains encoded by the germline gene segments DP47 and DPK9. Preferably, FW1, FW2 and FW3 of theVH region orVL region have the sequence of FW1, FW2 or FW3 from DP47 or DPK9. The human framework may optionally contain mutations, for example up to about 5 amino acid changes or up to about 10 amino acid changes in common in the human frameworks used in the ligands of the invention.
根据本发明的第二格局，多特异性配体中的可变区可排列成开放或闭合构象；也就是说，它们可以这样排列：使得可变区可独立和同时结合其关联配体，或者使得仅一个可变区可在任何时候结合其关联配体。According to a second aspect of the invention, the variable domains in the multispecific ligand can be arranged in an open or closed conformation; that is, they can be arranged such that the variable domains can independently and simultaneously bind their cognate ligands, or This allows only one variable domain to bind its cognate ligand at any one time.
本发明人知道，在某些结构条件下，非互补的可变区(例如两个轻链可变区或两个重链可变区)可以存在于配体中，使得第一表位与第一可变区的结合抑制第二表位与第二可变区的结合，甚至这样的非互补区不作为关联对一起作用。The inventors are aware that, under certain structural conditions, non-complementary variable domains (e.g. two light chain variable domains or two heavy chain variable domains) may be present in the ligand such that the first epitope is associated with the second epitope. Binding of one variable domain inhibits binding of a second epitope to the second variable domain, even if such non-complementary domains do not function together as a cognate pair.
最好配体包含两对或更多对可变区；也就是说，它包含至少4个可变区。最好这4个可变区都包含人源构架。Preferably the ligand comprises two or more pairs of variable domains; that is, it comprises at least 4 variable domains. Preferably, all four variable domains contain human frameworks.
在一个优选的实施方案中，人构架和人种系序列的构架相同。In a preferred embodiment, the human framework and the framework of the human germline sequence are the same.
本发明人认为，这样的抗体可特别用于配体结合测定，用于治疗用途和其它用途。The inventors believe that such antibodies are particularly useful in ligand binding assays, for therapeutic and other uses.
在本发明的第二格局的一个实施方案中，可变区来自针对第一和/或第二抗原或表位的抗体。在一个优选的实施方案中，可变区来自抗体单可变区库。在一个实例中，库是在动物或合成库中不会产生的库。在另一实例中，单可变区没有通过动物免疫而分离(至少部分)。因此，单域可以从天然文库中分离出来。In one embodiment of the second format of the invention, the variable region is from an antibody directed against the first and/or second antigen or epitope. In a preferred embodiment, the variable domains are from a repertoire of antibody single variable domains. In one example, the library is one not produced in animal or synthetic libraries. In another example, the single variable domain is not isolated (at least in part) by immunization of the animal. Thus, single domains can be isolated from natural libraries.
另一方面，本发明的第二格局提供多特异性配体，它包含具有第一表位结合特异性的第一表位结合域和具有第二表位结合特异性的非互补第二表位结合域。第一和第二结合特异性可以相同或不同。In another aspect, the second aspect of the invention provides a multispecific ligand comprising a first epitope binding domain having a first epitope binding specificity and a non-complementary second epitope having a second epitope binding specificity binding domain. The first and second binding specificities may be the same or different.
另一方面，本发明提供闭合构象多特异性配体，它包含具有第一表位结合特异性的第一表位结合域和具有第二表位结合特异性的非互补第二表位结合域，其中第一和第二结合特异性能够竞争表位结合，使得闭合构象多特异性配体不能同时结合两个表位。In another aspect, the invention provides a closed conformation multispecific ligand comprising a first epitope binding domain having a first epitope binding specificity and a non-complementary second epitope binding domain having a second epitope binding specificity , wherein the first and second binding specificities are capable of competing for epitope binding such that the closed conformation multispecific ligand cannot bind both epitopes simultaneously.
又一方面，本发明提供包含非互补结合域的开放构象配体，其中所述结构域对同一靶标上的不同表位具有特异性。所述配体与靶标的结合具有增加的亲合力。同样，本发明提供包含非互补结合域的多价配体，所述结合域对相同表位具有特异性并针对包含所述表位的多拷贝靶标，所述表位例如IL-5、PDGF-AA、PDGF-BB、TGFβ、TGFβ2、TGFβ3和TNFeα，例如人TNF受体1和人TNFα。In yet another aspect, the invention provides open conformation ligands comprising non-complementary binding domains, wherein said domains are specific for different epitopes on the same target. The ligand binds the target with increased affinity. Likewise, the present invention provides multivalent ligands comprising non-complementary binding domains specific for the same epitope, such as IL-5, PDGF- AA, PDGF-BB, TGFβ, TGFβ2, TGFβ3 and TNFeα, such as
在类似方面，本发明的配体可以低亲和力结合单个表位，使得与单个表位的结合没有治疗意义；但是与两个表位结合而导致的亲合力可提供治疗益处。在一个具体实例中，可以针对单独存在于正常细胞类型中、但却一起存在于异常或患病细胞(例如肿瘤细胞)中的表位。在这样的情况下，仅异常或患病细胞是本发明双特异性配体的有效靶标。In a similar aspect, the ligands of the invention may bind a single epitope with low affinity such that binding to a single epitope is of no therapeutic interest; however the resulting avidity from binding to two epitopes may provide a therapeutic benefit. In one specific example, epitopes that are present alone in normal cell types but are present together in abnormal or diseased cells (eg, tumor cells) can be targeted. In such cases, only abnormal or diseased cells are valid targets for the dual specific ligands of the invention.
对同一靶上多拷贝的相同表位或相邻表位具有特异性的配体(称为螯合dAb)，也可以是三聚体或多聚体(四聚体或更高聚集体)的配体，其包含3、4或更多个非互补结合域。例如，可以构建包含3或4个VH区或VL区的配体。Ligands specific for multiple copies of the same epitope or adjacent epitopes on the same target (known as chelating dAbs), which can also be trimers or multimers (tetramers or higher aggregates) A ligand comprising 3, 4 or more non-complementary binding domains. For example, ligands can be constructed that contain 3 or 4VH orVL domains.
此外，提供能结合多个亚单位靶标的配体，其中每个结合域对所述靶标亚单位具有特异性。配体可以是二聚体、三聚体或多聚体。In addition, ligands are provided that bind multiple subunit targets, wherein each binding domain is specific for said target subunit. Ligands can be dimers, trimers or multimers.
优选本发明以上方面的多特异性配体是通过本发明第一方面的方法而得到。Preferably the multispecific ligand of the above aspect of the invention is obtained by the method of the first aspect of the invention.
根据本发明第二格局的以上方面，最好第一表位结合域和第二表位结合域是本文所述的非互补免疫球蛋白可变区。也就是说，可以是VH-VH或VL-VL可变区。According to the above aspect of the second aspect of the invention, preferably the first epitope binding domain and the second epitope binding domain are non-complementary immunoglobulin variable regions as described herein. That is, it can be aVH -VH orVL -VL variable region.
具体地讲，可以按照本发明优选方面制备螯合dAb，即采用锚定dAb，其中采用载体来构建二聚体、三聚体或多聚体dAb的文库，所述载体包含接头序列上游或下游的恒定dAb，其具有插入到接头另一侧的第二、第三和更多dAb的库。例如，锚定或导向dAb可以是TAR1-5(Vκ)、TAR1-27(Vκ)、TAR2h-5(VH)或TAR2h-6(Vκ)。In particular, chelating dAbs can be prepared according to a preferred aspect of the invention, i.e. using anchor dAbs, wherein a library of dimeric, trimeric or multimeric dAbs is constructed using a vector comprising a linker sequence upstream or downstream A constant dAb with a repertoire of second, third and further dAbs inserted on the other side of the linker. For example, the anchor or targeting dAb can be TAR1-5(VK), TAR1-27(VK), TAR2h-5(VH) or TAR2h-6(VK).
在替代方法中，可以避免采用接头，例如通过使用结合域例如VH和Vκ之间的非共价键或天然亲和力。因此，本发明提供制备螯合多聚体配体的方法，该方法包括以下步骤：In an alternative approach, the use of linkers can be avoided, for example by using non-covalent bonds or natural affinity between binding domains such asVH and VK. Therefore, the present invention provides the method for preparing chelated multimeric part, and this method comprises the following steps:
(a)提供载体，该载体包含编码对靶标上的第一表位具有特异性的单结合域的核酸序列；(a) providing a vector comprising a nucleic acid sequence encoding a single binding domain specific for a first epitope on the target;
(b)提供载体，该载体编码包含对所述靶标上的第二表位具有特异性的第二结合域的库，所述表位可以与第一表位相同或不同，所述第二表位与所述第一表位相邻；和(b) providing a vector encoding a library comprising a second binding domain specific for a second epitope on said target, which epitope may be the same as or different from the first epitope, said second epitope is adjacent to said first epitope; and
(c)表达所述第一和第二结合域；和(c) expressing said first and second binding domains; and
(d)分离结合在一起的第一和第二结合域的组合，得到结合靶标的二聚体。(d) isolating the combination of the first and second binding domains bound together, resulting in a dimer that binds the target.
第一和第二表位相邻，使得多聚体配体能同时结合这两个表位。这提供了在结合时具有增加亲合力优势的配体。当表位相同时，可通过靶标上存在的多拷贝表位，而得到增加的亲合力，允许同时结合至少两个拷贝，以便亲和力效果增加。The first and second epitopes are adjacent such that the multimeric ligand can bind both epitopes simultaneously. This provides a ligand with an increased avidity advantage upon binding. When the epitopes are the same, increased avidity can be obtained by the presence of multiple copies of the epitope on the target, allowing simultaneous binding of at least two copies so that the avidity effect is increased.
结合域可通过若干方法并采用接头而结合。例如，结合域可包含cys残基、抗生物素蛋白和链霉抗生物素基团或其它在合成后用于非共价连接的方式；有效结合靶标的这些组合可以被分离出来。或者，接头可存在于第一和第二结合域之间，它们可以作为一条多肽从一个载体上表达，例如，如上所述，该多肽包含第一结合域、接头和第二结合域库。Binding domains can be bound by several methods and employing linkers. For example, binding domains may contain cys residues, avidin and streptavidin groups, or other means for non-covalent attachment after synthesis; such combinations that effectively bind the target can be isolated. Alternatively, a linker may be present between the first and second binding domains and they may be expressed as one polypeptide from a vector, eg, comprising the first binding domain, the linker and a repertoire of second binding domains, as described above.
在一个优选的方面，当与抗原结合时，第一和第二结合域就天然连接在一起；例如，VH区和Vκ区，当结合相邻表位时，以三种相互作用方式天然连接，形成稳定二聚体。这样的连接蛋白在靶结合测定中可以被分离出来。该方法的一个优势就是：只有以正确构象结合紧相邻表位的结合域才可以连接，并可因其对靶标的亲合力增加而分离。In a preferred aspect, the first and second binding domains are naturally linked together when bound to an antigen; for example, theVH region and the Vκ domain are naturally linked in three interactions when binding adjacent epitopes , forming a stable dimer. Such connexins can be isolated in target binding assays. An advantage of this approach is that only binding domains that bind immediately adjacent epitopes in the correct conformation can be joined and separated due to their increased affinity for the target.
在本发明的第二格局的以上方面的一个替代实施方案中，至少一个表位结合域包含本文所述的非免疫球蛋白“蛋白质支架”或“蛋白质骨架”。合适的非免疫球蛋白蛋白质支架包括但不限于选自以下的任何以上列出的支架：SpA、纤连蛋白、GroEL和其它陪伴分子、脂质运载蛋白、CCTLA4和亲和体。In an alternative embodiment of the above aspect of the second aspect of the invention, at least one epitope binding domain comprises a non-immunoglobulin "protein scaffold" or "protein backbone" as described herein. Suitable non-immunoglobulin protein scaffolds include, but are not limited to, any of the above-listed scaffolds selected from the group consisting of SpA, Fibronectin, GroEL and other chaperones, Lipocalin, CCTLA4 and Affibodies.
按照本发明的第二格局的以上方面，最好表位结合域结合蛋白质骨架。最好本发明的蛋白质骨架是免疫球蛋白骨架。According to the above aspect of the second aspect of the present invention, it is preferred that the epitope binding domain binds to the protein backbone. Preferably, the protein backbone of the present invention is an immunoglobulin backbone.
按照本发明，术语“免疫球蛋白骨架”是指如上所述的包含至少一个免疫球蛋白折叠并起到一个或多个表位结合域核心作用的蛋白质。According to the present invention, the term "immunoglobulin backbone" refers to a protein as described above comprising at least one immunoglobulin fold and serving as the core of one or more epitope binding domains.
本文所述的优选免疫球蛋白骨架包括任何一个或多个选自以下的骨架：至少包含以下部分的免疫球蛋白分子：(i)抗体的CL(κ或λ亚类)区；或(ii)抗体重链的CH1区；包含抗体重链CH1区和CH2区的免疫球蛋白分子；包含抗体重链CH1区、CH2区和CH3区的免疫球蛋白分子；或任何(ii)亚类以及抗体的CL(κ或λ亚类)区。铰链区也可包含在其中。这样的各区组合可以是例如例如模拟天然抗体，例如IgG或IgM或其片段，例如Fv、scFv、Fab或F(ab′)2分子。本领域技术人员知道，该名单并非是详尽而无遗漏的。Preferred immunoglobulin frameworks described herein include any one or more frameworks selected from the group consisting of immunoglobulin molecules comprising at least (i) the CL (kappa or lambda subclass) region of an antibody; or (ii) The CH1 region of an antibody heavy chain; an immunoglobulin molecule comprising a CH1 region and a CH2 region of an antibody heavy chain; an immunoglobulin molecule comprising a CH1 region, a CH2 region, and a CH3 region of an antibody heavy chain; or any (ii) subclass and of an antibody CL (kappa or lambda subclass) region. A hinge region may also be included. Such combinations of domains may for example mimic natural antibodies, eg IgG or IgM or fragments thereof eg Fv, scFv, Fab or F(ab')2 molecules. Those skilled in the art appreciate that this list is not intended to be exhaustive and exhaustive.
本文所述的骨架与表位结合域的连接可在多肽水平上得到，是在编码骨架和/或表位结合域的核酸表达之后。或者，连接步骤可以在核酸水平上进行。本发明的蛋白质骨架与一个或多个表位结合域的连接方法包括采用蛋白质化学和/或分子生物学技术，这些技术是本领域技术人员熟知的并在本文中有描述。Linkage of the backbone to the epitope binding domain described herein can be achieved at the polypeptide level, following expression of the nucleic acid encoding the backbone and/or the epitope binding domain. Alternatively, the ligation step can be performed at the nucleic acid level. Methods for linking the protein backbone of the invention to one or more epitope binding domains include the use of protein chemistry and/or molecular biology techniques, which are well known to those skilled in the art and described herein.
最好闭合构象多特异性配体可包含能够结合靶分子的第一区和能够结合延长配体半衰期的分子或基团的第二区。例如，分子或基团可以是膨胀剂(bulky agent)，例如HSA或细胞基质蛋白。本文所用的术语“延长配体半衰期的分子或基团”是指这样的分子或化学基团：当给予动物时，相对于不结合分子或基团的配体来说，当所述分子或化学基团结合本文所述的双特异性配体时，它们延长所述双特异性配体的体内半衰期。延长配体半衰期的分子或基团描述如下。在一个优选的实施方案中，仅在半衰期延长分子或基团替代时，闭合构象多特异性配体能够结合靶分子。因此，例如，闭合构象多特异性配体可以通过大体积的分子(例如HSA)而在患者血液循环中得以保持。当遇到靶分子时，闭合构象多特异性配体的结合域之间的竞争导致HAS的替代和靶的结合。Preferably, the closed conformation multispecific ligand may comprise a first region capable of binding a target molecule and a second region capable of binding a molecule or group that extends the half-life of the ligand. For example, the molecule or group can be a bulky agent such as HSA or a cell matrix protein. As used herein, the term "molecule or group that extends the half-life of a ligand" refers to a molecule or chemical group that, when administered to an animal, is When the groups bind the dual specific ligands described herein, they increase the half-life of the dual specific ligands in vivo. Molecules or groups that extend the half-life of ligands are described below. In a preferred embodiment, the closed conformation multispecific ligand is capable of binding the target molecule only upon substitution of the half-life extending molecule or group. Thus, for example, closed conformation multispecific ligands can be maintained in the blood circulation of a patient by bulky molecules such as HSA. When a target molecule is encountered, competition between the binding domains of the closed conformation multispecific ligand results in displacement of the HAS and binding of the target.
在本发明的第二格局的另一方面，本发明提供一种或多种核酸分子，所述核酸至少编码本文所述的多特异性配体。在一个实施方案中，所述配体是闭合构象配体。在另一个实施方案中，它是开放构象配体。多特异性配体可以由一条核酸分子编码；或者，各表位结合域可以由各自的核酸分子编码。当配体由一条核酸分子编码时，各区可表达为融合多肽，或者可以分别表达，再随后连接在一起，例如使用化学连接剂。各自的核酸所表达的配体可通过合适方法连接在一起。In another aspect of the second aspect of the invention, the invention provides one or more nucleic acid molecules encoding at least a multispecific ligand as described herein. In one embodiment, the ligand is a closed conformation ligand. In another embodiment, it is an open conformation ligand. A multispecific ligand can be encoded by one nucleic acid molecule; alternatively, each epitope binding domain can be encoded by a separate nucleic acid molecule. When the ligand is encoded by one nucleic acid molecule, the domains can be expressed as fusion polypeptides, or can be expressed separately and subsequently linked together, eg, using chemical linkers. The ligands expressed by the respective nucleic acids can be linked together by suitable methods.
核酸还可编码信号序列，用于在表达后将多肽从宿主细胞中输出，而且在表达后还可与丝状噬菌体颗粒的表面组分(或选择展示系统的其它组分)融合。可用于细菌表达和/或噬菌体或噬菌粒展示的前导序列包括pelB、stII、ompA、phoA、bla和pelA。The nucleic acid may also encode a signal sequence for export of the polypeptide from the host cell after expression and may also be fused to a surface component of the filamentous phage particle (or other components of the selection display system) after expression. Useful leader sequences for bacterial expression and/or phage or phagemid display include pelB, stII, ompA, phoA, bla, and pelA.
在本发明的第二格局的另一方面，本发明提供包含本发明核酸的载体。In another aspect of the second aspect of the invention, the invention provides a vector comprising a nucleic acid of the invention.
再一方面，本发明提供用本发明载体转染的宿主细胞。In yet another aspect, the invention provides host cells transfected with a vector of the invention.
从这样的载体表达，可以例如在噬菌体颗粒表面产生表位结合域，用于选择。这允许选择所展示的结构域，因此用本发明的方法选择“多特异性配体”。Expression from such vectors makes it possible, for example, to generate epitope binding domains on the surface of phage particles for selection. This allows selection of the displayed domains, thus the selection of "multispecific ligands" using the method of the invention.
本发明还提供试剂盒，所述试剂盒至少包括本发明的多特异性配体，所述配体可以是开放构象或闭合构象配体。本发明的试剂盒可以是例如诊断试剂盒、治疗用试剂盒、化学或生物标本的检测试剂盒等。The present invention also provides a kit comprising at least the multispecific ligand of the present invention, and the ligand may be an open conformation or a closed conformation ligand. The kits of the present invention may be, for example, diagnostic kits, therapeutic kits, detection kits for chemical or biological specimens, and the like.
本发明提供制备多特异性配体的方法，该方法包括以下步骤：The invention provides a method for preparing a multispecific ligand, the method comprising the following steps:
a)选择能够结合第一表位的第一表位结合域，a) selecting a first epitope binding domain capable of binding the first epitope,
b)选择能够结合第二表位的第二表位结合域，b) selecting a second epitope binding domain capable of binding the second epitope,
c)连接表位结合域；和c) linking the epitope binding domain; and
d)选择能够结合所述第一第二表位和所述第二表位的闭合构象多特异性配体。d) selecting a closed conformation multispecific ligand capable of binding said first second epitope and said second epitope.
在第二格局的另一方面，本发明提供制备闭合构象多特异性配体的方法，所述配体包含具有第一表位结合特异性的第一表位结合域和具有第二表位结合特异性的非互补第二表位结合域，其中第一和第二结合特异性竞争表位结合，使得闭合构象多特异性配体不同时结合这两个表位；该方法包括以下步骤：In another aspect of the second format, the invention provides a method of making a closed conformational multispecific ligand comprising a first epitope-binding domain having a first epitope-binding specificity and a domain having a second epitope-binding A specific non-complementary second epitope binding domain, wherein the first and second binding specificities compete for epitope binding such that the closed conformation multispecific ligand does not bind both epitopes simultaneously; the method comprising the steps of:
a)选择能够结合第一表位的第一表位结合域，a) selecting a first epitope binding domain capable of binding the first epitope,
b)选择能够结合第二表位的第二表位结合域，b) selecting a second epitope binding domain capable of binding the second epitope,
c)连接表位结合域，使得各结构域呈闭合构象；和c) linking the epitope binding domains such that each domain is in a closed conformation; and
d)选择能够结合所述第一第二表位和所述第二表位、但不同时结合所述第一和第二表位的闭合构象多特异性配体。d) selecting a closed conformation multispecific ligand capable of binding said first second epitope and said second epitope, but not simultaneously binding said first and second epitope.
本发明的第二格局的以上方面的一个替代实施方案任选还包括步骤(b1)，该步骤包括选择第三或更多表位结合域。这样，所产生的多特异性配体(无论是开放还是闭合构象)包含不止两个表位结合特异性。在本发明的第二格局的优选方面，当多特异性配体包含不止两个表位结合域时，至少两个所述结构域呈闭合构象并竞争结合；其它区可竞争结合或者可以独立于其关联表位而任意连接。An alternative embodiment of the above aspect of the second configuration of the invention optionally further comprises a step (b1 ) comprising selecting a third or more epitope binding domains. In this way, the resulting multispecific ligands (whether in open or closed conformation) contain more than two epitope binding specificities. In a preferred aspect of the second configuration of the invention, when the multispecific ligand comprises more than two epitope binding domains, at least two of said domains are in a closed conformation and compete for binding; other domains may compete for binding or may be independent of Its associated epitope can be linked arbitrarily.
在本发明的第二格局中，优选第一和第二表位是不同的。它们可以是天然存在或合成的多肽、蛋白质或核酸、或其部分。在该方面，本发明的配体能结合表位或抗原并起到拮抗剂或激动剂(例如EPO受体激动剂)的作用。在一个实施方案中，所述配体的表位结合域具有相同表位特异性，并且可以例如同时结合其表位，当多拷贝的表位存在于同一抗原上时。在另一个实施方案中，在不同抗原上提供这些表位，使得配体能结合表位并连接抗原。本领域技术人员将会理解，表位和抗原的选择是庞大的而不同的。它们可以是例如人或动物蛋白、细胞因子、细胞因子受体、酶的辅因子或DNA结合蛋白。合适的细胞因子和生长因子包括但不限于：ApoE、Apo-SAA、BDNF、心肌营养蛋白-1、EGF、EGF受体、ENA-78、嗜酸性粒细胞趋化因子、嗜酸性粒细胞趋化因子-2、Exodus-2、EpoR、FGF(酸性)、FGF(碱性)、成纤维细胞生长因子-10、FLT3配体、CXXXC趋化因子(CX3C)、GDNF、G-CSF、GM-CSF、GF-β1、胰岛素、IFN-γ、IGF-I、IGF-II、IL-1α、IL-1β、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8(72a.a.)、IL-8(77a.a.)、IL-9、IL-10、IL-11、IL-12、IL-13、IL-15、IL-16、IL-17、IL-18(IGIF)、抑制素α、抑制素β、EP-10、角质形成细胞生长因子-2(KGF-2)、KGF、瘦素、LIF、淋巴细胞趋化因子、米勒管(Mullerian)抑制物质、单核细胞集落抑制因子、单核细胞趋化蛋白、M-CSF、MDC(67a.a.)、MDC(69a.a.)、MCP-1(MCAF)、MCP-2、MCP-3、MCP-4、MDC(67a.a.)、MDC(69a.a.)、MIG、MIP-1α、MIP-1β、MIP-3α、MIP-3β、MIP-4、髓性祖细胞抑制因子-1(MPIF-1)、NAP-2、神经营养因子、神经生长因子、β-NGF、NT-3、NT-4、抑瘤素M、PDGF-AA、PDGF-AB、PDGF-BB、PF-4、RANTES、SDF1α、SDF1β、SCF、SCGF、干细胞因子(SCF)、TARC、TGF-α、TGF-β、TGF-β2、TGF-β3、肿瘤坏死因子(TNF)、TNF-α、TNF-β、TNF受体I、TNF受体II、TNIL-1、TPO、VEGF、VEGF受体1、VEGF受体2、VEGF受体3、GCP-2、GRO/MGSA、GRO-β、GRO-γ、HCC1、1-309、HER1、HER2、HER3、HER4、TACE识别位点、TNF BP-I和TNF BP-II、以及公开于附录2或附录3的任何靶标，无论是以附录中列出的组合、不同组合形式还是单独形式。细胞因子受体包含前述细胞因子的受体，例如IL-1R1；IL-6R；IL-10R；IL-18R，以及附录2或附录3中列出的细胞因子的受体以及附录2和附录3中公开的受体。可以理解，该名单并非是详尽而无遗漏的。当多特异性配体结合两个表位(在相同或不同抗原上)时，抗原可以从该名单中选出。在具体的实施方案中，所述配体包含能结合TNFR1的dAb和能结合任一这样的抗原的第二dAb或表位结合域。在这样的实施方案中，多特异性配体可包括免疫球蛋白可变区的任何组合(例如VHVH、VHVL、VLVL)。In the second aspect of the invention it is preferred that the first and second epitopes are different. They may be naturally occurring or synthetic polypeptides, proteins or nucleic acids, or portions thereof. In this regard, a ligand of the invention is capable of binding an epitope or antigen and acting as an antagonist or agonist (eg, an EPO receptor agonist). In one embodiment, the epitope binding domains of the ligands have the same epitope specificity and can, for example, bind their epitopes simultaneously, when multiple copies of the epitope are present on the same antigen. In another embodiment, the epitopes are provided on different antigens such that the ligand can bind the epitope and bind the antigen. Those skilled in the art will understand that the choice of epitopes and antigens is vast and varied. They may be, for example, human or animal proteins, cytokines, cytokine receptors, cofactors for enzymes or DNA binding proteins. Suitable cytokines and growth factors include, but are not limited to: ApoE, Apo-SAA, BDNF, cardiotrophin-1, EGF, EGF receptor, ENA-78, eotaxin, eotaxin Factor-2, Exodus-2, EpoR, FGF (acidic), FGF (basic), Fibroblast Growth Factor-10, FLT3 ligand, CXXXC chemokine (CX3C), GDNF, G-CSF, GM-CSF , GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 , IL-8(72a.a.), IL-8(77a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL -17, IL-18 (IGIF), inhibin alpha, inhibin beta, EP-10, keratinocyte growth factor-2 (KGF-2), KGF, leptin, LIF, lymphotactin, Miller Tube (Mullerian) inhibitory substance, monocyte colony inhibitory factor, monocyte chemoattractant protein, M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP- 2. MCP-3, MCP-4, MDC (67a.a.), MDC (69a.a.), MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid Progenitor inhibitory factor-1 (MPIF-1), NAP-2, neurotrophic factor, nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF -BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumor necrosis factor (TNF), TNF- α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF,
基于免疫球蛋白的多特异性配体的制备Preparation of immunoglobulin-based multispecific ligands
本发明的双特异性配体，在本发明所需格局的构象上无论是开放的还是闭合的，都可以按照用于抗体工程领域的先前建立的技术来制备，用于制备scFv、“噬菌体”抗体和其它改造的抗体分子。抗体、尤其是双特异性抗体的制备技术例如参见以下综述及其中所引用的参考文献：Winter和Milstein，(1991)Nature 349：293-299；Pluckthun(1992)Immunological Reviews 130：151-188；Wright等(1992)Crti.Rev.Immunol.，12：125-168；Holliger，P.和Winter，G.(1993)Curr.Op.Biotechn.，4，446-449；Carter等(1995)J.Hernatother.，4，463-470；Chester，K.A.和Hawkins，R.E.(1995)Trends Biotechn.，13，294-300；Hoogenboom，H.R.(1997)Nature Biotechnol.，15，125-126；Fearon，D.(1997)Nature Biotechnol.，15，618-619；Plückthun，A.和Pack，P.(1997)Immunotechnology3，83-105；Carter，P.和Merchant，A.M.(1997)Curr.Opin.Biotechnol.，8，449-454；Holliger，P.和Winter，G.(1997)CancerImmunol.Immunother.，45，128-130。The dual specific ligands of the invention, whether open or closed in the conformation of the desired pattern of the invention, can be prepared according to previously established techniques used in the field of antibody engineering for the preparation of scFv, "phages" Antibodies and other engineered antibody molecules. For techniques for preparing antibodies, especially bispecific antibodies, see for example the following review and references cited therein: Winter and Milstein, (1991) Nature 349:293-299; Pluckthun (1992) Immunological Reviews 130:151-188; Wright (1992) Crti. Rev. Immunol., 12: 125-168; Holliger, P. and Winter, G. (1993) Curr. Op. Biotechn., 4, 446-449; Carter et al. (1995) J. Hernatother ., 4, 463-470; Chester, K.A. and Hawkins, R.E. (1995) Trends Biotechn., 13, 294-300; Hoogenboom, H.R. (1997) Nature Biotechnol., 15, 125-126; Fearon, D. (1997 ) Nature Biotechnol., 15, 618-619; Plückthun, A. and Pack, P. (1997)
本发明提供用于针对两个不同抗原或表位的可变区选择和随后的可变区组合。The present invention provides for variable region selection and subsequent variable region combination against two different antigens or epitopes.
可变区选择所用的技术采用的是本领域已知的文库和选择方法。采用从人B细胞收获的重排V基因的天然文库(Marks等(1991)J.Mol.Biol.，222：581；Vaughan等(1996)Nature Biotech.，14：309)是本领域技术人员众所周知的。合成文库(Hoogenboom和Winter(1992)J.Mol.Biol.，227：381；Barbas等(1992)Proc.Natl.Acad.Sci.U.S.A.，89：4457；Nissim等(1994)EMBO J.，13：692；Griffiths等(1994)EMBO J.，13：3245；De Kruif等(1995)J.Mol.Biol.，248：97)则是通常采用PCR、通过克隆免疫球蛋白V基因而制备的。PCR过程中出现的错误可导致高度随机化。可以分别针对靶抗原或表位选择VH和/或VL文库，在这种情况下，直接选择或一起选择单域结合。The techniques used for variable region selection employ libraries and selection methods known in the art. The use of natural libraries of rearranged V genes harvested from human B cells (Marks et al. (1991) J. Mol. Biol., 222:581; Vaughan et al. (1996) Nature Biotech., 14:309) is well known to those skilled in the art. of. Synthetic libraries (Hoogenboom and Winter (1992) J.Mol.Biol., 227:381; Barbas et al. (1992) Proc.Natl.Acad.Sci.USA, 89:4457; Nissim et al. (1994) EMBO J., 13: 692; Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97) are usually prepared by cloning the immunoglobulin V gene by PCR. Errors in the PCR process can lead to a high degree of randomization. TheVH and/orVL libraries can be selected against the target antigen or epitope separately, in which case either directly or together for single domain binding.
本发明双特异性配体的优选制备方法包括采用选择系统，其中选择结合第一抗原或表位的可变区库，并选择结合第二抗原或表位的可变区库。再将所选的第一和第二可变区结合在一起并选择结合第一和第二抗原或表位的双特异性配体。选择单独、而非同时结合第一和第二抗原或表位的闭合构象配体。A preferred method of preparing the dual specific ligands of the invention involves the use of a selection system in which a repertoire of variable regions is selected for binding a first antigen or epitope and a repertoire of variable regions is selected for binding a second antigen or epitope. The selected first and second variable domains are then brought together and a dual specific ligand is selected that binds the first and second antigen or epitope. Closed conformation ligands are selected that bind the first and second antigens or epitopes individually, but not simultaneously.
A.文库载体系统A. Library vector system
各种选择系统是本领域已知的，适用于本发明。这些系统的实例如下所述。Various selection systems are known in the art and are suitable for use in the present invention. Examples of these systems are described below.
可以直接筛选噬菌体λ表达系统的噬菌斑或溶原体的菌落，这两者先前都有描述(Huse等(1989)Science，246：1275；Caton和Koprowski(1990)Proc.Natl.Acad.Sci.U.S.A.，87；Mullinax等(1990)Proc.Natl.Acad.Sci.U.S.A.，87：8095；Persson等(1991)Proc.Natl.Acad.Sci.U.S.A.，88：2432)并且用于本发明。尽管这样的表达系统可以用于筛选多达106个不同文库成员，但是它们不适合筛选更大数量(大于106个成员)。The phage lambda expression system can be directly screened for plaques or colonies of lysogens, both of which have been previously described (Huse et al. (1989) Science, 246:1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci. USA, 87; Mullinax et al. (1990) Proc. Natl. Acad. Sci. USA, 87:8095; Persson et al. (1991) Proc. Natl. Although such expression systems can be used to screen up to106 different library members, they are not suitable for screening larger numbers (greater than106 members).
文库构建中特别有用的是选择展示系统，所述系统使核酸连接到它所表达的多肽上。本文所用的选择展示系统是允许通过单个文库成员结合通用配体和/或靶配体的合适展示方法而选择的系统。Particularly useful in library construction are selection display systems which link a nucleic acid to the polypeptide it expresses. A selection display system as used herein is a system that allows selection by appropriate display methods of binding of a single library member to a generic ligand and/or target ligand.
在大文库中分离出所需成员的选择方案是本领域已知的，以噬菌体展示技术为代表。这种将多样化肽序列展示在丝状噬菌体表面的系统(Scott和Smith(1990)Science，249：386)，已经证明可用于产生抗体片段(及其编码它们的核苷酸序列)文库，用于体外选择和结合靶抗原的特异性抗体片段的扩增(McCafferty等，WO 92/01047)。编码VH区和VL区的核苷酸序列连接到编码前导信号的基因片段上，这样的信号可指导它们到达大肠杆菌细胞的壁膜间隙，结果所得抗体片段可展示在噬菌体表面，通常是与噬菌体外壳蛋白(例如pIII或pVIII)融合。或者，抗体片段向外展示在λ噬菌体衣壳(噬菌体)上。基于噬菌体的展示系统的一个优点就是：因为它们是生物系统，所以可以将含有所选文库成员的噬菌体在细菌细胞中进行培养，而简单扩增所选文库成员。此外，因为编码多肽文库成员的核苷酸序列被噬菌体或噬菌粒载体所包含，所以测序、表达和其后的遗传操作相对直接。Selection protocols for isolating desired members in large libraries are known in the art, represented by phage display technology. This system of displaying diverse peptide sequences on the surface of filamentous bacteriophage (Scott and Smith (1990) Science, 249:386) has been shown to be useful for generating libraries of antibody fragments (and the nucleotide sequences encoding them) using In vitro selection and amplification of specific antibody fragments that bind the target antigen (McCafferty et al., WO 92/01047). The nucleotide sequences encoding theVH andVL regions are linked to a gene segment encoding a leader signal that directs them to the periplasmic space of E. coli cells, resulting in antibody fragments that can be displayed on the surface of the phage, usually Fusion to phage coat protein (eg pill or pVIII). Alternatively, antibody fragments are externally displayed on lambda phage capsids (phages). One advantage of phage-based display systems is that because they are biological systems, selected library members can be simply amplified by culturing phage containing selected library members in bacterial cells. Furthermore, because the nucleotide sequences encoding polypeptide library members are contained by phage or phagemid vectors, sequencing, expression and subsequent genetic manipulation are relatively straightforward.
噬菌体抗体展示文库和λ噬菌体表达文库的构建方法是本领域众所周知的(McCafferty等(1990)Nature，348：552；Kang等(1991)Proc.Natl.Acad.Sci.U.S.A.，88：4363；Clackson等(1991)Nature，352：624；Lowman等(1991)Biochemistry，30：10832；Burton等(1991)Proc.Natl.Acad.Sci.U.S.A.，88：10134；Hoogenboom等(1991)Nucleic Acids Res.，19：4133；Chang等(1991)J.Immunol.，147：3610；Breitling等(1991)Gene，104：147；Marks等(1991)，出处同上；Barbas等(1992)，出处同上；Hawkins和Winter(1992)J.Immunol.，22：867；Marks等，1992，J.Biol.Chem.，267：16007；Lerner等(1992)Science，258：1313，所述文献通过引用结合到本文中)。The construction methods of phage antibody display library and lambda phage expression library are well known in the art (McCafferty et al. (1990) Nature, 348:552; Kang et al. (1991) Proc.Natl.Acad.Sci.U.S.A., 88:4363; (1991) Nature, 352:624; Lowman et al. (1991) Biochemistry, 30:10832; Burton et al. (1991) Proc.Natl.Acad.Sci.U.S.A., 88:10134; Hoogenboom et al. : 4133; Chang et al. (1991) J. Immunol., 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991), supra; Barbas et al. (1992), supra; Hawkins and Winter ( 1992) J. Immunol., 22:867; Marks et al., 1992, J. Biol. Chem., 267:16007; Lerner et al. (1992) Science, 258:1313, which are incorporated herein by reference).
一个特别具有优势的方法是使用scFv噬菌体-文库(Huston等，1988，Proc.Natl.Acad.Sci.U.S.A.，85：5879-5883；Chaudhary等(1990)Proc.Natl.Acad.Sci.U.S.A.，87：1066-1070；McCafferty等(1990)，出处同上；Clackson等(1991)Nature，352：624；Marks等(1991)J.Mol.Biol.，222：581；Chiswell等(1992)Trends Biotech.，10：80；Marks等(1992)J.Biol.Chem.，267)。在噬菌体外壳蛋白上展示的scFv文库的各种实施方案已有描述。噬菌体展示方法的改进也是已知的，例如参见WO96/06213和WO92/01047(Medical Research Council等)和WO97/08320(Morphosys)，所述文献通过引用结合到本文中。A particularly advantageous method is the use of scFv phage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883; Chaudhary et al. : 1066-1070; McCafferty et al. (1990), cited above; Clackson et al. (1991) Nature, 352: 624; Marks et al. (1991) J.Mol.Biol., 222: 581; Chiswell et al. (1992) Trends Biotech., 10:80; Marks et al. (1992) J. Biol. Chem., 267). Various embodiments of scFv libraries displayed on phage coat proteins have been described. Improvements to phage display methods are also known, see eg WO96/06213 and WO92/01047 (Medical Research Council et al) and WO97/08320 (Morphosys), which are incorporated herein by reference.
产生多肽文库的其它系统包括使用无细胞酶促机制，用于体外合成文库成员。在一个方法中，通过针对靶配体的多轮选择和PCR扩增来选择RNA分子(Tuerk和Go1d(1990)Science，249：505；Ellington和Szostak(1990)Nature，346：818)。一项类似技术可用于鉴定结合预定人转录因子的DNA序列(Thiesen和Bach(1990)Nucleic Acids Res.，18：3203；Beaudry和Joyce(1992)Science，257：635；WO92/05258和WO92/14843)。以类似方式，体外翻译可用于合成多肽，这作为产生大文库的方法。这些通常包括稳定的多核糖体复合物的方法进一步参见WO88/08453、WO90/05785、WO90/07003、WO91/02076、WO91/05058和WO92/02536。并非基于噬菌体的替代展示系统例如那些公开于WO95/22625和WO95/11922(Affymax)的系统，采用多核糖体来展示多肽，用于选择。Other systems for generating polypeptide libraries include the use of cell-free enzymatic mechanisms for the in vitro synthesis of library members. In one approach, RNA molecules are selected by multiple rounds of selection against target ligands and PCR amplification (Tuerk and Gold (1990) Science, 249:505; Ellington and Szostak (1990) Nature, 346:818). A similar technique can be used to identify DNA sequences that bind predetermined human transcription factors (Thiesen and Bach (1990) Nucleic Acids Res., 18:3203; Beaudry and Joyce (1992) Science, 257:635; WO92/05258 and WO92/14843 ). In a similar manner, in vitro translation can be used to synthesize polypeptides as a means of generating large libraries. These methods generally involving stable polysome complexes are further described in WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058 and WO92/02536. Alternative display systems that are not phage-based, such as those disclosed in WO95/22625 and WO95/11922 (Affymax), employ polysomes to display polypeptides for selection.
还有一项技术包括对人工区室中的库的选择，这允许将基因与其基因产物结合起来。例如，通过在油包水乳剂形成的微胶囊中选择编码所需基因产物的核酸的选择系统，参见WO99/02671、WO00/40712；Tawfik和Griffiths(1998)Nature Biotechnol 16(7)，652-6。将编码具有所需活性的基因产物的遗传元件在微胶囊中区室化，然后在微胶囊内转录和/或翻译以产生它们相应的基因产物(RNA或蛋白质)。随后再分选产生具有所需活性的基因产物的遗传元件。该方法通过各种方法检测所需活性而选择目标基因产物。Yet another technique involves selection of repertoires in artificial compartments, which allow combining genes with their gene products. For example, selection systems by selecting nucleic acids encoding desired gene products in microcapsules formed from water-in-oil emulsions, see WO99/02671, WO00/40712; Tawfik and Griffiths (1998) Nature Biotechnol 16(7), 652-6 . Genetic elements encoding gene products with desired activities are compartmentalized in microcapsules, and then transcribed and/or translated within the microcapsules to produce their corresponding gene products (RNA or protein). Genetic elements that produce gene products with the desired activity are subsequently sorted. This method selects the gene product of interest by detecting the desired activity by various methods.
B.文库构建B. Library Construction
可以采用本领域已知技术，构建例如以上提出的文库用于选择，或者从商业来源购买。可用于本发明的文库参见例如WO99/20749。一旦选择载体系统并将一个或多个编码目标多肽的核酸序列克隆到文库载体中，通过在表达前进行诱变，就可以在克隆分子内产生多样性；或者，可以在如上所述的诱变和多轮选择进行之前，表达并选择所编码的蛋白质。按照标准分子方法，对编码结构优化多肽的核酸序列进行诱变。特别有用的是聚合酶链式反应即PCR(Mullis和Faloona(1987)Methods Enzymol，155：335，所述文献通过引用结合到本文中)。PCR是本领域众所周知的，它是采用由热稳定的DNA依赖性DNA聚合酶催化的多轮DNA复制，以扩增目标靶序列。各种抗体文库的构建参见Winter等(1994)Ann.Rev.Immunology，12，433-55及其中引用的参考文献。Libraries such as those set forth above can be constructed for selection using techniques known in the art, or purchased from commercial sources. Libraries that can be used in the present invention are described eg in WO99/20749. Once a vector system is selected and one or more nucleic acid sequences encoding a polypeptide of interest are cloned into a library vector, diversity can be generated within the cloned molecules by performing mutagenesis prior to expression; alternatively, mutagenesis can be performed as described above The encoded protein is expressed and selected before multiple rounds of selection are performed. Nucleic acid sequences encoding structurally optimized polypeptides are subjected to mutagenesis according to standard molecular methods. Particularly useful is the polymerase chain reaction, or PCR (Mullis and Faloona (1987) Methods Enzymol, 155:335, which is hereby incorporated by reference). PCR is well known in the art and employs multiple rounds of DNA replication catalyzed by a thermostable DNA-dependent DNA polymerase to amplify a target sequence of interest. For the construction of various antibody libraries, see Winter et al. (1994) Ann. Rev. Immunology, 12, 433-55 and references cited therein.
用模板DNA(至少1fg；更有用的为1-1000ng)和至少25pmol寡核苷酸引物，进行PCR；当引物库(primer pool)非常不均一时，最好采用较大量引物，因为各序列仅有少量引物库分子作为代表，而且数量在随后的扩增循环中成为限制。通常反应混合物包括：2μl DNA、25pmol寡核苷酸引物、2.5μl 10X PCR缓冲液1(Perkin-Elmer，FosterCity，CA)，0.4μl 1.25μM dNTP、0.15μl(或2.5单位)Taq DNA聚合酶(Perkin Elmer，Foster City，CA)和去离子水加至总体积为25μl。覆盖矿物油，进行PCR，用程序加热循环仪。PCR循环中各步骤的长度和温度以及循环次数，按照结果所需的严格性来调整。退火温度和时间由引物预计与模板退火的效率和可耐受的错配程度而定；显然，当核酸分子同时扩增和诱变的话，错配是必需的，至少在合成的第一轮。优化引物退火条件的严格性的能力是本领域普通技术人员的知识范围之内。采用的退火温度在30℃和72℃之间。模板分子开始变性通常发生在92℃和99℃之间达4分钟，再是20-40次循环，循环是由变性(94-99℃15秒至1分钟)、退火(按照上述温度；1-2分钟)和延伸(72℃1-5分钟，取决于扩增产物的长度)组成。最终延伸通常72℃4分钟，再接着是不确定(0-24小时)步骤，在4℃。Perform PCR with template DNA (at least 1 fg; more usefully 1-1000 ng) and at least 25 pmol oligonucleotide primers; when the primer pool is very heterogeneous, it is best to use a larger number of primers because each sequence is only A small number of primer library molecules are represented, and the number becomes limiting in subsequent amplification cycles. Usually the reaction mixture includes: 2 μl DNA, 25 pmol oligonucleotide primers, 2.5 μl 10X PCR buffer 1 (Perkin-Elmer, Foster City, CA), 0.4 μl 1.25 μM dNTP, 0.15 μl (or 2.5 units) Taq DNA polymerase ( Perkin Elmer, Foster City, CA) and deionized water were added to a total volume of 25 μl. Cover with mineral oil and perform PCR with programmed heating cycler. The length and temperature of each step in the PCR cycle, as well as the number of cycles, are adjusted according to the stringency required for the result. The annealing temperature and time depend on the efficiency with which the primers are expected to anneal to the template and the degree of mismatches that can be tolerated; clearly, when nucleic acid molecules are simultaneously amplified and mutagenized, mismatches are required, at least in the first round of synthesis. The ability to optimize the stringency of primer annealing conditions is within the knowledge of one of ordinary skill in the art. The annealing temperature employed was between 30°C and 72°C. The initial denaturation of template molecules usually occurs between 92°C and 99°C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94-99°C for 15 seconds to 1 minute), annealing (according to the above temperatures; 1- 2 minutes) and extension (1-5 minutes at 72°C, depending on the length of the amplified product). The final extension is typically 4 minutes at 72°C, followed by an indeterminate (0-24 hour) step at 4°C.
C.结合单可变区C. Binding to single variable domains
本发明所用的结构域，一旦选出就可以通过本领域已知的各种方法连接，包括共价和非共价方法。The domains used in the invention, once selected, can be linked by various methods known in the art, including covalent and non-covalent methods.
优选的方法包括采用如上所述的多肽接头，例如连接scFv分子(Bird等(1988)Science 242；423-426)。有关合适接头的讨论参见Bird等，Science 242，423-426；Hudson等，Journal Immunol Methods 231(1999)177-189；Hudson等，Proc.Nat.Acad.Sci.USA，85，5879-5883。接头优选是柔性的，允许两个单域相互作用。一个接头的实例是(Gly4Ser)n接头，其中n＝1-8，例如2、3、4、5或7。也可采用双链抗体中使用的接头(其柔性较差)(Holliger等(1993)PNAS(USA)90：6444-6448)。A preferred method involves the use of polypeptide linkers as described above, for example linking scFv molecules (Bird et al. (1988)
在一个实施方案中，所用的接头不是免疫球蛋白铰链区。In one embodiment, the linker used is not an immunoglobulin hinge region.
可变区可采用不用接头的方法而结合。例如，可以开发二硫桥的用途(所述二硫桥是通过天然存在的或改造的半胱氨酸残基而提供的)，以便稳定VH-VH、VL-VL或VH-VL二聚体(Reiter等(1994)ProtemEng.，7：697-704)或者通过可变区间界面的重构以改善“适合性(fit)”，因而稳定相互作用(Ridgeway等(1996)Protein Eng.7：617-621；Zhu等(1997)Protein Science 6：781-788)。Variable regions can be combined using methods that do not use linkers. For example, the use of disulfide bridges provided by naturally occurring or engineered cysteine residues can be exploited to stabilizeVH -VH ,VL -VL orVH - VL dimer (Reiter et al. (1994) ProtemEng., 7:697-704) or by remodeling of the variable interval interface to improve "fit" and thus stabilize the interaction (Ridgeway et al. (1996) Protein Eng. 7:617-621; Zhu et al. (1997) Protein Science 6:781-788).
如果适当的话，可以使用用于连接或稳定免疫球蛋白可变区、尤其是抗体VH区的其它技术。Other techniques for linking or stabilizing immunoglobulin variable regions, particularly antibodyVH regions, may be used, as appropriate.
根据本发明，双特异性配体在溶液中可以呈“闭合”构象。“闭合”构型是指：其中两个区(例如VH和VL)以连接形式存在，例如连接的VH-VL对形成抗体结合位点。例如，scFv可以呈闭合构象，这取决于用于连接VH区和VL区的接头的排列。如果它具有足够柔性让这两区连接，或者刚性地将它们保持在连接位置上，则这两区可能采取闭合构象。According to the invention, the dual specific ligand may assume a "closed" conformation in solution. A "closed" configuration is one in which the two regions (eg,VH andVL ) exist in linked form, eg, the linkedVH -VL pair forms the antibody binding site. For example, a scFv can assume a closed conformation, depending on the arrangement of the linker used to connect theVH andVL regions. The two domains may adopt a closed conformation if it is flexible enough to allow the two domains to link, or to hold them rigidly in the linked position.
同样，VH区对和VL区对可存在闭合构象。通常，这将随配体分子中的两区通过例如刚性接头紧密连接而变。闭合构象中的配体不能同时结合延长配体的半衰期的分子和第二靶分子。因此，配体通常只结合从延长配体半衰期的分子上解离下来的第二靶分子。Likewise, pairs ofVH domains andVL domains can exist in closed conformations. Typically, this will vary with the two domains in the ligand molecule being tightly linked, eg by a rigid linker. Ligands in the closed conformation are unable to simultaneously bind the ligand's half-life-extending molecule and the second target molecule. Thus, a ligand typically only binds a second target molecule dissociated from the molecule that extends the half-life of the ligand.
此外，无需接头的VH/VH、VL/VL或VH/VL二聚体的构建为两区之间带来竞争。Furthermore, theconstruction of VH/VH, VL/VLorVH/VLdimers without a linker introduces competition between the two domains.
而且，本发明的配体也可以呈开放构象。在这样的构象中，所述配体能同时结合延长配体的半衰期的分子和第二靶分子。通常，开放构型中的可变区(就VH-VL对而言)与以下结构域保持足够远的距离：所述结构域不会相互作用而形成抗体结合位点、而且也不会竞争性结合它们各自的表位。就VH/VH或VL/VL二聚体而言，这些结构域并不通过刚性接头结合在一起。当然，这样的结构域配对也不竞争抗原结合或形成抗体结合位点。Furthermore, the ligands of the invention may also be in an open conformation. In such a conformation, the ligand is capable of binding both the ligand's half-life-extending molecule and the second target molecule. Typically, the variable region in the open configuration (in the case of aVH -VL pair) is sufficiently distant from domains that do not interact to form an antibody binding site and that do not Competitively bind their respective epitopes. In the case ofVH /VH orVL /VL dimers, these domains are not held together by a rigid linker. Of course, such domain pairings also do not compete for antigen binding or form antibody binding sites.
Fab片段和完整抗体将会主要以闭合构象存在，尽管可以理解，开放和闭合双特异性配体在不同情况下可能以各种均衡存在。配体与靶的结合可能会改变开放构型均衡的平衡。因此，本发明的某些配体在溶液中可存在两种构象，其一(开放形式)可独立结合两个抗原或表位，同时另一构象(闭合形式)仅能结合一个抗原或表位；因此，在该构象中，抗原或表位竞争性结合配体。Fab fragments and intact antibodies will primarily exist in a closed conformation, although it will be appreciated that open and closed dual specific ligands may exist in various equilibria under different circumstances. Binding of a ligand to a target may alter the balance of the open conformation equilibrium. Thus, certain ligands of the invention can exist in two conformations in solution, one (the open form) which can independently bind two antigens or epitopes, while the other conformation (the closed form) can only bind one antigen or epitope ; thus, in this conformation, the antigen or epitope competes for ligand binding.
尽管双特异性配体的开放形式因此在溶液中存在具有闭合形式的均衡，但是考虑到均衡将有利于闭合形式；此外，开放形式可以被靶向闭合构象的结合而掩蔽。因此，优选本发明的某些双特异性配体存在两个(开放和闭合)构象间的平衡。Although the open form of the dual specific ligand therefore has an equilibrium in solution with the closed form, consideration of the equilibrium will favor the closed form; moreover, the open form can be masked by binding to the closed conformation. Thus, it is preferred that certain dual specific ligands of the invention exist in equilibrium between two (open and closed) conformations.
可以对本发明的双特异性配体进行修饰，以便有利于开放或闭合构象。例如，VH-VL的相互关系因二硫键而稳定，从而稳定了闭合构象。此外，可以构建用于结合各区(包括VH区和VL区对)的接头，以有利于开放形式；例如，接头可在空间上阻碍各区的结合，例如通过在合适位置上结合大的氨基酸残基，或者设计能物理性分离各区的合适的刚性结构。The dual specific ligands of the invention can be modified to favor an open or closed conformation. For example, theVH -VL relationship is stabilized by disulfide bonds, thereby stabilizing the closed conformation. In addition, linkers for joining domains (including pairs ofVH andVL domains) can be constructed to favor the open format; for example, linkers can sterically hinder the joining of domains, for example by incorporating large amino acids at appropriate positions residues, or design suitable rigid structures that physically separate the regions.
D.双特异性配体的表征D. Characterization of Dual Specific Ligands
双特异性配体与其特异性抗原或表位的结合可以用本领域技术人员熟知的方法来测定，该方法包括ELISA。在本发明的一个优选的实施方案中，采用单克隆噬菌体ELISA来测定结合。Binding of a dual specific ligand to its specific antigen or epitope can be assayed by methods well known to those skilled in the art, including ELISA. In a preferred embodiment of the invention, binding is determined using a monoclonal phage ELISA.
可以按照任何合适方法，进行噬菌体ELISA：示例性方案如下。Phage ELISA can be performed according to any suitable method: an exemplary protocol follows.
可以通过ELISA筛选每轮选择中产生的、与所选抗原或表位结合的噬菌体群体，以鉴定“多克隆”噬菌体抗体。然后可通过ELISA筛选来自单个受感染细菌菌落的这些群体的噬菌体，以鉴定“单克隆”噬菌体抗体。最好也筛选结合抗原或表位的可溶性抗体片段，这也可通过ELISA、采用针对C-端或N-端标记等试剂来进行(参见例如Winter等(1994)Ann.Rev.Immunology 12，433-55及其中引用的参考文献。The population of phage produced in each round of selection for binding to a selected antigen or epitope can be screened by ELISA to identify "polyclonal" phage antibodies. Phage from these populations of single infected bacterial colonies can then be screened by ELISA to identify "monoclonal" phage antibodies. Preferably also soluble antibody fragments are screened for antigen or epitope binding, which can also be done by ELISA, using reagents such as labeling for the C-terminus or N-terminus (see e.g. Winter et al. (1994) Ann.
可以通过PCR产物的凝胶电泳(Marks等1991，出处同上；Nissim等1994，出处同上)、探测(Tomlinson等，1992)J.Mol.Biol.227，776)或通过载体DNA测序，来评价所选噬菌体单克隆抗体的多样性。The presence of PCR products can be assessed by gel electrophoresis of PCR products (Marks et al. 1991, supra; Nissim et al. 1994, supra), probing (Tomlinson et al., 1992) J. Mol. Biol. 227, 776) or by vector DNA sequencing. Diversity of selected phage monoclonal antibodies.
E.双特异性配体的结构E. Structure of Dual Specific Ligands
如上所述，在本文中，抗体定义为抗体(例如IgG、IgM、IgA、IgA、IgE)或其片段(Fab、Fv、二硫键连接的Fv、scFv、双链抗体)，它包含至少一个重链和轻链可变区、至少两个重链可变区或至少两个轻链可变区。它可以至少部分来源于任何物种天然产生的抗体，或通过重组DNA技术产生的抗体；无论是从下列哪种样品中分离的：血清、B细胞、杂交瘤、转染瘤、酵母或细菌。As mentioned above, herein an antibody is defined as an antibody (e.g. IgG, IgM, IgA, IgA, IgE) or a fragment thereof (Fab, Fv, disulfide-linked Fv, scFv, diabody) comprising at least one Heavy and light chain variable regions, at least two heavy chain variable regions, or at least two light chain variable regions. It may be derived at least in part from antibodies naturally produced by any species, or antibodies produced by recombinant DNA techniques; whether isolated from any of the following samples: serum, B cells, hybridomas, transfectomas, yeast or bacteria.
在本发明的一个优选实施方案中，双特异性配体包含抗体的至少一个单重链可变区和抗体的一个单轻链可变区、或两个单重链可变区或轻链可变区。例如，配体可包含一对VH/VL、一对VH区或一对VL区。In a preferred embodiment of the invention, the dual specific ligand comprises at least one single heavy chain variable region of an antibody and one single light chain variable region of an antibody, or two single heavy chain variable regions or light chain variable regions. Variable area. For example, a ligand may comprise a pair ofVH /VL , a pair ofVH domains, or a pair ofVL domains.
所述配体的第一和第二可变区可以在同一多肽链上。或者它们可在不同多肽链上。就它们在同一多肽链上而言，它们可以通过接头而连接，所述接头优选为如上所述的肽序列。The first and second variable regions of the ligand may be on the same polypeptide chain. Or they can be on different polypeptide chains. To the extent that they are on the same polypeptide chain, they may be linked by a linker, preferably a peptide sequence as described above.
第一和第二可变区可以共价或非共价结合。在它们是共价结合的情况下，共价键可以是二硫键。The first and second variable regions can be covalently or non-covalently associated. Where they are covalently bound, the covalent bond may be a disulfide bond.
在采用例如本文所述的噬菌体展示技术从V基因库中选出可变区的情况下，这些可变区可包含通用构架区，使得它们可以被本文所述的特异性通用配体所识别。有关通用构架、通用配体等的用途参见WO 99/20749。Where variable regions are selected from V gene repertoires using, for example, phage display techniques as described herein, these variable regions may comprise common framework regions such that they can be recognized by specific universal ligands as described herein. See WO 99/20749 for use of general frameworks, general ligands, etc.
当使用V基因库时，多肽序列中的变异优选位于可变区结构环内。各可变区的多肽序列可以通过DNA改组或通过突变而改变，以便增强各可变区与其互补对的相互作用。DNA改组是本领域已知的，参见例如Stemmer，Nature 370：389-391(1994)和美国专利第6,297,053号，所述文献都通过引用结合到本文中。其它诱变方法是本领域技术人员众所周知的。When using a repertoire of V genes, variations in the polypeptide sequence are preferably located within the variable region structural loops. The polypeptide sequence of each variable domain can be altered by DNA shuffling or by mutation in order to enhance the interaction of each variable domain with its complementary pair. DNA shuffling is known in the art, see, eg, Stemmer, Nature 370:389-391 (1994) and US Patent No. 6,297,053, both of which are incorporated herein by reference. Other methods of mutagenesis are well known to those skilled in the art.
在本发明的一个优选实施方案中，“双特异性配体”是单链Fv片段。在本发明的一个替代实施方案中，“双特异性配体”由Fab形式组成。In a preferred embodiment of the invention the "dual specific ligand" is a single chain Fv fragment. In an alternative embodiment of the invention the "dual specific ligand" consists of a Fab format.
另一方面，本发明提供核酸分子，所述核酸至少编码本文所述的“双特异性配体”。In another aspect, the invention provides nucleic acid molecules encoding at least a "dual specific ligand" as described herein.
本领域技术人员应该理解，根据本发明该方面，抗原或表位可同时结合同一抗体分子。或者，它们可以竞争性结合同一抗体分子。例如，当两个表位同时结合时，双特异性配体的两个可变区能独立结合它们的靶表位。当这些结构域竞争时，一个可变区能结合其靶标，但是不在其它可变区结合其关联靶标的同时；或者第一可变区能结合其靶标，但不在第二可变区结合其关联靶标的同时。It will be appreciated by those skilled in the art that, according to this aspect of the invention, antigens or epitopes can be bound simultaneously to the same antibody molecule. Alternatively, they can compete for binding to the same antibody molecule. For example, the two variable domains of a dual specific ligand can independently bind their target epitopes when the two epitopes are bound simultaneously. When these domains compete, one variable domain can bind its target, but not at the same time as the other variable domain binds its cognate target; or the first variable domain can bind its target, but not its cognate target at the second variable domain target at the same time.
可变区可来自针对靶抗原或表位的抗体。或者，它们可来自单域抗体库，例如在丝状噬菌体表面表达的那些。可以如下所述进行选择。The variable regions can be from antibodies raised against the target antigen or epitope. Alternatively, they may be derived from single domain antibody repertoires, such as those expressed on the surface of filamentous phage. Selections can be made as described below.
一般而言，可以按照例如以下标准实验室手册的方法，构建并操作实现本发明所需的核酸分子和载体构建体：Sambrook等(1989)Molecular Cloning：A Laboratory Manual，Cold Spring Harbor，USA。In general, the nucleic acid molecules and vector constructs required for the practice of the present invention can be constructed and manipulated, for example, according to the methods of the following standard laboratory manual: Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, USA.
本发明所用的核酸的操作通常在重组载体中进行。Manipulation of nucleic acids used in the present invention is usually carried out in recombinant vectors.
因此，另一方面，本发明提供载体，所述载体包含至少编码本文所述的“双特异性配体”的核酸。Thus, in another aspect, the present invention provides a vector comprising at least a nucleic acid encoding a "dual specific ligand" as described herein.
本文所用的载体是指这样的分离的元件：所述元件用于将异源DNA引入细胞，用于表达和/或复制异源DNA。所述载体的选择或构建以及随后的使用方法是本领域技术人员众所周知的。大量载体是公众可得的，包括细菌质粒、噬菌体、人工染色体和附加型载体。这类载体可用于简单克隆和诱变；或者采用基因表达载体。可以选择本发明所用的载体，以适应所需大小的多肽编码序列，长度通常为0.25千碱基对(kb)至40kb或更大。体外克隆操作后，用载体转化合适的宿主细胞。各载体含有不同的功能性组件，通常包括克隆(或“多接头”)位点、复制起点和至少一个选择标记基因。如果给定的载体是表达载体，它还可具有一个或多个以下元件：增强子元件、启动子、转录终止和信号序列，每个都位于克隆位点附近，使得它们与编码本发明配体的基因有效连接。A vector as used herein refers to an isolated element useful for introducing heterologous DNA into a cell for expressing and/or replicating the heterologous DNA. Methods for selection or construction of such vectors and subsequent use are well known to those skilled in the art. A large number of vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, and episomal vectors. Such vectors can be used for simple cloning and mutagenesis; or gene expression vectors. Vectors used in the present invention can be selected to accommodate polypeptide coding sequences of the desired size, typically 0.25 kilobase pairs (kb) to 40 kb or greater in length. After in vitro cloning operations, appropriate host cells are transformed with the vector. Each vector contains different functional components, usually including a cloning (or "polylinker") site, an origin of replication and at least one selectable marker gene. If a given vector is an expression vector, it may also have one or more of the following elements: an enhancer element, a promoter, a transcription termination and a signal sequence, each located near the cloning site such that they are compatible with encoding the ligands of the invention The genes are effectively linked.
克隆载体和表达载体一般都含有使载体在一种或多种所选宿主细胞中能复制的核酸序列。通常在克隆载体中，该序列使载体独立于宿主染色体DNA而复制，并且还包含复制起点或自主复制序列。这类序列众所周知可用于多种多样的细菌、酵母和病毒。质粒pBR322的复制起点适合于大多数革兰氏阴性菌，2μ质粒起点适合于酵母，而各种病毒起点(例如SV40、腺病毒)可用于哺乳动物细胞中的克隆载体。通常，复制起点对哺乳动物表达载体而言是不需要的，除非这些起点用于能高水平复制DNA的哺乳动物细胞，例如COS细胞。Cloning and expression vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence enables the vector to replicate independently of the host chromosomal DNA and also contains an origin of replication or an autonomously replicating sequence. Such sequences are well known for a wide variety of bacteria, yeast and viruses. The origin of replication of the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (eg SV40, adenovirus) can be used as cloning vectors in mammalian cells. Generally, origins of replication are not required for mammalian expression vectors unless these origins are used in mammalian cells capable of high levels of DNA replication, such as COS cells.
最好克隆载体或表达载体可含有选择基因，也称为选择标记。该基因编码转化宿主细胞在选择培养基中存活或生长所需的蛋白质。因此，没有被含有选择基因的载体转化的宿主细胞将不能在培养基中存活。典型的选择基因编码这样的蛋白质：所述蛋白质赋予抗生素抗性(例如氨苄青霉素、新霉素、甲氨蝶呤或四环素)和其它毒素抗性、补充生长培养基中所没有的营养缺陷或补充关键性营养物。Preferably the cloning or expression vector may contain a selectable gene, also known as a selectable marker. This gene encodes a protein required for the transformed host cell to survive or grow in a selective medium. Thus, host cells that have not been transformed with a vector containing the selection gene will not survive in the culture. Typical selection genes encode proteins that confer antibiotic resistance (such as ampicillin, neomycin, methotrexate, or tetracycline) and other toxin resistance, complement auxotrophs not present in the growth medium, or supplement key nutrients.
因为编码本发明配体的载体的复制是在大肠杆菌中常规进行的，所以采用大肠杆菌选择标记，例如赋予抗生素氨苄青霉素抗性的β-内酰胺酶基因。这些可以得自大肠杆菌质粒，例如pBR322或pUC质粒，例如pUC18或pUC19。Since replication of vectors encoding ligands of the invention is routinely performed in E. coli, an E. coli selectable marker is employed, such as the beta-lactamase gene that confers resistance to the antibiotic ampicillin. These can be obtained from E. coli plasmids such as pBR322 or pUC plasmids such as pUC18 or pUC19.
表达载体通常含有宿主生物可识别的启动子，并且该启动子与目标编码序列操作性连接。这样的启动子可以是诱导型或组成型的。术语“操作性连接”是指并置方式使所述组件的关系允许它们以既定方式起作用。控制序列与编码序列“操作性连接”是指连接方式使得在控制序列相容的条件可进行编码序列的表达。Expression vectors usually contain a promoter recognizable by the host organism, and the promoter is operably linked to the coding sequence of interest. Such promoters may be inducible or constitutive. The term "operably linked" means that described components are juxtaposed such that their relationship permits them to function in an intended manner. "Operatively linked" between a control sequence and a coding sequence means that they are linked in such a way that expression of the coding sequence can be performed under conditions compatible with the control sequences.
适用于原核宿主的启动子包括例如β-内酰胺酶和乳糖启动子系统、碱性磷酸酶、色氨酸(trp)启动子系统和杂合启动子(例如tac启动子)。用于细菌系统的启动子一般还含有与编码序列操作性连接的Shine-Delgarno序列。Promoters suitable for use with prokaryotic hosts include, for example, the beta-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters (eg, the tac promoter). Promoters for use in bacterial systems also typically contain a Shine-Delgarno sequence operably linked to the coding sequence.
优选的载体是能表达对应于多肽文库成员的核苷酸序列的表达载体。因此，可以通过表达多肽文库成员的单克隆的单独增殖和表达或者通过使用任何选择展示系统，用第一和/或第二抗原或表位进行选择。如上所述，优选的选择展示系统是噬菌体展示。因此，可以使用噬菌体或噬菌粒载体，例如pIT1或pIT2。用于本发明的前导序列包括pelB、stII、ompA、phoA、bla和pelA。一个实例是具有大肠杆菌复制起点(用于双链复制)以及噬菌体复制起点(用于产生单链DNA)的噬菌粒载体。这类载体的操作和表达是本领域众所周知的(Hoogenboom和Winter(1992)，出处同上；Nissim等(1994)，出处同上)。简而言之，载体含有β-内酰胺酶基因(以便在噬菌粒上赋予选择性)和表达盒上游的lac启动子，后者由以下部分组成(N端→C端)：pelB前导序列(指导表达多肽到壁膜间隙)、多克隆位点(用于核苷酸形式的克隆文库成员)、任选一个或多个肽标记(用于检测)、任选一个或多个TAG终止密码子和噬菌体蛋白pIII。因此，使用大肠杆菌的不同抑制菌株和非抑制菌株，并且添加葡萄糖、异丙基硫代-β-D-半乳糖苷(IPTG)或辅助噬菌体(例如VCS M13)，载体能够作为质粒进行复制，但却不表达，仅产生大量多肽文库成员或产生噬菌体，它们中的一些在其表面含有至少一个拷贝的多肽-pIII融合体。Preferred vectors are expression vectors capable of expressing nucleotide sequences corresponding to members of the polypeptide library. Thus, selection with the first and/or second antigen or epitope can be performed by individual propagation and expression of single clones expressing polypeptide library members or by use of any selection display system. As mentioned above, the preferred display system for selection is phage display. Thus, phage or phagemid vectors such as pIT1 or pIT2 can be used. Leader sequences useful in the present invention include pelB, stll, ompA, phoA, bla and pelA. An example is a phagemid vector with an E. coli origin of replication (for double-stranded replication) and a bacteriophage origin of replication (for production of single-stranded DNA). The manipulation and expression of such vectors is well known in the art (Hoogenboom and Winter (1992), supra; Nissim et al. (1994), supra). Briefly, the vector contains the β-lactamase gene (to confer selectivity on the phagemid) and the lac promoter upstream of the expression cassette consisting of (N-terminal → C-terminal): pelB leader sequence (directs expression of polypeptides to the periplasmic space), multiple cloning site (for cloning library members in nucleotide form), optionally one or more peptide tags (for detection), optionally one or more TAG stop codons sub and phage protein pIII. Thus, using different suppressor and non-suppressor strains of E. coli with the addition of glucose, isopropylthio-β-D-galactoside (IPTG) or a helper phage (e.g. VCS M13), the vector is able to replicate as a plasmid, Instead of expression, only large numbers of polypeptide library members are produced or phage are produced, some of which contain at least one copy of the polypeptide-pill fusion on their surface.
编码本发明配体的载体的构建采用常规连接技术。分离的载体或DNA片段被切割、剪裁并重新连接成产生所需载体的理想形式。如有必要，可以用已知方式进行分析，证实所构建的载体中存在正确序列。用于构建表达载体、制备体外转录物、将DNA引入宿主细胞、以及进行评价表达和功能的分析所用的合适方法，是本领域技术人员已知的。通过常规方法，检测样品中存在的基因序列、或其扩增和/或表达量，所述常规方法例如例如DNA印迹分析或RNA印迹分析、蛋白质印迹、DNA、RNA或蛋白质斑点印迹、原位杂交、免疫细胞化学或者核酸或蛋白质分子的测序分析。如有必要，本领域技术人员会容易地知道如何改进这些方法。The construction of the vector encoding the ligand of the present invention adopts conventional ligation techniques. Isolated vectors or DNA fragments are cut, tailored and religated into the desired form to generate the desired vector. If necessary, analysis can be performed in a known manner to confirm the presence of the correct sequence in the constructed vector. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing assays to assess expression and function are known to those skilled in the art. The gene sequence present in the sample, or its amplification and/or expression level, is detected by conventional methods, such as, for example, Southern blot analysis or Northern blot analysis, Western blot, DNA, RNA or protein dot blot, in situ hybridization , immunocytochemistry, or sequencing analysis of nucleic acid or protein molecules. Those skilled in the art will readily know how to modify these methods, if necessary.
闭合构象多特异性配体的结构Structure of a closed conformational multispecific ligand
根据本发明第二格局的一方面，将两个或更多个非互补表位结合域连接在一起，使它们呈现本文所述的闭合构象。最好除了本文所述的接头之外，它们还可以连接骨架，以便形成和/或维持表位结合位点彼此的闭合构象。According to an aspect of the second configuration of the invention, two or more non-complementary epitope binding domains are linked together such that they assume the closed conformation as described herein. Preferably, in addition to the linkers described herein, they may also be attached to the backbone so as to form and/or maintain the closed conformation of the epitope binding sites with each other.
(I)骨架(I) Skeleton
骨架可以基于免疫球蛋白分子或者可以是如上所述来源的非免疫球蛋白。本文所述的优选免疫球蛋白骨架包括任何一个或多个选自以下的骨架：至少包含以下部分的免疫球蛋白分子：(i)抗体的CL(κ或λ亚类)区；或(ii)抗体重链的CH1区；包含抗体重链CH1区和CH2区的免疫球蛋白分子；包含抗体重链CH1区、CH2区和CH3区的免疫球蛋白分子；或任何(ii)亚类以及抗体的CL(κ或λ亚类)区。也可以包含铰链区域。这样的各区组合可以是例如例如模拟天然抗体，例如IgG或IgM或其片段，例如Fv、scFv、Fab或F(ab′)2分子。本领域技术人员知道，该名单并非是详尽而无遗漏的。The backbone may be based on an immunoglobulin molecule or may be of non-immunoglobulin origin as described above. Preferred immunoglobulin frameworks described herein include any one or more frameworks selected from the group consisting of immunoglobulin molecules comprising at least (i) the CL (kappa or lambda subclass) region of an antibody; or (ii) The CH1 region of an antibody heavy chain; an immunoglobulin molecule comprising a CH1 region and a CH2 region of an antibody heavy chain; an immunoglobulin molecule comprising a CH1 region, a CH2 region, and a CH3 region of an antibody heavy chain; or any (ii) subclass and of an antibody CL (kappa or lambda subclass) region. Hinge areas can also be included. Such combinations of domains may for example mimic natural antibodies, eg IgG or IgM or fragments thereof eg Fv, scFv, Fab or F(ab')2 molecules. Those skilled in the art appreciate that this list is not intended to be exhaustive and exhaustive.
(II)蛋白质支架(II) Protein Scaffold
各表位结合域包含蛋白质支架和一个或多个CDR，它们参与结构域与一个或多个表位的特异性相互作用。最好本发明的表位结合域包含3个CDR。合适的蛋白质支架包括选自以下的任何支架：基于免疫球蛋白结构域的支架、基于纤连蛋白的支架、基于亲和体的支架、基于CTLA4的支架、基于陪伴分子例如GroEL的支架、基于脂质运载蛋白的支架和基于细菌Fc受体SpA和SpD的支架。本领域技术人员可以理解，该名单并非是详尽而无遗漏的。Each epitope-binding domain comprises a protein scaffold and one or more CDRs that are involved in the domain's specific interaction with one or more epitopes. Preferably the epitope binding domain of the invention comprises 3 CDRs. Suitable protein scaffolds include any scaffold selected from the group consisting of immunoglobulin domain-based scaffolds, fibronectin-based scaffolds, affibody-based scaffolds, CTLA4-based scaffolds, chaperone-based scaffolds such as GroEL, lipid-based Scaffolds for plasmid proteins and scaffolds based on the bacterial Fc receptors SpA and SpD. Those skilled in the art will understand that this list is not exhaustive and exhaustive.
F：用于构建双特异性配体的支架F: Scaffolds for construction of dual-specific ligands
i.主链构象的选择i. Choice of Main Chain Conformation
免疫球蛋白超家族成员都共享其多肽链的类似折叠。例如，尽管抗体在其一级序列上具有高度多样性，但是序列比较和晶体结构表明，与预期相反，抗体的6个抗原结合环中有5个(H1、H2、L1、L2、L3)采用数目有限的主链构象或正则结构(Chothia和Lesk(1987)J.Mol.Biol.，196：901；Chothia等(1989)Nature，342：877)。因此，对环长度和关键残基进行分析，能够预测在大多数人抗体中存在的H1、H2、L1、L2和L3的主链构象(Chothia等(1992)J.Mol.Biol.，227：799；Tomlinson等(1995)EMBO J.，14：4628；Williams等(1996)J.Mol.Biol.，264：220)。尽管H3区在序列、长度和结构上更具多样性(因为使用D区段)，但是它也构成数目有限的短环长度的主链构象，这取决于环及抗体构架的关键位置上特定残基的长度和存在与否，或残基种类(Martin等(1996)J.Mol.Biol.，263：800；Shirai等(1996)FEBS Letters，399：1)。Members of the immunoglobulin superfamily all share a similar fold of their polypeptide chains. For example, despite the high diversity of antibodies in their primary sequences, sequence comparisons and crystal structures show that, contrary to expectations, five of the six antigen-binding loops of antibodies (H1, H2, L1, L2, L3) employ There are a limited number of backbone conformations or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196:901; Chothia et al. (1989) Nature, 342:877). Thus, analysis of loop lengths and key residues enables prediction of the main-chain conformations of H1, H2, L1, L2 and L3 present in most human antibodies (Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14:4628; Williams et al. (1996) J. Mol. Biol., 264:220). Although the H3 region is more diverse in sequence, length, and structure (due to the use of the D segment), it also constitutes a limited number of backbone conformations of short loop lengths, depending on specific residues at key positions in the loop and antibody framework. The length and presence or absence of the base, or the type of residue (Martin et al. (1996) J. Mol. Biol., 263:800; Shirai et al. (1996) FEBS Letters, 399:1).
本发明的双特异性配体最好从结构域文库中装配，例如VH区文库和/或VL区文库。此外，本发明的双特异性配体本身可以以文库形式提供。在本发明的一方面，设计双特异性配体和/或结构域文库，其中可以选择出某些环长度和关键残基，以保证各成员的主链构象是已知的。如上所述，最好这些是天然存在的免疫球蛋白超家族分子的真实构象，以将它们是非功能性的机会降至最低。种系V基因区段起到一个合适的基本构架的作用，用于构建抗体或T细胞受体文库；也可使用其它序列。可以低频率发生变异，使得少量功能性成员具有改变的主链构象，这样的改变并不影响其功能。The dual specific ligands of the invention are preferably assembled from a library of domains, eg a library ofVH regions and/or a library of VLregions . Furthermore, the dual specific ligands of the invention may themselves be provided in library form. In one aspect of the invention, libraries of dual specific ligands and/or domains are designed in which certain loop lengths and key residues are selected to ensure that the main chain conformation of each member is known. As mentioned above, preferably these are true conformations of naturally occurring immunoglobulin superfamily molecules, to minimize the chance that they are non-functional. Germline V gene segments serve as a suitable basic framework for constructing antibody or T cell receptor libraries; other sequences may also be used. Mutations can occur at low frequency so that a small number of functional members have altered backbone conformations that do not affect their function.
正则结构理论也用于评价配体所编码的不同主链构象的数目，以预测基于主链构象的配体序列并选择用于多样化的残基，同时又不影响正则结构。已经知道，在人Vκ区中，L1环可采用4种正则结构之一，L2环具有一种正则结构，而且90％的人Vκ区的L3环采用4或5种正则结构之一(Tomlinson等(1995)，出处同上)；因此，仅在Vκ区，不同正则结构可组合产生大量的不同主链构象。假定Vλ区为L1、L2和L3环编码各种不同正则结构，Vκ区和Vλ区可与任何VH区(其可为H1和H2环编码几种正则结构)配对，则这5个环所观察到的正则结构的组合数量将会非常庞大。这表明主链构象多样性的产生对产生各种结合特异性来说是必不可少的。然而，通过构建基于抗体文库的一种已知主链构象，已经发现，与预期相反，主链构象的多样性并非产生足够的针对几乎所有抗原的多样性所必需的。甚至更令人吃惊的是，一种主链构象不一定是共有结构-一种天然存在的构象可以用作整个文库的基础。因此，在优选的方面，本发明的双特异性配体具有一种已知主链构象。Canonical structure theory is also used to evaluate the number of different main-chain conformations encoded by ligands to predict ligand sequences based on main-chain conformations and to select residues for diversification without affecting canonical structures. It is known that in the human Vκ region, the L1 loop can adopt one of 4 canonical structures, the L2 loop has a canonical structure, and the L3 loop of 90% of the human Vκ regions adopts one of 4 or 5 canonical structures (Tomlinson et al. (1995), supra); thus, different canonical structures can be combined to generate a large number of different backbone conformations only in the Vκ region. Assuming that the Vλ regions encode various canonical structures for the L1, L2, and L3 loops, and that the Vκ and Vλ regions can be paired with anyVH region (which can encode several canonical structures for the H1 and H2 loops), then the five loops The number of combinations of observed canonical structures would be enormous. This suggests that the generation of backbone conformational diversity is essential to generate a variety of binding specificities. However, by constructing antibody libraries based on one known backbone conformation, it has been found that, contrary to expectations, diversity in the backbone conformation is not necessary to generate sufficient diversity against nearly all antigens. Even more surprising, one backbone conformation is not necessarily the consensus structure—one naturally occurring conformation can be used as the basis for an entire library. Thus, in preferred aspects, the dual specific ligands of the invention have a known backbone conformation.
所选的一种主链构象优选在所述免疫球蛋白超家族类型的分子中是常见的。当观察到大量天然存在的分子采用该构象时，该构象是常见的。因此，在本发明的优选方面，单独考虑免疫球蛋白结构域的各结合环的不同主链构象的天然发生率，然后选择具有不同环的主链构象所需组合的天然存在的可变区。如果没有可用的，就可选择最接近的等同物。优选不同环的主链构象的所需组合是由所选种系基因区段(其编码所需主链构象)产生的。更优选所选种系基因区段在自然界频繁表达，最优选它们在所有天然种系基因区段中是最频繁表达的。The selected one backbone conformation is preferably common among molecules of the immunoglobulin superfamily type. A conformation is common when it is observed for a large number of naturally occurring molecules to adopt that conformation. Thus, in a preferred aspect of the invention, the natural occurrence of different backbone conformations for each binding loop of an immunoglobulin domain is considered individually, and then naturally occurring variable regions are selected to have the desired combination of backbone conformations for the different loops. If none is available, the closest equivalent is chosen. Preferably, the desired combination of main-chain conformations of the different loops is produced by selected germline gene segments that encode the desired main-chain conformations. More preferably the selected germline gene segments are frequently expressed in nature, most preferably they are the most frequently expressed of all natural germline gene segments.
在设计双特异性配体或其文库过程中，可以单独考虑6个抗原结合环中每一个的不同主链构象的发生率。对于H1、H2、L1、L2和L3，选择被20％和100％的天然分子抗原结合环所采用的给定构象。通常，所观察到的它的发生率35％以上(即介于35％和100％之间)、更理想的是50％以上或甚至65％以上。因为绝大多数H3环没有正则结构，所以优先选择在可展示正则结构的环中常见的主链构象。因此，对于每个环来说，选择天然库中最常见的构象。在人抗体中，各环最常见的正则结构(CS)如下：H1-CS 1(79％表达库)、H2-CS 3(46％)、Vκ的L1-CS 2(39％)、L2-CS 1(100％)、Vκ的L3-CS 1(36％)(计算假定κ∶λ的比例为70∶30，Hood等(1967)Cold Spring Harbor Symp.Quant.Biol.，48：133)。对于具有正则结构的H3环而言，具有从残基94到残基101的盐桥的7个残基的CDR3长度(Kabat等(1991)Sequences of Proteins of immunological interest，美国健康和人类服务部)看来是最常见的。EMBL数据文库中有至少16个人类抗体序列具有所需H3长度和关键残基，构成该构象，并且在蛋白质数据库中至少有两个晶体结构可用作抗体建模的基础(2cgr和1tet)。最常表达的种系基因区段的正则结构组合是VH区段3-23(DP-47)、JH区段JH4b、Vκ区段O2/O12(DPK9)和Jκ区段Jκ1。VH区段DP45和DP38也是合适的。因此，这些区段可用于组合，作为构建具有所需单一主链构象的文库的基础。The occurrence of different backbone conformations for each of the six antigen-binding loops can be considered independently during the design of dual-specific ligands or libraries thereof. For H1, H2, L1, L2 and L3, a given conformation adopted by 20% and 100% of the native molecule's antigen-binding loop was chosen. Typically, it is observed at an incidence of greater than 35% (ie between 35% and 100%), more desirably greater than 50% or even greater than 65%. Because the vast majority of H3 loops do not have canonical structures, a main-chain conformation that is common in loops that can exhibit canonical structures is preferred. Therefore, for each loop, the most common conformation in the natural library was chosen. In human antibodies, the most common canonical structures (CS) of each loop are as follows: H1-CS 1 (79% expression library), H2-CS 3 (46%), L1-
或者，并非根据分离的各结合环的不同主链构象的天然发生率来选择单一主链构象，而是采用主链构象组合的天然发生率作为选择单一主链构象的基础。就抗体而言，例如，可以确定任何2、3、4、5或所有6个抗原结合环的正则结构组合的天然发生率。在此，优选所选构象在天然存在的抗体中是常见的，最优选它是天然库中所观察到的最常见的。因此，在人抗体中，例如当考虑H1、H2、L1、L2和L3五个抗原结合环的天然组合时，确定最常见的正则结构的组合，然后结合最常见的H3环构象，作为选择单一主链构象的基础。Alternatively, rather than selecting a single main-chain conformation based on the natural occurrence of different main-chain conformations for each of the isolated binding loops, the natural occurrence of combinations of main-chain conformations is used as the basis for selecting a single main-chain conformation. In the case of antibodies, for example, the natural occurrence of canonical structural combinations of any 2, 3, 4, 5 or all 6 antigen binding loops can be determined. Here, it is preferred that the selected conformation is common in naturally occurring antibodies, most preferably it is the most common observed in natural repertoires. Thus, in human antibodies, for example, when considering the natural combination of the five antigen-binding loops H1, H2, L1, L2, and L3, the combination of the most common canonical structures is determined, and then the combination of the most common H3 loop conformations is selected as a single Basis of Main Chain Conformation.
ii.正则序列的多样化ii. Diversification of regular sequences
如果具有所选几种已知的主链构象、或者优选单一已知主链构象，可以通过改变分子的结合位点，构建本发明的双特异性配体或本发明所用的文库，以便产生具有结构和/或功能性多样性的库。这表明，产生变异体，使得它们在其结构和/或功能上具有足够的多样性，使得它们能够提供各种活性。If there are several known main chain conformations selected, or a single known main chain conformation is preferred, the dual specific ligand of the present invention or the library used in the present invention can be constructed by changing the binding site of the molecule, so as to generate Libraries of structural and/or functional diversity. This suggests that variants are produced such that they are sufficiently diverse in their structure and/or function that they can confer a variety of activities.
所需多样性通常是通过在一个或多个位置上改变所选分子而产生的。可以随机或优选选择所改变的位置。然后，可以通过以下方式达到变异：通过随机化(其间残余氨基酸被任何氨基酸或其类似物(天然或合成的)所取代)，产生非常大数量的变异体；或者通过用一个或多个氨基酸的限定亚类来取代残余氨基酸，产生更数目有限的变异体。Desired diversity is typically created by altering selected molecules at one or more positions. The altered position may be chosen randomly or preferentially. Variation can then be achieved either by randomization, during which residual amino acids are replaced by any amino acid or its analogue (natural or synthetic), producing a very large number of variants; or by Subclasses are defined to substitute residual amino acids, resulting in a more limited number of variants.
已经报道了各种方法，用于引入这样的多样性。易错PCR(Hawkins等(1992)J.Mol.Biol.，226：889)、化学诱变(Deng等(1994)J.Biol.Chem.，269：9533)或细菌突变菌株(Low等(1996)J.Mol.Biol.，260：359)可用于将随机突变引入编码分子的基因内。使所选位置发生突变的方法也是本领域众所周知的，它包括使用错配寡核苷酸或简并寡核苷酸，使用或不使用PCR。例如，通过抗原结合环的定向突变，已经产生了一些合成抗体文库。使人破伤风类毒素结合Fab的H3区随机化，以便产生各种新的结合特异性(Barbas等(1992)Proc.Natl.Acad.Sci.U.S.A.，89：4457)。随机或半随机H3和L3区已经附加到种系V基因区段上，产生具有未突变构架区的大文库(Hoogenboom和Winter(1992)J.Mol.Biol.，227：381；Barbas等(1992)Proc.Natl.Acad.Sci.U.S.A.，89：4457；Nissim等(1994)EMBO J.，13：692；Griffiths等(1994)EMBO J.，13：3245；De Kruif等(1995)J.Mol.Biol.，248：97)。这样的多样化可以延伸至包括某些或所有其它抗原结合环(Crameri等(1996)Nature Med.，2：100；Riechmann等(1995)Bio/Technology，13：475；Morphosys，WO97/08320，出处同上)。Various methods have been reported for introducing such diversity. Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol., 226:889), chemical mutagenesis (Deng et al. (1994) J. Biol. Chem., 269:9533) or bacterial mutant strains (Low et al. (1996) ) J. Mol. Biol., 260:359) can be used to introduce random mutations into genes encoding molecules. Methods of mutating selected positions are also well known in the art and include the use of mismatched or degenerate oligonucleotides, with or without PCR. For example, some synthetic antibody libraries have been generated by targeted mutagenesis of the antigen-binding loop. The H3 region of the human tetanus toxoid binding Fab was randomized in order to generate various new binding specificities (Barbas et al. (1992) Proc. Natl. Acad. Sci. U.S.A., 89:4457). Random or semi-random H3 and L3 regions have been appended to germline V gene segments, generating large libraries with unmutated framework regions (Hoogenboom and Winter (1992) J. Mol. Biol., 227:381; Barbas et al. (1992 ) Proc.Natl.Acad.Sci.U.S.A., 89:4457; Nissim et al. (1994) EMBO J., 13:692; Griffiths et al. (1994) EMBO J., 13:3245; De Kruif et al. (1995) J.Mol Biol., 248:97). Such diversification can be extended to include some or all other antigen-binding loops (Crameri et al. (1996) Nature Med., 2:100; Riechmann et al. (1995) Bio/Technology, 13:475; Morphosys, WO97/08320, in ibid).
因为环随机化仅对H3就具有产生约超过1015种结构的潜力，而对其它5个环也具有产生类似的大数量变异体的潜力，所以不能通过使用现有的转化技术或使用无细胞系统来产生代表所有可能组合的文库。例如，在迄今为止所构建的最大文库之一中，产生6×1010个不同抗体，这仅仅是该设计文库的潜在多样性的一部分(Griffiths等(1994)，出处同上)。Because loop randomization has the potential to generate approximately over1015 structures for H3 alone and a similarly large number of variants for the other five loops, it cannot be achieved by using existing transformation techniques or using cell-free system to generate libraries representing all possible combinations. For example, in one of the largest libraries constructed to date, 6 x1010 different antibodies were generated, which is only a fraction of the potential diversity of this designed library (Griffiths et al. (1994), supra).
在一个优选的实施方案中，只有直接参与产生或修饰分子所需功能的残基，才被多样化。对于许多分子而言，功能是结合靶标，因此，多样性应该集中在靶结合位点中，而避免改变那些对分子总体包装或保持所选主链构象来说必不可少的残基。In a preferred embodiment, only residues that are directly involved in creating or modifying the desired function of the molecule are diversified. For many molecules, the function is to bind the target, so diversity should be concentrated in the target binding site while avoiding altering residues that are essential for the overall packaging of the molecule or for maintaining a chosen backbone conformation.
正则序列的多样化，当它用于抗体区时Diversification of canonical sequences, as it applies to antibody domains
就抗体双特异性配体而言，靶结合位点是最常见的抗原结合位点。因此，在高度优选的方面，本发明提供抗体双特异性配体的文库或用于装配它们的文库，其中只有在抗原结合位点的残基才改变。在人抗体库中，这些残基极端多样性，已知让其接触高分辨率抗体/抗原复合物。例如，在L2中，已知位置50和53在天然存在的抗体中是不同的，并且观察到它们与抗原接触。相比之下，常规方法已经改变了相应互补决定区(CDR1)中的所有残基(定义参见Kabat等，1991，出处同上)、本发明所用的文库中多样化的两个相比的某七个残基。这显示了产生各种抗原结合特异性所需的功能多样性的显著改善。In the case of antibody dual specific ligands, the target binding site is the most common antigen binding site. Thus, in a highly preferred aspect, the invention provides libraries of antibody dual specific ligands, or libraries used to assemble them, wherein only residues in the antigen binding site are varied. In the human antibody repertoire, these residues are extremely diverse and are known to make them accessible to high-resolution antibody/antigen complexes. For example, in L2, positions 50 and 53 are known to be different in naturally occurring antibodies, and they are observed to contact the antigen. In contrast, conventional methods have altered all residues in the corresponding complementarity-determining region (CDR1) (as defined see Kabat et al., 1991, supra), and the two compared certain seven residues that were diversified in the library used in the present invention residues. This shows a dramatic improvement in the functional diversity required to generate various antigen-binding specificities.
在自然界，抗体多样性是两个过程的结果：种系V、D和J基因区段的体细胞重组，产生首次用于实验(naive)初级库(所谓的种系和连接多样性)；以及所得重排V基因的体细胞高变。对人类抗体序列进行分析表明，初级库中的多样性集中在抗原结合位点的中心，而体细胞高变则将多样性扩散到抗原结合位点周围区，而这些区在初级库中是高度保守的(参见Tomlinson等(1996)J.Mol.Biol.，256：813)。这一互补性可能逐渐成为检索序列空间的有效策略，而且，尽管显然是抗体所特有的，但是它也可容易地用于其它多肽库。改变的残基是构成靶的结合位点的残基的亚类。如有必要，在选择期间的不同时期，可以改变靶结合位点中残基的不同(包括重叠)亚类。In nature, antibody diversity is the result of two processes: somatic recombination of germline V, D, and J gene segments, generating naive primary repertoires (so-called germline and junctional diversity); and Somatic hypermutation of the resulting rearranged V gene. Analysis of human antibody sequences revealed that diversity in the primary repertoire is concentrated in the center of the antigen-binding site, whereas somatic hypermutation spreads diversity to regions surrounding the antigen-binding site that are highly concentrated in the primary repertoire Conserved (see Tomlinson et al. (1996) J. Mol. Biol., 256:813). This complementarity may emerge as an effective strategy for searching sequence space, and, although apparently antibody-specific, it could readily be applied to other polypeptide repertoires as well. The altered residues are a subclass of residues that make up the binding site for the target. Different (including overlapping) subsets of residues in the target binding site can be altered, if necessary, at different times during the selection.
就抗体库而言，产生起始“幼稚”库(initial“naive”repertoire)，其中改变抗原结合位点的某些、而不是全部的残基。在这种情况下，本文所用的术语“幼稚(naive)”是指没有预定靶标的抗体分子。这些分子类似于没有经历免疫多样化的个体(例如在胎儿和新生儿个体的情况下)的免疫球蛋白基因所编码的那些分子，所述个体的免疫系统尚未受到各种抗原性刺激的攻击。然后，针对各种抗原或表位来选择该库。如有必要，还可在起始库改变的结构域之外引入更多多样性。可以选择具有修饰功能、特异性或亲和力的这种成熟的库。In the case of antibody repertoires, an initial "naive" repertoire is generated in which some, but not all, residues of the antigen binding site are altered. In this context, the term "naive" as used herein refers to an antibody molecule that has no intended target. These molecules are similar to those encoded by the immunoglobulin genes of individuals who have not undergone immune diversification (eg, in the case of fetal and neonatal individuals), whose immune systems have not yet been challenged by various antigenic stimuli. This library is then selected against various antigens or epitopes. If necessary, more diversity can also be introduced beyond the domains where the starting repertoire was changed. Such mature repertoires can be selected for modified function, specificity or affinity.
本发明提供两个不同的幼稚结合域库，用于构建双特异性配体或双特异性配体的幼稚文库，其中改变了抗原结合位点中的某些或所有残基。“初级”文库模拟天然初级库，其多样性限制在抗原结合位点中心的残基，它们在种系V基因区段中具有多样性(种系多样性)，或者在重组过程中被赋予多样性(连接多样性)。这些多样化的残基包括但不限于H50、H52、H52a、H53、H55、H56、H58、H95、H96、H97、H98、L50、L53、L91、L92、L93、L94和L96。在“体细胞”文库中，多样性局限于重组过程中改变的残基(连接多样性)或者是高度体细胞突变的)。这些多样化的残基包括但不限于H31、H33、H35、H95、H96、H97、H98、L30、L31、L32、L34和L96。已知让适于这些文库多样性的以上列出的所有残基接触一个或多个抗体-抗原复合物。因为在这两种文库中，抗原结合位点中并非所有残基都改变，在选择中，通过改变剩余残基而结合另外的多样性，如果需要这样的话。本领域技术人员显而易见的是，任何这些残基(或者包括抗原结合位点的其它残基)的任何亚类都可以用于抗原结合位点的起始和/或后来的多样化。The invention provides two different repertoires of naive binding domains for the construction of dual specific ligands or naive libraries of dual specific ligands wherein some or all residues in the antigen binding site are altered. The 'primary' library mimics the natural primary repertoire with diversity restricted to residues central to the antigen-binding site that are diverse among germline V gene segments (germline diversity) or that have been endowed with diversity during recombination sex (connection diversity). These diverse residues include, but are not limited to, H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91 , L92, L93, L94, and L96. In 'somatic' libraries, diversity is limited to residues that are changed during recombination (joint diversity or are highly somatically mutated). These diverse residues include, but are not limited to, H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34, and L96. All of the residues listed above that are suitable for the diversity of these libraries are known to contact one or more antibody-antigen complexes. Since in both libraries not all residues in the antigen binding site were changed, in selection additional diversity was incorporated by changing the remaining residues, if so desired. It will be apparent to those skilled in the art that any subgroup of any of these residues (or other residues comprising antigen binding sites) may be used for initial and/or subsequent diversification of antigen binding sites.
在用于本发明的文库构建中，所选位置的多样化通常是在核酸水平上进行，即通过改变指定多肽序列的编码序列，使得许多可能的氨基酸(所有20种或其亚类)可以结合在该位置。采用IUPAC命名法，最通用的密码子是NNK，它编码所有氨基酸以及TAG终止密码子。NNK密码子优选用于引入所需多样性。也可以使用达到相同末端的其它密码子，包括NNN密码子，该密码子导致产生额外的终止密码子TGA和TAA。In library construction for use in the present invention, diversification of selected positions is usually performed at the nucleic acid level, i.e., by altering the coding sequence of a given polypeptide sequence so that many possible amino acids (all 20 or subclasses thereof) can be combined at that location. Using IUPAC nomenclature, the most common codon is NNK, which encodes all amino acids, as well as the TAG stop codon. NNK codons are preferably used to introduce the desired diversity. Other codons to the same end can also be used, including the NNN codon which results in the generation of additional stop codons TGA and TAA.
人抗体抗原结合位点中侧链多样性的特征明显对某些氨基酸残基有偏倚。如果统计每个VH、Vκ和Vλ区中的10个最多变的位置的氨基酸组成的话，超过76％的侧链多样性来自仅7个不同残基，它们是丝氨酸(24％)、酪氨酸(14％)、天冬酰胺(11％)、甘氨酸(9％)、丙氨酸(7％)、天冬氨酸(6％)和苏氨酸(6％)。这种倾向于可提供主链柔性的亲水性残基和小残基的偏倚，可能反应了表面进化，该进化倾向于结合各种抗原或表位，并且有助于解释初级库中抗体的所需混杂。The signature of side-chain diversity in human antibody antigen-binding sites is clearly biased towards certain amino acid residues. If the amino acid composition of the 10 most variable positions in each of theVH , Vκ, and Vλ domains is counted, over 76% of the side chain diversity comes from just 7 different residues, which are serine (24%), tyrosine, acid (14%), asparagine (11%), glycine (9%), alanine (7%), aspartic acid (6%) and threonine (6%). This bias toward hydrophilic residues and small residues that provide backbone flexibility may reflect surface evolution that favors binding to various antigens or epitopes and helps explain the diversity of antibodies in primary repertoires. Mixed required.
因为优选模拟这样的氨基酸分布，所以在有待改变位置上的氨基酸分布优选模拟在抗体的抗原结合位点所见到的那些。这种允许针对各种靶抗原来选择某些多肽(不仅是抗体多肽)的氨基酸取代中的偏倚，易于用于任何多肽库。有各种方法，用于在有待改变的位置上使氨基酸分布发生偏倚(包括使用三核苷酸诱变，参见WO97/08320)，其中优选的方法，由于容易合成，是采用常规简并密码子。通过比较由简并密码子所有组合所编码的氨基酸分布型(在各位置上具有等比例的单、双、三和四重简并性)与天然氨基酸的使用，可以计算出最具代表性的密码子。密码子(AGT)(AGC)T、(AGT)(AGC)C和(AGT)(AGC)(CT)，也就是说，分别用IUPAC命名法命名的DVT、DVC和DVY-就是最接近所需氨基酸分布型的密码子：它们编码22％丝氨酸和11％酪氨酸、天冬酰胺、甘氨酸、丙氨酸、天冬氨酸、苏氨酸和半胱氨酸。因此，优选的文库是在各多样化位置上用DVT、DVC或DVY密码子构建的。As it is preferable to mimic such an amino acid distribution, the amino acid distribution at the position to be altered preferably mimics that seen at the antigen binding site of an antibody. This bias in amino acid substitutions that allows selection of certain polypeptides (not just antibody polypeptides) against various target antigens is readily applicable to any polypeptide library. There are various methods for biasing the distribution of amino acids at positions to be altered (including the use of trinucleotide mutagenesis, see WO97/08320), of which the preferred method, due to ease of synthesis, is the use of conventional degenerate codons . By comparing the amino acid profile encoded by all combinations of degenerate codons (with equal proportions of single, double, triple and quadruple degeneracy at each position) to the usage of natural amino acids, the most representative a. The codons (AGT)(AGC)T, (AGT)(AGC)C, and (AGT)(AGC)(CT), that is, DVT, DVC, and DVY respectively in IUPAC nomenclature - are the closest to the required Codons for amino acid profile: they code for 22% serine and 11% tyrosine, asparagine, glycine, alanine, aspartic acid, threonine and cysteine. Thus, preferred libraries are constructed using DVT, DVC or DVY codons at each variegated position.
G：能够延长配体半衰期的抗原G: Antigen that can prolong the half-life of the ligand
本发明的双特异性配体(呈现它的一个格局)能够结合一个或多个在体内延长配体半衰期的分子。通常，这类分子是体内天然存在的多肽，它抵抗通过内源机制所致的降解和/或清除，所述机制能够从生物体中除去不想要的物质。例如，可从以下物质中选出延长生物体半衰期的分子：A dual specific ligand of the invention (in one of its configurations) is capable of binding one or more molecules that extend the half-life of the ligand in vivo. Typically, such molecules are naturally occurring polypeptides in the body that resist degradation and/or clearance by endogenous mechanisms capable of removing unwanted substances from the organism. For example, molecules that extend the half-life of organisms can be selected from:
来自胞外基质的蛋白质；例如胶原、层粘连蛋白、整联蛋白和纤连蛋白。胶原是胞外基质的主要蛋白质。目前已经知道约15种胶原分子，在身体的不同部位发现，例如I型胶原(占身体总胶原的90％)在骨、皮肤、腱、韧带、角膜、内脏中发现，而II型胶原在软骨、椎间盘(invertebral disc)、脊索、眼睛的玻璃体中发现。Proteins from the extracellular matrix; eg, collagen, laminin, integrin, and fibronectin. Collagen is the major protein of the extracellular matrix. About 15 collagen molecules are known, found in different parts of the body, for example, type I collagen (accounting for 90% of the total body collagen) is found in bone, skin, tendons, ligaments, cornea, internal organs, and type II collagen is found in cartilage , intervertebral disc (invertebral disc), notochord, vitreous of the eye.
血液中发现的蛋白质，包括：Proteins found in blood, including:
血浆蛋白，例如血纤蛋白、α-2巨球蛋白、血清白蛋白、血纤蛋白原A、血纤蛋白原B、血清淀粉样蛋白A、珠蛋白、抑制蛋白、泛蛋白、子宫球蛋白和β-2-微球蛋白；Plasma proteins such as fibrin, alpha-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid A, globin, arrestin, ubiquitin, uteroglobulin, and beta-2-microglobulin;
酶和抑制剂，例如血纤蛋白溶解酶原、溶菌酶、半胱氨酸蛋白酶抑制剂C、α-1-抗胰蛋白酶和胰腺胰蛋白酶抑制剂。血纤蛋白溶解酶原是胰蛋白酶样丝氨酸蛋白酶纤溶酶的无活性前体。它通常在血流循环中存在。当血纤蛋白溶解酶原活化时，就转化成纤溶酶，它打开强效酶促区，溶解血凝块中缠住血细胞的血纤蛋白原纤维。这称为血纤蛋白溶解。Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, alpha-1-antitrypsin, and pancreatic trypsin inhibitor. Plasminogen is the inactive precursor of the trypsin-like serine protease plasmin. It is usually present in the blood circulation. When plasminogen is activated, it is converted into plasmin, which opens the powerful enzymatic domain and dissolves the fibrin fibrils that entangle blood cells in the clot. This is called fibrinolysis.
免疫系统蛋白质，例如IgE、IgG、IgM。Immune system proteins such as IgE, IgG, IgM.
转运蛋白，例如视黄醇结合蛋白、α-1微球蛋白。Transport proteins such as retinol binding protein, alpha-1 microglobulin.
防卫素，例如β-防卫素1、嗜中性粒细胞防卫素1、2和3。Defensins such as β-
血脑屏障或神经组织中发现的蛋白质，例如黑皮质素受体、髓磷脂、抗坏血酸转运蛋白。Proteins found in the blood-brain barrier or nervous tissue, eg, melanocortin receptor, myelin, ascorbate transporter.
转铁蛋白受体特异性配体-神经药物(neuropharmaceutical agent)融合蛋白(参见US5977307)；Transferrin receptor specific ligand-neuropharmaceutical agent fusion protein (see US5977307);
脑毛细血管内皮细胞受体、转铁蛋白、转铁蛋白受体、胰岛素、胰岛素样生长因子1(IGF1)受体、胰岛素样生长因子2(IGF2)受体、胰岛素受体。Brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin-like growth factor 1 (IGF1) receptor, insulin-like growth factor 2 (IGF2) receptor, insulin receptor.
局限于肾脏中的蛋白质，例如多囊蛋白、IV型胶原、有机阴离子转运蛋白K1、Heymann抗原。Proteins localized in the kidney, eg polycystin, collagen type IV, organic anion transporter K1, Heymann antigen.
局限于肝脏中的蛋白质，例如醇脱氢酶、G250。Proteins confined to the liver, eg alcohol dehydrogenase, G250.
血液凝固X因子blood coagulation factor X
α1抗胰蛋白酶
HNF1αHNF1α
局限于肺部的蛋白质，例如分泌成分(结合IgA)。Proteins localized to the lungs, such as secretory components (binding IgA).
局限于心脏中的蛋白质，例如HSP27。它与扩张型心肌病相关。Proteins confined to the heart, such as HSP27. It is associated with dilated cardiomyopathy.
局限于皮肤的蛋白质，例如角蛋白。Proteins confined to the skin, such as keratin.
骨特异性蛋白，例如骨形态发生蛋白质(BMP)，它们是表现出成骨活性(osteogenic activity)的转化生长因子β超家族亚类。实例包括BMP-2、BMP-4、BMP-5、BMP-6、BMP-7(也称为成骨蛋白(OP-1)和成骨蛋白-8(OP-2)。Bone-specific proteins, such as bone morphogenetic proteins (BMPs), which are a subclass of the transforming growth factor beta superfamily that exhibit osteogenic activity. Examples include BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein (OP-1 ) and osteogenic protein-8 (OP-2).
肿瘤特异性蛋白，包括人滋养层抗原、herceptin受体、雌激素受体、组织蛋白酶例如组织蛋白酶B(在肝和脾中发现)。Tumor-specific proteins, including human trophoblast antigen, herceptin receptors, estrogen receptors, cathepsins such as cathepsin B (found in liver and spleen).
疾病特异性蛋白，例如仅在活化T细胞上表达的抗原：包括Disease-specific proteins, such as antigens expressed only on activated T cells: include
LAG-3(淋巴细胞活化基因)、护骨蛋白(osteoprotegerin)配体(OPGL)，参见Nature 402，304-309；1999，OX40(TNF受体家族成员，在活化T细胞上表达，已知只有共刺激T细胞分子在人T细胞白血病病毒I型(HTLV-I)产生细胞中被特异性上调)。参见J.Immunol.2000年7月1日；165(1)：263-70；金属蛋白酶(与关节炎/癌症有关)，包括CG6512果蝇(Drosophila)、人截瘫蛋白、人FtsH、人AFG3L2、鼠ftsH；血管生成生长因子，包括酸性成纤维细胞生长因子(FGF-1)、碱性成纤维细胞生长因子(FGF-2)、血管内皮生长因子/血管通透因子(VE-GF/VPF)、转化生长因子-α(TGFα)、肿瘤坏死因子-α(TNF-α)、血管生成素、白介素-3(IL-3)、白介素-8(IL-8)、血小板衍生内皮生长因子(PD-ECGF)、胎盘生长因子(PlGF)、中期因子(midkine)血小板衍生生长因子-BB(PDGF)、CXXXC趋化因子。LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL), see
应激蛋白(热激蛋白)Stress protein (heat shock protein)
HSP通常在胞内发现。当在胞外发现它们时，就表明细胞已经死亡并溢出其内容物。这样的非程序性细胞死亡(坏死)仅当作为创伤、疾病或损伤的结果时才发生，因此，在体内，胞外HSP触发免疫系统对抗感染和疾病的反应。结合胞外HSP的双特异性蛋白可以局限于疾病部位。HSPs are usually found intracellularly. When they are found outside the cell, it is a sign that the cell has died and spilled its contents. Such unprogrammed cell death (necrosis) occurs only as a result of trauma, disease or injury, thus, in vivo, extracellular HSPs trigger the immune system's response against infection and disease. Bispecific proteins that bind extracellular HSPs can be localized to disease sites.
参与Fc转运的蛋白质Proteins involved in Fc transport
Brambell受体(也称为FcRB)Brambell receptors (also known as FcRB)
该Fc受体具有两个功能，这两者可潜在用于递送。The Fc receptor has two functions, both of which are potentially useful for delivery.
其功能是Its function is
跨越胎盘将IgG从母亲转运给孩子Transport of IgG from mother to child across the placenta
保护IgG免遭降解，因而延长IgG的血清半衰期。Protects IgG from degradation, thus prolonging the serum half-life of IgG.
认为受体从核内体中再循环IgG。The receptor is thought to recycle IgG from the endosome.
参见Holliger等，Nat Biotechnol 1997年7月；15(7)：632-6。See Holliger et al., Nat Biotechnol 1997 Jul;15(7):632-6.
可以设计本发明的配体，使它对以上靶标具有特异性，而不需要延长体内半衰期。例如，本发明的配体可以对选自前述的组织特异性靶标具有特异性，因此使结合组织特异性治疗相关靶标的双特异性配体或dAb单体具有组织特异性，无论半衰期怎么延长，尽管这可导致半衰期的延长。此外，当配体或dAb单体靶向肾脏或肝脏时，这可使配体或dAb单体改变体内替代清除途径(例如，配体可从肝脏清除改为肾脏清除)。Ligands of the invention can be designed to be specific for the above targets without requiring increased half-life in vivo. For example, a ligand of the invention may be specific for a tissue-specific target selected from the foregoing, thus rendering a dual-specific ligand or dAb monomer that binds a tissue-specific therapeutically relevant target tissue-specific, regardless of extended half-life, Although this can lead to a prolongation of the half-life. In addition, when the ligand or dAb monomer is targeted to the kidney or liver, this allows the ligand or dAb monomer to alter an alternative clearance pathway in vivo (eg, the ligand can be cleared from the liver to the kidney).
H：本发明第二格局的多特异性配体的用途H: Use of the Multispecific Ligand of the Second Format of the Invention
本发明第二格局的多特异性配体可用于体内治疗和预防用途、体外和体内诊断用途、体外测定和试剂用途等。例如，按照本领域技术人员已知的方法，抗体分子可用于基于抗体的测定技术，例如ELISA技术。The multispecific ligands of the second configuration of the present invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. For example, antibody molecules can be used in antibody-based assay techniques, such as ELISA techniques, according to methods known to those skilled in the art.
如上所述，本发明的多特异性配体可用于诊断、预防和治疗方法。本发明的多特异性抗体在诊断中可用于蛋白质印迹分析和通过标准免疫组织化学方法进行的原位蛋白质检测；对于这些用途而言，可以采用本领域已知技术，对配体进行标记。另外，这类抗体多肽可制备性地用于亲和色谱方法，当与色谱支持物例如树脂结合时。所有这些方法都是本领域技术人员众所周知的。As noted above, the multispecific ligands of the invention are useful in diagnostic, prophylactic and therapeutic methods. The multispecific antibodies of the invention are useful diagnostically in Western blot analysis and in situ protein detection by standard immunohistochemical methods; for these uses, ligands can be labeled using techniques known in the art. In addition, such antibody polypeptides can be used preparatively in affinity chromatography methods when bound to a chromatographic support such as a resin. All of these methods are well known to those skilled in the art.
本发明的闭合构象多特异性配体的诊断应用包括均质测定，用于测定这样的分析物：所述分析物能利用闭合构象多特异性配体与两个靶标竞争性结合的能力，使得两个靶标不能同时结合(闭合构象)，或者它们能够同时结合两个靶标(开放构象)。Diagnostic applications of the closed conformation multispecific ligands of the invention include homogeneous assays for assaying analytes that utilize the ability of the closed conformation multispecific ligands to bind to two targets competitively such that Either the two targets cannot bind simultaneously (closed conformation), or they are able to bind both targets simultaneously (open conformation).
在本发明第二格局的又一方面，本发明提供均质免疫测定，采用本发明的配体。In yet another aspect of the second aspect of the invention, the invention provides a homogeneous immunoassay employing a ligand of the invention.
诊断试剂和研究测定系统制造商一直在热切寻找真正的均质免疫测定形式，用于药物发现和开发。主要的诊断试剂市场包括在医院、医生办公室和诊所、商业参考实验室、血库和家庭中进行的人体检测，非人体诊断(例如食品检验、水质化验、环境监测、生物防制(bio-defence)和兽医检查)，以及最终研究(包括药物开发；基础研究和学术研究)。Manufacturers of diagnostic reagents and research assay systems have been eagerly seeking truly homogeneous immunoassay formats for drug discovery and development. The major diagnostic reagents market includes human testing in hospitals, doctors' offices and clinics, commercial reference laboratories, blood banks, and homes, non-human diagnostics (e.g., food testing, water testing, environmental monitoring, bio-defence) and veterinary examinations), and ultimately research (including drug development; basic research and academic research).
目前，所有这些市场都采用免疫测定系统，这些都是围绕化学发光、ELISA、荧光或(在很少的情况下)放免测定技术而建立起来的。这些测定形式的每一种都需要分离步骤(将结合物与未结合物分开)。在某些情况下，需要几个分离步骤。添加这些附加步骤增加了试剂和自动化仪器、花费时间并且影响最终的测定结果。在人体诊断中，分离步骤可以是自动化的，这掩盖了问题、但并未消除问题。机器人技术、额外的试剂、额外的孵育时间等等都增加了相当大的成本和复杂性。在药物开发例如高通量筛选中，当同时检测数百万份样品且待测分子水平非常低时，增加额外的分离步骤可消除进行筛选的能力。然而，避免分离则在读出时产生太多噪声。因此，需要真正的均质形式，所述形式在现有检测形式所得范围内提供灵敏度。最好测定具有完整定量读出，并具有高灵敏度和大的动态范围。灵敏度是重要要求，因为降低了所需样品数量。这些特征都是均质系统提供的。在样品非常珍贵的医学检验(care testing)和药物开发中，这一点非常重要。本领域目前采用的异质系统需要大量样品和昂贵试剂。All of these markets currently employ immunoassay systems, which are built around chemiluminescence, ELISA, fluorescence or (in rare cases) radioimmunoassay techniques. Each of these assay formats requires a separation step (separation of bound from unbound). In some cases, several isolation steps are required. Adding these additional steps adds reagents and automated instrumentation, takes time, and affects the final assay results. In human diagnostics, the separation step can be automated, which masks the problem but does not eliminate it. Robotics, additional reagents, additional incubation times, and more add considerable cost and complexity. In drug development, such as high-throughput screening, when millions of samples are being tested simultaneously and the levels of the analyte molecule are very low, adding an extra separation step can eliminate the ability to perform the screen. However, avoiding separation creates too much noise in readout. Therefore, there is a need for a truly homogeneous format that provides sensitivity within the range obtained with existing assay formats. Desirable assays have a full quantitative readout with high sensitivity and a large dynamic range. Sensitivity is an important requirement because the required sample quantity is reduced. These features are provided by homogeneous systems. This is important in care testing and drug development, where samples are at a premium. Heterogeneous systems currently employed in the field require large amounts of sample and expensive reagents.
均质测定的应用包括癌症检查，其中最大的测定是前列腺特异性抗原，用于筛选男人的前列腺癌。其它应用包括生育检查，它为想怀孕的妇女提供一系列检查，包括α-hcg，用于妊娠。感染性疾病的检测，包括肝炎、HIV、风疹和其它病毒和微生物和性传播疾病。检测可用于血库，特别是检测HIV、甲型肝炎、乙型肝炎、丙型肝炎、非甲非乙型肝炎。治疗药物监控试验包括监控处方药在患者体内的水平，以便达到功效和避免毒性，例如地高辛对心律失常的功效、以及精神病患者体内的苯巴比妥水平；茶碱对哮喘的功效。诊断测定还可用于药物滥用测定，例如测定可卡因、大麻等。代谢测定用于测定甲状腺功能、贫血和其它生理性障碍和功能。Applications of homogeneous assays include cancer screening, with the largest assay being prostate-specific antigen, used to screen men for prostate cancer. Other applications include Fertility Screening, which offers women trying to conceive a range of tests, including alpha-hcg, for pregnancy. Testing for infectious diseases, including hepatitis, HIV, rubella and other viral and microbial and sexually transmitted diseases. Tests are available in blood banks, specifically for HIV, Hepatitis A, Hepatitis B, Hepatitis C, Non-A Non-B Hepatitis. Therapeutic drug monitoring trials include monitoring levels of prescribed drugs in patients to achieve efficacy and avoid toxicity, eg digoxin for arrhythmias, phenobarbital levels in psychotic patients; theophylline for asthma. Diagnostic assays can also be used for drug abuse assays such as assays for cocaine, marijuana, etc. Metabolic assays are used to measure thyroid function, anemia, and other physiological disorders and functions.
均质免疫测定形式还用于标准临床化学测定的制造。免疫测定和化学测定都在同一仪器上进行，对诊断测定是非常有利的。合适的化学测定包括葡萄糖、胆固醇、钾等测定。Homogeneous immunoassay formats are also used in the manufacture of standard clinical chemistry assays. Both immunoassays and chemical assays are performed on the same instrument, which is very advantageous for diagnostic assays. Suitable chemical assays include glucose, cholesterol, potassium, etc. assays.
均质免疫测定的另一个主要用途就是药物发现和开发：高通量筛选包括以超大体积、针对靶标测定组合化学文库。检测信号，再将阳性组分为更小的组，最终在细胞中、继而在动物体内进行测定。均质测定可用于所有这些类型的测定。在药物开发、尤其是动物研究和临床试验中，大量采用免疫测定。均质测定极大地促进和简化了这些方法。其它应用还包括食品饮料检测：检测肉类和其它食品中的大肠杆菌、沙门氏菌(salmonella)等；水质化验，包括在水厂检测所有类型的污染物，包括大肠杆菌；和兽医检验。Another major use of homogeneous immunoassays is in drug discovery and development: high-throughput screening involves assaying combinatorial chemical libraries against a target in very large volumes. The signal is detected, the positive fraction is divided into smaller groups, and finally assayed in cells and then in animals. Homogeneous assays can be used for all of these types of assays. Immunoassays are heavily used in drug development, especially in animal studies and clinical trials. Homogeneous assays greatly facilitate and simplify these methods. Other applications include food and beverage testing: detection of E. coli, salmonella, etc. in meat and other food products; water quality testing, including detection of all types of contaminants in water plants, including E. coli; and veterinary testing.
在一个广泛的实施方案中，本发明提供包含检测试剂的结合测定，所述试剂结合本发明的闭合构象多特异性配体，其检测特性因分析物与所述闭合构象多特异性配体的结合而改变。这样的测定可以几种不同方式进行，每种方式采用闭合构象多特异性配体的以上特性。In a broad embodiment, the invention provides a binding assay comprising a detection reagent that binds a closed conformation multispecific ligand of the invention, the detection properties of which are determined by the interaction of the analyte with said closed conformation multispecific ligand. combine to change. Such assays can be performed in several different ways, each of which exploits the above properties of closed conformational multispecific ligands.
测定依赖于试剂直接或间接地被分析物取代，导致试剂的检测特性发生变化。例如，当试剂是能够催化反应(所述反应具有可检测终点)的酶，而配体可以结合所述酶，以阻碍例如其活性部位，从而使酶失活。闭合构象多特异性配体也可以结合分析物，以取代酶，通过释放活性部位而使它活化。然后，酶与底物反应，引起可检测事件。在一个替代实施方案中，所述配体可在活性部位之外与酶结合，影响酶的构象，因而改变其活性。例如，活性部位的结构可因结合配体而受到限制，或者可以阻止活性所必需的辅因子的结合。Assays rely on reagents being directly or indirectly displaced by analytes, resulting in a change in the detection properties of the reagents. For example, when the reagent is an enzyme capable of catalyzing a reaction with a detectable end point, a ligand can bind to the enzyme to block, eg, its active site, thereby inactivating the enzyme. Closed conformation multispecific ligands can also bind analytes to displace the enzyme and activate it by releasing the active site. The enzyme then reacts with the substrate, causing a detectable event. In an alternative embodiment, the ligand can bind the enzyme outside the active site, affecting the conformation of the enzyme and thus altering its activity. For example, the structure of the active site may be restricted by binding ligands, or may prevent the binding of cofactors necessary for activity.
测定的实际操作可以采用本领域任何已知形式。例如，可以在测试条上提供闭合构象多特异性配体/酶复合物；可以在测试条的不同区提供底物，并提供含有分析物的溶剂，允许分析物通过配体/酶复合物而迁移，取代酶，并携带它到达底物区，产生信号。或者，可在测试棒或其它固相上提供配体/酶复合物，再浸泡到分析物/底物溶液中，使酶释放到溶液中，以响应存在的分析物。The actual operation of the assay can take any form known in the art. For example, a closed conformation multispecific ligand/enzyme complex can be provided on a test strip; substrates can be provided in different regions of the test strip, and a solvent containing the analyte can be provided to allow the analyte to pass through the ligand/enzyme complex. Migrates, displaces the enzyme, and carries it to the substrate area, generating a signal. Alternatively, the ligand/enzyme complex can be provided on a test stick or other solid phase, which is soaked in the analyte/substrate solution, allowing the enzyme to be released into solution in response to the analyte present.
因为每个分析物分子潜在释放一个酶分子，所以测定是定量的，在给定时间内产生的其信号强度依赖于溶液中分析物浓度。Because each analyte molecule potentially releases one enzyme molecule, the assay is quantitative, the strength of its signal produced at a given time dependent on the analyte concentration in solution.
采用闭合构象的分析物的其它格局也是可能的。例如，闭合构象多特异性配体可以在变构位点结合酶，由此活化酶。在这样的实施方案中，在分析物不存在时，酶是有活性的。加入分析物取代酶，去除变构激活作用，从而使酶失活。Other configurations of the analyte in a closed conformation are also possible. For example, a closed conformation multispecific ligand can bind an enzyme at an allosteric site, thereby activating the enzyme. In such embodiments, the enzyme is active in the absence of analyte. Addition of analyte displaces the enzyme, removing allosteric activation and thus inactivating the enzyme.
就使用酶活性作为分析物浓度的度量的以上实施方案而论，酶的活化或失活是指酶活性的增加或降低，是以酶催化产生信号的反应的能力而定。例如，酶可催化无法检测的底物转化成为其可检测形式。例如，辣根过氧化物酶与显色底物或化学发光底物一起广泛用于本领域，它们都是市售可得的。酶活性的增加或下降水平可以介于10％和100％之间，例如20％、30％、40％、50％、60％、70％、80％或90％；在活性增加的情况下，增加可超过100％，即200％、300％、500％以上，或者，如果抑制酶的基线活性无法检测的话，就不能测定其增加百分比。With respect to the above embodiments that use enzyme activity as a measure of analyte concentration, activation or inactivation of an enzyme refers to an increase or decrease in enzyme activity, as determined by the ability of the enzyme to catalyze a signal-generating reaction. For example, an enzyme can catalyze the conversion of an undetectable substrate into its detectable form. For example, horseradish peroxidase is widely used in the art together with chromogenic or chemiluminescent substrates, both of which are commercially available. The level of increase or decrease in enzyme activity may be between 10% and 100%, for example 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; in the case of increased activity, The increase may be greater than 100%, ie 200%, 300%, 500% or more, or the percent increase cannot be determined if the baseline activity of the inhibitory enzyme is not detectable.
在另一格局，闭合构象多特异性配体能结合酶/底物对中的底物、而不是酶。因此，底物不能利用酶，直到通过分析物结合而使酶从闭合构象多特异性配体中释放出来。这一格局的实施与结合酶的格局是一样的。In another configuration, a closed conformation multispecific ligand can bind the substrate of an enzyme/substrate pair, but not the enzyme. Thus, the substrate cannot utilize the enzyme until the enzyme is released from the closed conformation multispecific ligand by analyte binding. The implementation of this configuration is the same as for the binding enzyme configuration.
此外，可以使测定结合荧光分子，例如荧光素或别的荧光团，其构象使得在结合配体后荧光被猝灭。在这种情况下，分析物与配体的结合将取代荧光分子，从而产生信号。本发明所用的荧光分子的替代物包括冷光试剂(例如萤光素/萤光素酶)和显色试剂(包括免疫测定中常用的试剂，例如HRP)。In addition, the assay can be made to incorporate a fluorescent molecule, such as fluorescein or another fluorophore, in a conformation such that fluorescence is quenched upon binding of the ligand. In this case, the binding of the analyte to the ligand displaces the fluorescent molecule, resulting in a signal. Alternatives to fluorescent molecules used in the present invention include luminescent reagents (such as luciferin/luciferase) and chromogenic reagents (including reagents commonly used in immunoassays, such as HRP).
治疗和诊断组合物及其用途Therapeutic and diagnostic compositions and uses thereof
本发明提供组合物以及使用本发明配体或组合物的治疗和诊断方法，所述组合物包含本发明的TNFR1拮抗剂(例如配体)(例如双特异性配体、多特异性配体、dAb单体)和药学上可接受的载体、稀释剂或赋形剂。本发明方法的拮抗剂和配体(例如双特异性配体、多特异性配体、dAb单体)可用于体内治疗和预防用途、体内诊断用途等。The invention provides compositions and methods of treatment and diagnosis using ligands of the invention or compositions comprising TNFRl antagonists (eg, ligands) of the invention (eg, dual-specific ligands, multispecific ligands, dAb monomer) and a pharmaceutically acceptable carrier, diluent or excipient. Antagonists and ligands (eg, dual specific ligands, multispecific ligands, dAb monomers) of the methods of the invention are useful for in vivo therapeutic and prophylactic applications, in vivo diagnostic applications, and the like.
本发明拮抗剂和配体(例如多特异性配体、双特异性配体、dAb单体)的治疗和预防用途包括将本发明拮抗剂和/或配体给予哺乳动物受体，例如人。双特异性和多特异性配体(例如双特异性抗体形式)以高亲和力结合多聚体抗原。双特异性或多特异性配体可使两个抗原交联，例如在募集的细胞毒T细胞中，从而介导肿瘤细胞系的杀伤。Therapeutic and prophylactic uses of the antagonists and ligands of the invention (eg, multispecific ligands, dual specific ligands, dAb monomers) include administering the antagonists and/or ligands of the invention to mammalian receptors, eg, humans. Bispecific and multispecific ligands (eg, bispecific antibody formats) bind multimeric antigens with high affinity. Bispecific or multispecific ligands can mediate killing of tumor cell lines by crosslinking two antigens, for example in recruited cytotoxic T cells.
优选将至少90-95％同质性(homogeneity)的基本纯化的配体或其结合蛋白(例如dAb单体)给予哺乳动物，最优选98-99％以上同质性供药用，尤其是当哺乳动物是人时。一旦部分纯化或纯化至所需同质性时，配体可用于诊断或治疗(包括离体)或用于开发和实施测定方法、免疫荧光染色等(Lefkovite和Pernis，(1979和1981)ImmunologicalMethods，第I和II卷，Academic Press，NY)。Substantially purified ligands or binding proteins thereof (e.g. dAb monomers) of at least 90-95% homogeneity are preferably administered to mammals, most preferably more than 98-99% homogeneity for pharmaceutical use, especially when when the mammal is a human. Once partially purified or purified to the desired homogeneity, the ligand can be used in diagnosis or therapy (including ex vivo) or in the development and implementation of assay methods, immunofluorescent staining, etc. (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).
例如，本发明的配体或其结合蛋白，例如dAb单体，通常可用于预防、抑制或治疗炎性状态，包括急性及慢性炎性疾病。例如，可以给予拮抗剂和/或配体，以治疗、抑制或预防慢性炎性疾病、变应性超敏反应、癌症、细菌性或病毒性感染、自身免疫性疾病(包括但不限于I型糖尿病、哮喘、多发性硬化、类风湿性关节炎、青少年类风湿性关节炎、银屑病性关节炎、脊椎关节病(spondylarthropathy)(例如关节强硬性脊椎炎)、系统性红斑狼疮、炎性肠病(例如节段性回肠炎(Crohn’s disease)、溃疡性结肠炎)、重症肌无力和Behcet氏综合征)、银屑病、子宫内膜异位症和腹部粘连(例如腹部手术后)。For example, the ligands of the invention or their binding proteins, such as dAb monomers, are generally useful for preventing, inhibiting or treating inflammatory conditions, including acute and chronic inflammatory diseases. For example, antagonists and/or ligands can be administered to treat, inhibit or prevent chronic inflammatory diseases, allergic hypersensitivity, cancer, bacterial or viral infections, autoimmune diseases (including but not limited to type I Diabetes, asthma, multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, spondylarthropathy (eg, ankylosing spondylitis), systemic lupus erythematosus, inflammatory Bowel disease (eg, Crohn's disease, ulcerative colitis), myasthenia gravis, and Behcet's syndrome), psoriasis, endometriosis, and abdominal adhesions (eg, after abdominal surgery).
能结合参与胞吞作用的胞外靶标(例如网格蛋白(Clathrin))的本发明拮抗剂(例如配体)(例如双特异性配体、多特异性配体、dAb单体)可以被胞吞，使它们能接近胞内靶标。此外，双特异性或多特异性配体可提供这样的方法：通过该方法将特异性结合胞内靶标的结合域(例如dAb单体)递送到胞内环境。该策略需要例如这样的双特异性配体：其物理特性能让它在细胞内保留功能性。或者，如果最终目的地的胞内区室是氧化态的，良好折叠的配体可能不需要游离二硫化物。Antagonists (e.g., ligands) of the invention (e.g., dual-specific ligands, multispecific ligands, dAb monomers) capable of binding extracellular targets involved in endocytosis (e.g., clathrin) can be cytosolic endocytosis, making them accessible to intracellular targets. In addition, bispecific or multispecific ligands can provide a means by which binding domains (eg, dAb monomers) that specifically bind an intracellular target are delivered to the intracellular environment. This strategy requires, for example, a dual-specific ligand whose physical properties allow it to retain functionality within the cell. Alternatively, well-folded ligands may not require free disulfides if the intracellular compartment of final destination is oxidized.
在本发明的应用中，术语“预防”包括在疾病诱发之前给予保护性组合物。“抑制”是指在诱导事件之后、而在临床上出现疾病之前给予组合物。“治疗”包括在疾病症状出现后给予保护性组合物。In the application of the present invention, the term "prevention" includes administration of a protective composition prior to the induction of a disease. "Inhibit" means that the composition is administered after the inducing event, but before the clinical appearance of disease. "Treatment"includes administration of a protective composition after disease symptoms have appeared.
最好双特异性或多特异性配体可用于针对在生物体内治疗环境中协同作用的细胞因子和其它分子。因此，本发明提供协同能结合细胞因子或其它分子的两个或更多个结合域(例如dAb)活性的方法，该方法包括给予能够结合所述两个或更多个分子(例如细胞因子)的双特异性或多特异性配体。在本发明的这一方面，双特异性或多特异性配体可以是任何双特异性或多特异性配体，包括由互补和/或非互补区组成的配体、开放构象的配体和闭合构象的配体。例如，本发明的这一方面涉及VH区和VL区的组合、仅有VH区的组合和仅有VL区的组合。Advantageously, bispecific or multispecific ligands can be used to target cytokines and other molecules that act synergistically in an in vivo therapeutic setting. Accordingly, the invention provides methods of synergizing the activity of two or more binding domains (e.g., dAbs) capable of binding a cytokine or other molecule, the method comprising administering bispecific or multispecific ligands. In this aspect of the invention, the bispecific or multispecific ligand may be any bispecific or multispecific ligand, including ligands consisting of complementary and/or non-complementary regions, ligands in open conformation and Ligands in a closed conformation. For example, this aspect of the invention pertains to combinations ofVH andVL domains, combinations ofVH only domains and combinations of onlyVL domains.
在治疗中可以多种方式达到协同。例如，仅当配体同时针对两个靶标时，靶标组合才具有治疗活性，而仅针对一个靶标则没有疗效。在另一个实施方案中，仅一个靶标可提供稍低或最小疗效，但是与第二靶标一起的组合协同性地增加了疗效。优选在本发明这一方面的双特异性或多特异性配体所结合的细胞因子选自附录2所示的列表。Synergy in therapy can be achieved in a variety of ways. For example, a target combination is therapeutically active only if the ligands target both targets simultaneously, whereas targeting only one target is ineffective. In another embodiment, only one target may provide slightly lower or minimal therapeutic effect, but combination with a second target synergistically increases the therapeutic effect. Preferably the cytokines to which the bispecific or multispecific ligands of this aspect of the invention bind are selected from the list shown in
此外，双特异性或多特异性配体可用于肿瘤学用途，所述配体的一个特异性是针对CD89(它是由细胞毒细胞表达的)，而其它特异性则是肿瘤特异性的。所针对的肿瘤抗原的实例见附录3。In addition, bispecific or multispecific ligands, one specificity of which is for CD89 (which is expressed by cytotoxic cells) and the other specificity of which is tumor specific, may be used in oncology. Examples of tumor antigens targeted are given in
可以采用动物模型系统，用于在保护机体免遭疾病或治疗疾病中筛选TNFR1拮抗剂(例如配体、抗体或其结合蛋白)的有效性。在易感小鼠中检测系统性红斑狼疮(SLE)的方法是本领域已知的(Knight等(1978)J.Exp.Med.，147：1653；Reinersten等(1978)New Eng.J.Med.，299：515)。在SJL/J雌性小鼠中，通过用来自另一物种的可溶性AchR蛋白诱发疾病，检测重症肌无力(MG)(Lindstrom等(1988)Adv.Immunol.，42：233)。在易感的小鼠品系中，通过注射II型胶原诱发关节炎(Stuart等(1984)Ann.Rev.Immunol.，42：233)。在易感大鼠中通过注射分枝杆菌热激蛋白而诱发佐剂关节炎的模型已有描述(Van Eden等(1988)Nature，331：171)。描述了通过给予甲状腺球蛋白，在小鼠中诱发甲状腺(Maron等(1980)J.Exp.Med.，152：1115)。在某些品系的小鼠中，胰岛素依赖型糖尿病(IDDM)可自然发生或者可以诱发产生，所述小鼠例如Kanasawa等(1984)Diabetologia，27：113描述的那些。小鼠和大鼠的EAE可作为模型，用于人类的MS。在该模型中，脱髓鞘疾病可以通过给予髓磷脂碱性蛋白而诱发(参见Paterson(1986)Textbook of Immunopathology，Mischer等(主编)，Grune和Stratton，NewYork，第179-213页；McFarlin等(1973)Science，179：478；Satoh等(1987)J.Immunol.，138：179)。Animal model systems can be employed for screening the effectiveness of antagonists of TNFRl (eg, ligands, antibodies, or binding proteins thereof) in protecting the body from disease or treating disease. Methods for detecting systemic lupus erythematosus (SLE) in susceptible mice are known in the art (Knight et al. (1978) J. Exp. Med., 147:1653; Reinersten et al. (1978) New Eng. J. Med ., 299:515). Myasthenia gravis (MG) was detected in SJL/J female mice by inducing disease with soluble AchR protein from another species (Lindstrom et al. (1988) Adv. Immunol., 42:233). In a susceptible mouse strain, arthritis was induced by injection of type II collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42:233). A model of adjuvant arthritis induced by injection of mycobacterial heat shock proteins in susceptible rats has been described (Van Eden et al. (1988) Nature, 331:171). Induction of the thyroid in mice by administration of thyroglobulin has been described (Maron et al. (1980) J. Exp. Med., 152:1115). Insulin-dependent diabetes mellitus (IDDM) can occur naturally or can be induced in certain strains of mice, such as those described by Kanasawa et al. (1984) Diabetologia, 27:113. EAE in mice and rats can be used as a model for MS in humans. In this model, demyelinating disease can be induced by administration of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et al. (eds.), Grune and Stratton, New York, pp. 179-213; McFarlin et al. ( 1973) Science, 179:478; Satoh et al. (1987) J. Immunol., 138:179).
通常，纯化形式的本发明拮抗剂(例如配体)可与药理学上合适的载体一起使用。通常，这些载体包括水性或醇/水溶液剂、乳剂或混悬剂，任意包括盐水和/或缓冲介质。胃肠外溶媒包括氯化钠溶液、林格氏葡萄糖、葡萄糖和氯化钠和乳酸化林格液。可以从增稠剂中选出合适的生理上可接受的辅料，如果需要在混悬剂中维持多肽复合物的话，这些增稠剂例如羧甲基纤维素、聚乙烯吡咯烷酮、明胶和藻酸盐。In general, antagonists (eg, ligands) of the invention in purified form will be employed together with a pharmacologically suitable carrier. Typically, such carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, optionally including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically acceptable excipients may be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates, if necessary to maintain the polypeptide complex in suspension. .
静脉内溶媒包括液体和营养补充剂和电解质补充剂，例如基于林格氏葡萄糖的那些。也可含有防腐剂和其它添加剂，例如抗微生物剂、抗氧化剂、螯合剂和惰性气体(Mack(1982)Remington′sPharmaceutical Sciences，第16版)。各种合适制剂都可采用，包括延长释放的制剂。Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases may also be included (Mack (1982) Remington's Pharmaceutical Sciences, 16th ed.). Various suitable formulations can be used, including extended release formulations.
可以单独给予的组合物形式使用本发明的拮抗剂(例如配体)，或者将其与其它药物联用。这些药物可包括各种免疫治疗药，例如环孢菌素、甲氨蝶呤、阿霉素或顺铂和免疫毒素。药物组合物可包含各种细胞毒剂或其它药物以及本发明拮抗剂(例如配体)、或者甚至是具有不同特异性的本发明配体(例如用不同靶抗原或表位所选择的配体)的组合的“合剂”，无论它们是否在给药之前混合。Antagonists (eg, ligands) of the invention can be used in compositions administered alone, or in combination with other drugs. These drugs can include various immunotherapeutics such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Pharmaceutical compositions may comprise various cytotoxic or other drugs and antagonists of the invention (eg, ligands), or even ligands of the invention with different specificities (eg, ligands selected with different target antigens or epitopes) "Cocktails" of combinations of drugs, whether or not they are mixed prior to administration.
本发明药物组合物的给药途径可以是本领域普通技术人员公知的任何途径。对于治疗(包括但不限于免疫治疗)来说，可以采用标准技术，将本发明所选配体给予任何患者。可通过任何合适方式给予，包括胃肠外、静脉内、肌内、腹膜内、经皮、通过肺部途径，或者合适的是，通过导管直接输注。给药剂量和频次将取决于患者年龄、性别和状况、同时给予的其它药物、对抗适应症(counterindication)和临床医生要考虑的其它参数。按照指示，可以局部给药(例如局部给予肺部，通过肺部给药，例如鼻内给药)或系统给药。The route of administration of the pharmaceutical composition of the present invention can be any route known to those of ordinary skill in the art. For therapy, including but not limited to immunotherapy, selected ligands of the invention can be administered to any patient using standard techniques. Administration may be by any suitable means, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, via the pulmonary route, or, where appropriate, direct infusion via a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, other drugs administered concomitantly, counter indications and other parameters to be considered by the clinician. Administration can be local (eg, topically, pulmonary, eg, intranasal) or systemic, as indicated.
可将本发明配体冻干后贮存备用，使用前重配在合适载体中。已知该技术对常规免疫球蛋白来说是有效的，可以采用本领域已知的冻干和重配技术。本领域技术人员可以理解，冻干和重配可导致不同程度的抗体活性丧失(例如对于常规免疫球蛋白，IgM抗体要比IgG抗体活性损失更高)，因此使用水平应调高以便进行补偿。The ligand of the present invention can be stored after freeze-drying and reconstituted in a suitable carrier before use. This technique is known to be effective for conventional immunoglobulins, and lyophilization and reconstitution techniques known in the art can be employed. Those skilled in the art can understand that lyophilization and reconstitution can lead to different degrees of loss of antibody activity (for example, for conventional immunoglobulins, IgM antibodies have higher activity loss than IgG antibodies), so the use level should be increased to compensate.
可以给予含有本发明拮抗剂(例如配体)或其合剂的组合物，用于预防和/或治疗性治疗。在某些治疗应用中，达到至少部分抑制、遏制、调节、杀伤或某些其它可检测参数的所选细胞群体的足够用量，定义为“治疗有效量”。尽管达到该剂量所需的用量将取决于疾病严重程度和患者自身免疫系统的一般状态，但是通常范围为每千克体重0.005-5.0mg配体(例如抗体、受体(例如T细胞受体)或其结合蛋白)，更常用的剂量为0.05-2.0mg/kg/剂量。对于预防应用而言，也可以类似或稍低剂量给予含有本发明配体或其合剂的组合物，以预防、抑制或延迟疾病发作(例如维持缓解或静止状态，或者预防急性期)。熟练的临床医生能够确定合适的给药间隔，以便治疗、抑制或预防疾病。当给予TNFR1拮抗剂(例如配体)以治疗、抑制或预防慢性炎性疾病时，可以给予最多4次/天，每周两次，每周一次，每两周一次，每月一次，每两月一次，其剂量例如为约10μg/kg至约80mg/kg、约100μg/kg至约80mg/kg、约1mg/kg至约80mg/kg、约1mg/kg至约70mg/kg、约1mg/kg至约60mg/kg、约1mg/kg至约50mg/kg、约1mg/kg至约40mg/kg、约1mg/kg至约30mg/kg、约1mg/kg至约20mg/kg、约1mg/kg至约10mg/kg、约10μg/kg至约10mg/kg、约10μg/kg至约5mg/kg、约10μg/kg至约2.5mg/kg、约1mg/kg、约2mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg或约10mg/kg。在具体的实施方案中，给予TNFR1拮抗剂(例如配体)以治疗、抑制或预防慢性炎性疾病，每两周一次或每月一次，剂量为约10μg/kg至约10mg/kg(例如约10μg/kg、约100μg/kg、约1mg/kg、约2mg/kg、约3mg/kg、约4mg/kg、约5mg/kg、约6mg/kg、约7mg/kg、约8mg/kg、约9mg/kg或约10mg/kg)。Compositions containing antagonists (eg, ligands) of the invention, or combinations thereof, may be administered for prophylactic and/or therapeutic treatment. In certain therapeutic applications, a sufficient amount of a selected cell population to at least partially inhibit, suppress, regulate, kill, or some other detectable parameter is defined as a "therapeutically effective amount". A typical range is 0.005-5.0 mg ligand (e.g. antibody, receptor (e.g. T cell receptor) or its binding protein), the more commonly used dosage is 0.05-2.0mg/kg/dose. For prophylactic applications, compositions containing the ligands of the invention or combinations thereof may also be administered in similar or slightly lower doses to prevent, inhibit or delay the onset of disease (eg, maintain remission or quiescence, or prevent the acute phase). A skilled clinician will be able to determine appropriate dosing intervals for the treatment, suppression or prevention of disease. When an antagonist of TNFR1 (e.g., a ligand) is administered to treat, inhibit, or prevent a chronic inflammatory disease, it can be administered up to 4 times/day, twice a week, once a week, once every two weeks, once a month, every two weeks Once a month, the dose is, for example, about 10 μg/kg to about 80 mg/kg, about 100 μg/kg to about 80 mg/kg, about 1 mg/kg to about 80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kg kg to about 60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg kg to about 10 mg/kg, about 10 μg/kg to about 10 mg/kg, about 10 μg/kg to about 5 mg/kg, about 10 μg/kg to about 2.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg /kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. In specific embodiments, an antagonist of TNFR1 (eg, a ligand) is administered to treat, inhibit or prevent chronic inflammatory diseases, once every two weeks or once a month, at a dose of about 10 μg/kg to about 10 mg/kg (eg, about 10 μg/kg, about 100 μg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg).
相对于治疗前所具有的所述症状、或者相对于未用所述组合物或其它合适对照治疗的个体(人或模型动物)的所述症状而言，当一种或多种症状减少(例如达至少10％或在临床评价量表中达至少一个点)时，就认为用本文所述的组合物进行的治疗是“有效”的。尽管症状明显不同，取决于所针对的疾病或障碍，但是可以由普通有技术的临床医生或技术人员进行测定。这些症状可以通过例如以下方法来测定：通过监测疾病或障碍的一种或多种生化指标水平(例如疾病相关的酶或代谢物水平，受累细胞数量等)、通过监测身体表现(例如炎症、肿瘤大小等)、或者通过可接受的临床评价量表(例如扩大失能状态量表(Expanded Disability Status Scale)(用于多发性硬化)、Irvine炎性肠病问卷调查表(32点评价表，评价有关肠功能、全身症状、社交功能和情感状态的生活质量-评分范围为32-224，评分越高表明生活质量越好)、类风湿性关节炎生活质量量表、或本领域已知的其它可接受的临床评价量表。疾病或障碍症状持续(例如一天以上，优选超过一天)下降达至少10％或者在给定临床量表上下降达一个点或多个点，就表明是“有效”治疗。同样，相对于未经组合物治疗的类似个体(人或动物模型)的所述症状而言，当一种或多种症状的发作或严重程度被延迟、减少或消除时，用本文所述的组合物进行预防就是“有效”的。When one or more symptoms are reduced (e.g., Treatment with a composition described herein is considered "effective" when the score reaches at least 10% or at least one point on a clinical evaluation scale. Symptoms, although distinctly different, depending on the disease or disorder addressed, can be determined by an ordinarily skilled clinician or technician. These symptoms can be measured, for example, by monitoring the levels of one or more biochemical markers of the disease or disorder (e.g., levels of disease-associated enzymes or metabolites, number of affected cells, etc.), by monitoring physical manifestations (e.g., inflammation, tumor size, etc.), or by an acceptable clinical assessment scale (e.g., Expanded Disability Status Scale (for multiple sclerosis), Irvine Inflammatory Bowel Disease Questionnaire (32-point scale, rating Quality of life for bowel function, general symptoms, social functioning, and emotional status—scores range from 32 to 224, with higher scores indicating better quality of life), the Rheumatoid Arthritis Quality of Life Scale, or other known in the art Acceptable Clinical Evaluation Scale. Sustained (e.g., more than one day, preferably more than one day) reduction in symptoms of the disease or disorder by at least 10% or by one or more points on a given clinical scale is considered "effective" Treatment. Likewise, when the onset or severity of one or more symptoms is delayed, reduced or eliminated relative to said symptoms in a similar individual (human or animal model) not treated with the composition, the Prophylaxis is "effective" with the composition described above.
含有本发明的拮抗剂(例如配体)或其合剂的组合物可用于预防和治疗装置，以帮助改变、钝化、杀伤或去除哺乳动物的所选靶细胞群体。另外，本文所述的所选多肽库可用于离体或体外选择性杀伤、耗尽或从异质细胞群体中有效去除靶细胞群体。可以按照标准技术，将来自哺乳动物的血在离体情况下与配体(例如抗体、细胞表面受体或其结合蛋白)混合，由此杀伤或从血液中除去不想要的细胞，再回输到哺乳动物体内。Compositions containing antagonists (eg, ligands) of the invention, or cocktails thereof, are useful in prophylactic and therapeutic devices to help alter, inactivate, kill or remove selected target cell populations in mammals. Additionally, selected polypeptide repertoires described herein can be used ex vivo or in vitro to selectively kill, deplete, or efficiently remove target cell populations from heterogeneous cell populations. Blood from mammals can be mixed ex vivo with ligands (such as antibodies, cell surface receptors, or their binding proteins) to kill or remove unwanted cells from the blood prior to reinfusion, according to standard techniques into mammals.
含有本发明的拮抗剂(例如配体)的组合物可用于预防和治疗装置，以帮助改变、钝化、杀伤或去除哺乳动物的所选靶细胞。Compositions containing antagonists (eg, ligands) of the invention are useful in prophylactic and therapeutic devices to help alter, inactivate, kill or remove selected target cells in mammals.
可以给予TNFR1拮抗剂(例如配体、dAb单体)和/或与一种或多种其它治疗药或活性药一起配制。当TNFR1拮抗剂(例如配体、dAb单体)与其它治疗药一起给予时，TNFR1拮抗剂可以在给予其它药物之前、同时或之后给予。通常，TNFR1拮抗剂(例如配体、dAb单体)和其它药物的给予方式能提供重叠疗效。TNFRl antagonists (eg, ligands, dAb monomers) can be administered and/or formulated with one or more other therapeutic or active agents. When an antagonist of TNFRl (eg, a ligand, a dAb monomer) is administered with other therapeutic agents, the antagonist of TNFRl can be administered before, simultaneously with, or after the administration of the other agent. Often, TNFRl antagonists (eg, ligands, dAb monomers) and other agents are administered in a manner that provides overlapping therapeutic effects.
在一个实施方案中，本发明是治疗、抑制或预防慢性炎性疾病的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In one embodiment, the invention is a method of treating, inhibiting or preventing a chronic inflammatory disease comprising administering to a mammal in need thereof a therapeutically effective amount of a TNFR1 antagonist (eg, a ligand comprising a dAb monomer that binds TNFR1 ).
在一个实施方案中，本发明是治疗、抑制或预防关节炎(例如类风湿性关节炎、青少年类风湿性关节炎、关节强硬性脊椎炎、银屑病性关节炎)的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In one embodiment, the invention is a method of treating, inhibiting or preventing arthritis (e.g. rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis) comprising A therapeutically effective amount of a TNFRl antagonist (eg, a ligand comprising a dAb monomer that binds TNFRl) is administered to a mammal in need thereof.
在另一个实施方案中，本发明是治疗、抑制或预防银屑病的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In another embodiment, the invention is a method of treating, inhibiting or preventing psoriasis comprising administering to a mammal in need thereof a therapeutically effective amount of a TNFR1 antagonist (eg, a ligand comprising a dAb monomer that binds TNFR1 ).
在另一个实施方案中，本发明是治疗、抑制或预防炎性肠病(例如节段性回肠炎、溃疡性结肠炎)的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In another embodiment, the present invention is a method of treating, inhibiting or preventing inflammatory bowel disease (e.g. Crohn's disease, ulcerative colitis) comprising administering to a mammal in need thereof a therapeutically effective amount of a TNFR1 antagonist An agent (eg, a ligand comprising a dAb monomer that binds TNFR1).
在另一个实施方案中，本发明是治疗、抑制或预防慢性阻塞性肺病(例如慢性支气管炎、慢性阻塞性支气管炎、肺气肿)的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In another embodiment, the present invention is a method of treating, inhibiting or preventing chronic obstructive pulmonary disease (e.g. chronic bronchitis, chronic obstructive bronchitis, emphysema) comprising administering to a mammal in need thereof a therapeutically effective amount of An antagonist of TNFR1 (eg, a ligand comprising a dAb monomer that binds TNFR1).
在另一个实施方案中，本发明是治疗、抑制或预防肺炎(例如细菌性肺炎，例如葡萄球菌性肺炎)的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In another embodiment, the invention is a method of treating, inhibiting or preventing pneumonia (e.g., bacterial pneumonia, such as staphylococcal pneumonia), the method comprising administering to a mammal in need thereof a therapeutically effective amount of a TNFR1 antagonist (e.g., comprising Ligands of dAb monomers that bind TNFR1).
本发明提供治疗、抑制或预防除了慢性阻塞性肺病以外的其它肺病和肺炎的方法。按照本发明可治疗、抑制或预防的其它肺病包括例如囊性纤维化和哮喘(例如甾体抵抗型哮喘)。因此，在另一个实施方案中，本发明是治疗、抑制或预防肺病(例如囊性纤维化、哮喘)的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。The present invention provides methods of treating, suppressing or preventing lung diseases and pneumonia other than chronic obstructive pulmonary disease. Other lung diseases that may be treated, inhibited or prevented according to the present invention include, for example, cystic fibrosis and asthma (eg, steroid-resistant asthma). Accordingly, in another embodiment, the present invention is a method of treating, inhibiting or preventing a lung disease (e.g. cystic fibrosis, asthma) comprising administering to a mammal in need thereof a therapeutically effective amount of a TNFR1 antagonist (e.g. comprising Ligands of dAb monomers that bind TNFR1).
在具体的实施方案中，TNFR1拮抗剂是通过肺部给予，例如通过吸入(例如支气管内、鼻内或口腔吸入、鼻内滴剂)或通过系统给予(例如胃肠外、静脉内、肌内、腹膜内、皮下)。In specific embodiments, the TNFRl antagonist is administered pulmonary, such as by inhalation (e.g., intrabronchial, intranasal or buccal inhalation, intranasal drops) or by systemic administration (e.g., parenteral, intravenous, intramuscular , intraperitoneal, subcutaneous).
在另一个实施方案中，本发明是治疗、抑制或预防脓毒性休克的方法，该方法包括给予有需要的哺乳动物治疗有效量的TNFR1拮抗剂(例如包含能结合TNFR1的dAb单体的配体)。In another embodiment, the invention is a method of treating, inhibiting or preventing septic shock comprising administering to a mammal in need thereof a therapeutically effective amount of an antagonist of TNFR1 (eg, a ligand comprising a dAb monomer that binds TNFR1 ).
在本发明的第二格局的再一方面，本发明提供组合物，所述组合物包含闭合构象多特异性配体和药学上可接受的载体、稀释剂或赋形剂，所述配体是通过本发明的方法获得的。In yet another aspect of the second aspect of the present invention, the present invention provides a composition comprising a closed conformation multispecific ligand and a pharmaceutically acceptable carrier, diluent or excipient, the ligand being Obtained by the method of the present invention.
此外，本发明提供用“闭合构象多特异性配体”或本发明的组合物治疗疾病的方法。Furthermore, the invention provides methods of treating diseases with "closed conformation multispecific ligands" or compositions of the invention.
在本发明的一个优选实施方案中，所述疾病是癌症或炎性疾病，例如类风湿性关节炎、哮喘或节段性回肠炎。In a preferred embodiment of the invention, the disease is cancer or an inflammatory disease, such as rheumatoid arthritis, asthma or Crohn's disease.
在本发明的第二格局的另一方面，本发明提供诊断方法，包括用本发明的闭合构象多特异性配体或组合物诊断疾病。因此，一般而言，可以利用分析物与闭合构象多特异性配体的结合来替代某种试剂(agent)，所述试剂在替代时导致信号产生。例如，分析物(第二抗原)的结合可替代酶(第一抗原)结合抗体，提供了免疫测定的基础，尤其是当酶通过其活性部位保持抗体时。In another aspect of the second aspect of the invention, the invention provides methods of diagnosis comprising the diagnosis of a disease using a closed conformation multispecific ligand or composition of the invention. Thus, in general, binding of an analyte to a closed conformation multispecific ligand can be used to displace an agent which, upon displacement, results in signal generation. For example, binding of an analyte (second antigen) may provide the basis for an immunoassay instead of binding of an enzyme (primary antigen) to an antibody, especially if the enzyme retains the antibody via its active site.
因此，本发明提供检测靶分子存在的方法，该方法包括：Accordingly, the present invention provides a method of detecting the presence of a target molecule, the method comprising:
(a)提供结合某种试剂的闭合构象多特异性配体，所述配体对靶分子和该试剂具有特异性，其中与配体结合的试剂导致可检测信号的产生，当替代配体时；(a) providing a closed conformation multispecific ligand that binds an agent that is specific for a target molecule and the agent, wherein the agent bound to the ligand results in the generation of a detectable signal, when displacing the ligand ;
(b)将闭合构象多特异性配体暴露给靶分子；和(b) exposing the closed conformation multispecific ligand to the target molecule; and
(c)检测试剂替代而产生的信号。(c) Signals generated by detection of reagent substitution.
根据本发明的第二格局的以上方面，最好试剂是酶，当与闭合构象多特异性配体结合时所述酶是无活性的。或者，试剂可以是选自以下的任何一种或多种物质：酶的底物和荧光分子、发光分子或显色分子，所述分子当与配体结合是无活性的或者被猝灭。According to the above aspect of the second aspect of the invention, it is preferred that the agent is an enzyme which is inactive when bound to a closed conformation multispecific ligand. Alternatively, the reagent may be any one or more selected from substrates for enzymes and fluorescent, luminescent or chromogenic molecules that are inactive or quenched when bound to a ligand.
另外，本文所述的所选多肽库可用于离体或体外选择性杀伤、耗尽或从异质细胞群体中有效去除靶细胞群体。可以按照标准技术，将来自哺乳动物的血在离体情况下与配体(例如抗体、细胞表面受体或其结合蛋白)混合，由此杀伤或从血液中除去不想要的细胞，再回输到哺乳动物体内。Additionally, selected polypeptide repertoires described herein can be used ex vivo or in vitro to selectively kill, deplete, or efficiently remove target cell populations from heterogeneous cell populations. Blood from mammals can be mixed ex vivo with ligands (such as antibodies, cell surface receptors, or their binding proteins) to kill or remove unwanted cells from the blood prior to reinfusion, according to standard techniques into mammals.
实施例Example
下面的实施例进一步描述了本发明，这些实施例仅用于说明目的。为了给dAb命名，本文所用的人TNFα称为TAR1，人TNFα受体1(p55受体)称为TAR2。The invention is further described by the following examples which are provided for illustrative purposes only. For the purpose of naming the dAbs, human TNFa receptor 1 (p55 receptor) is referred to as TAR2 as used herein.
实施例1.针对人血清白蛋白(HSA)和β-半乳糖苷酶(β-gal)的双特异性scFv抗体(K8)的选择Example 1. Selection of bispecific scFv antibody (K8) against human serum albumin (HSA) and β-galactosidase (β-gal)
该实施例说明了制备针对β-gal和HAS的双特异性抗体的方法，其中选择了与种系(dummy)VH区连接的Vκ可变区库用于结合β-gal，并且选择了与种系(dummy)Vκ区连接的VH可变区库，用于结合HAS。然后将所选的可变VH HSA和Vκ β-gal区结合在一起，选择结合β-gal和HSA的抗体。HAS是人血中存在的半衰期延长蛋白。This example illustrates a method for making a bispecific antibody directed against β-gal and HAS, wherein a repertoire of Vκ variable regions linked to a germline (dummy)VH region is selected for binding β-gal and selected for binding to β-gal. Repertoire ofVH variable domains linked by dummy VK domains for binding to HAS. The selected variableVH HSA and Vκ β-gal regions are then combined and antibodies are selected for binding β-gal and HSA. HAS is a half-life extending protein present in human blood.
本实验中使用了4个人类噬菌体抗体文库。Four human phage antibody libraries were used in this experiment.
文库1：种系Vκ/DVT VH 8.46×107Library 1: Germline Vκ/DVTVH 8.46×107
文库2：种系Vκ/NNK VH 9.64×107Library 2: Germline Vκ/NNKVH 9.64×107
文库3：种系VH/DVT Vκ 1.47×108Library 3: GermlineVH /DVT Vκ 1.47×108
文库4：种系VH/NNK Vκ 1.45×108Library 4: GermlineVH /NNK Vκ 1.45×108
所有文库都基于VH(V3-23/DP47和JH4b)和Vκ(O12/O2/DPK9和Jκ1)的单一人构架，其中侧链多样性结合到互补决定区(CDR2和CDR3)中。All libraries are based on a single human framework ofVH (V3-23/DP47 andJH4b ) and Vκ (O12/O2/DPK9 and Jκ1) with side chain diversity incorporated into complementarity determining regions (CDR2 and CDR3).
文库1和文库2含有dummy Vκ序列，而VH序列在位置H50、H52、H52a、H53、H55、H56、H58、H95、H96、H97和H98(DVT或NNK分别编码)上具有多样性(图1)。文库3和文库4含有dummy VH序列，Vκ序列在位置L50、L53、L91、L92、L93、L94和L96 (DVT或NNK分别编码)上具有多样性(图1)。文库呈现噬菌粒pIT2/ScFv形式(图2)并且已经预选出了所述文库对蛋白A和蛋白L通用配体的结合性，使得非选择性文库中的大部分克隆都是功能性的。以上所示的文库大小对应于预选后的大小。在针对抗原进行选择前，将文库1和文库2混合，得到单一VH/dummy Vκ文库，将文库3和文库4混合，形成单一Vκ/dummy VH文库。
对β-gal进行3轮选择，使用Vκ/Dummy VH文库，并对HSA进行3轮选择，使用VH/dummy Vκ文库。就β-gal而言，噬菌体效价从第一轮的1.1×106到第三轮的2.0×108。就HAS而言，噬菌体效价从第一轮的2×104到第三轮的1.4×109。按照Griffith等(1993)所述，进行选择，除了使用KM13辅助噬菌体(其含有蛋白酶切位点在D2和D3区的pIII蛋白)而且噬菌体用1mg/ml胰蛋白酶的PBS进行洗脱以外。加入胰蛋白酶，通过在c-myc标记的切割，切割来自辅助噬菌体(而不是来自噬菌粒)的pIII蛋白，洗脱结合scFv-噬菌体融合体(图2)，因而进一步富集了噬菌体表达的功能性scFvs，并相应减低了背景(Kristensen和Winter，Folding&Design 3：321-328，1998年7月9日)。使用浓度100μg/ml的HSA或β-gal包被的免疫管(Immunotube)，进行选择。Three rounds of selection were performed on β-gal using the VK/DummyVH library, and three rounds of selection were performed on HSA using theVH /dummy VK library. For β-gal, the phage titers ranged from 1.1×106 in the first round to 2.0×108 in the third round. For HAS, the phage titer ranged from 2×104 in the first round to 1.4×109 in the third round. Selection was performed as described by Griffith et al. (1993), except that the KM13 helper phage (which contains the pill protein with protease sites in the D2 and D3 regions) was used and the phage were eluted with 1 mg/ml trypsin in PBS. Addition of trypsin elutes bound scFv-phage fusions by cleaving the pIII protein from the helper phage (rather than from the phagemid) by cleavage at the c-myc marker (Figure 2), thus further enriching for phage-expressed Functional scFvs with corresponding background reduction (Kristensen and Winter, Folding & Design 3:321-328, Jul. 9, 1998). Selection was performed using HSA or β-gal-coated immunotubes (Immunotube) at a concentration of 100 μg/ml.
为了检查结合，用单克隆噬菌体ELISA筛选来自各选择的第三轮的24个菌落。按照Harrison等(Methods Enzymol.1996；267：83-109)所述，产生噬菌体颗粒。96孔ELISA板用100μl 10μg/ml HSA或β-gal的PBS，在4℃包被过夜。按照标准ELISA方案(Hoogenboom等，1991)，采用结合噬菌体与抗M13-HRP缀合物的检测。对克隆进行选择，得到50μl上清液中的ELISA信号大于1.0。To check binding, 24 colonies from the third round of each selection were screened by monoclonal phage ELISA. Phage particles were generated as described by Harrison et al. (Methods Enzymol. 1996; 267:83-109). A 96-well ELISA plate was coated with 100 μl of 10 μg/ml HSA or β-gal in PBS overnight at 4°C. Detection of bound phage and anti-M13-HRP conjugate was employed following standard ELISA protocols (Hoogenboom et al., 1991). Clones were selected for an ELISA signal greater than 1.0 in 50 [mu]l supernatant.
然后，采用QIAprep Spin Miniprep试剂盒(Qiagen)，用针对HAS而选择的VH/dummy Vκ文库以及针对β-gal而选择的Vκ/dummy VH文库制备DNA制备物。为了得到大部分多样性，从3轮选择的每一轮制备DNA制备物，再合在一起用于每种抗原。再将DNA制备物用SalI/NotI在37℃消化过夜。接着对片段进行凝胶纯化，将来自针对β-gal而选择的Vκ/dummy VH文库的Vκ链，连接在针对HAS而选择的VH/dummy Vκ文库的dummy Vκ链的位置上，得到3.3×109个克隆的文库。DNA preps were then prepared using theVH /dummy VK library selected for HAS and the VK/dummy VH library selected for β-galusing the QIAprep Spin Miniprep kit (Qiagen). To get the most diversity, DNA preparations from each of the 3 rounds of selection were prepared and pooled together for each antigen. The DNA preparation was then digested overnight at 37°C with SalI/NotI. Then the fragment was gel purified, and the Vκ chain from the Vκ/dummyVH library selected for β-gal was connected to the position of the dummy Vκ chain of theVH /dummy Vκ library selected for HAS to obtain 3.3 Library of ×109 clones.
然后，针对HSA(第一轮)和β-gal(第二轮)对该文库进行HSA/β-gal选择，或者针对β-gal(第一轮)和HSA(第二轮)对该文库进行β-gal/HSA选择。如上所述进行选择。在第二轮后的每种情况下，通过单克隆噬菌体ELISA(如上所述)并通过可溶性scFv片段的ELISA，测定48个克隆与HSA和β-gal的结合。按照Harrison等(1996)和标准ELISA方案(Hoogenboom等(1991)Nucleic Acids Res.，19：4133)所述，产生可溶性抗体片段，只是采用2％吐温(Tween)/PBS作为封闭缓冲液，以及用蛋白L-HRP检测结合scFv。来自HSA/β-gal选择的3个克隆(E4、E5和E8)和来自β-gal/HSA选择的2个克隆(K8和K10)能结合这两种抗原。按照Ignatovich等(1999)J.Mol.Biol.，1999年11月26日；294(2)：457-65所述，使用引物LMB3和pHENseq，对来自这些克隆的scFv进行PCR扩增并测序。序列分析表明，所有克隆都是相同的。因此，只选择一个编码双特异性抗体的克隆(K8)进行进一步工作(图3)。This library was then subjected to HSA/β-gal selection against HSA (first round) and β-gal (second round) or against β-gal (first round) and HSA (second round) β-gal/HSA selection. Select as above. After the second round in each case, 48 clones were assayed for binding to HSA and [beta]-gal by monoclonal phage ELISA (as described above) and by ELISA of soluble scFv fragments. Soluble antibody fragments were generated as described by Harrison et al. (1996) and the standard ELISA protocol (Hoogenboom et al. (1991) Nucleic Acids Res., 19:4133), except that 2% Tween/PBS was used as blocking buffer, and Bound scFvs were detected with protein L-HRP. Three clones (E4, E5 and E8) from the HSA/β-gal selection and 2 clones (K8 and K10) from the β-gal/HSA selection were able to bind both antigens. scFv from these clones were PCR amplified and sequenced as described by Ignatovich et al. (1999) J. Mol. Biol., 1999
实施例2.K8抗体结合特性的表征Example 2. Characterization of K8 Antibody Binding Properties
首先，K8抗体的结合特性通过单克隆噬菌体ELISA来表征。96孔板用100μl 10μg/ml HSA和β-gal以及碱性磷酸酶(APS)、牛血清白蛋白(BSA)、花生凝集素、溶菌酶和细胞色素c(以检查交叉反应)的PBS在4℃包被过夜。按照Harrison等(1996)所述，将来自K8克隆的噬菌粒用KM13拯救，含有噬菌体的上清液(50μl)直接进行测定。按照标准ELISA方案(Hoogenboom等，1991)，用抗M13-HRP缀合物检测结合噬菌体。当在噬菌体表面展示时，发现双特异性K8抗体能结合HSA和β-gal，其吸收信号大于1.0(图4)。也观察到与BSA的强烈结合(图4)。因为HSA和BSA在氨基酸水平上具有76％同源性，所以毫不奇怪，K8抗体能识别这些结构相关的蛋白质。未检出与其它蛋白质的交叉反应性(图4)。First, the binding properties of the K8 antibody were characterized by monoclonal phage ELISA. 96-well plate with 100 μl of 10 μg/ml HSA and β-gal along with alkaline phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin, lysozyme and cytochrome c (to check for cross-reactivity) in PBS at 4 °C overnight. Phagemids from the K8 clone were rescued with KM13 as described by Harrison et al. (1996), and the phage-containing supernatants (50 [mu]l) were assayed directly. Bound phage were detected with anti-M13-HRP conjugate following standard ELISA protocol (Hoogenboom et al., 1991). When displayed on the surface of phage, the bispecific K8 antibody was found to bind HSA and β-gal with an absorbance signal greater than 1.0 (Figure 4). Strong binding to BSA was also observed (Figure 4). Since HSA and BSA share 76% homology at the amino acid level, it is not surprising that the K8 antibody recognizes these structurally related proteins. No cross-reactivity with other proteins was detected (Figure 4).
其次，在可溶性scFv ELISA中检测了K8抗体的结合特性。按照Harrison等(1996)，可溶性scFv片段是由IPTG诱导产生的。为了测定K8 scFv的表达水平，按照Harlow和Lane(Antibodies：aLaboratory Manual，(1988)Cold Spring Harbor)所述，用蛋白A-琼脂糖Seph柱，从50ml诱导的上清液中纯化出可溶性抗体片段。按照Sambrook等(1989)所述，测定OD280并计算蛋白质浓度。上清液中产生的K8 scFv为19mg/L。Second, the binding properties of the K8 antibody were tested in a soluble scFv ELISA. According to Harrison et al. (1996), soluble scFv fragments were induced by IPTG. To determine expression levels of K8 scFv, soluble antibody fragments were purified from 50 ml of induced supernatant using protein A-sepharose Seph columns as described by Harlow and Lane (Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor) .OD280 was determined and protein concentration calculated as described by Sambrook et al. (1989). The K8 scFv produced in the supernatant was 19 mg/L.
再用已知浓度的K8抗体片段，进行可溶性scFv ELISA。96孔板用100μl HSA、BSA和β-gal以10μg/ml和100μl蛋白A的1μg/ml浓度包被。采用50μl K8 scFv的系列稀释液，结合抗体片段用蛋白L-HRP检测。ELISA结果证实K8抗体的双特异性(图5)。Soluble scFv ELISA was performed with known concentration of K8 antibody fragment. A 96-well plate was coated with 100 μl of HSA, BSA and β-gal at a concentration of 10 μg/ml and 100 μl of protein A at a concentration of 1 μg/ml. Using 50 μl of serial dilutions of K8 scFv, bound antibody fragments were detected with protein L-HRP. ELISA results confirmed the bispecificity of the K8 antibody (Figure 5).
为了证实与β-gal的结合是由Vκ区确定，与HSA/BSA的结合是由K8 scFv抗体的VH区确定，经SalI/NotI消化，将Vκ区从K8 scFvDNA上切下，并连接到经SalI/NotI消化的含有dummy VH链的pIT2载体中(图1和2)。所得克隆K8 Vκ/dummy VH的结合表征通过可溶性scFv ELISA进行分析。按照Harrison等(1996)，可溶性scFv片段是由IPTG诱导产生的，含有scFv的上清液(50μ)直接进行测定。按照实施例1所述，进行可溶性scFv ELISA，结合scFv用蛋白L-HRP测定。ELISA结果表明，该克隆仍能结合β-gal，而与BSA的结合已被消除掉(图6)。In order to confirm that the binding to β-gal is determined by the Vκ region, and the binding to HSA/BSA is determined by theVH region of the K8 scFv antibody, after digestion with SalI/NotI, the Vκ region was excised from the K8 scFvDNA and ligated into In the pIT2 vector containing the dummy VH chain digested by SalI/NotI (Figures 1 and 2). Binding characterization of the resulting clone K8 VK/dummyVH was analyzed by soluble scFv ELISA. According to Harrison et al. (1996), soluble scFv fragments were induced by IPTG and scFv-containing supernatants (50[mu]) were directly assayed. Soluble scFv ELISA was performed as described in Example 1, and binding scFv was determined with protein L-HRP. ELISA results showed that the clone could still bind to β-gal, but the binding to BSA had been eliminated (Fig. 6).
实施例3.针对抗原A和B的VH单域抗体的选择，以及针对抗原C和D的Vκ单域抗体的选择Example 3. Selection ofVH single domain antibodies against antigens A and B, and selection of Vκ single domain antibodies against antigens C and D
该实施例描述了针对抗原A和B的VH单域抗体，以及针对抗原C和D的Vκ单域抗体的制备方法，即通过在互补可变区不存在时，选择处女(virgin)单抗体可变区库对这些抗原的结合。This example describes the preparation ofVH single domain antibodies against antigens A and B, and Vκ single domain antibodies against antigens C and D, by selecting virgin single domain antibodies in the absence of complementary variable regions Binding of variable region repertoires to these antigens.
按照前述方法(参见实施例5，PCT/GB02/003014)，进行结合克隆的选择和表征。选择4个克隆进行进一步工作：Selection and characterization of binding clones were performed as described previously (see Example 5, PCT/GB02/003014). 4 clones were selected for further work:
VH1-抗A VHVH1-anti-A VH
VH2-抗B VHVH2-anti-BVH
VK1-抗C VκVK1-anti-C Vκ
VK2-抗D VκVK2-anti-DVκ
可以采用以上实施例1-3所述的方法，按照所述类似方式，产生包含VH区组合(即VH-VH配体)和VL区组合(VL-VL配体)的二聚体分子。The methods described in the above Examples 1-3 can be used to produce a combination ofVH regions (i.e.VH -VH ligands) andVL regions (VL -VL ligands) in a similar manner. dimer molecule.
实施例4.双特异性ScFv抗体的产生和表征(针对抗原A和B的VH1/VH2以及针对抗原C和D的VK1/VK2)Example 4. Generation and characterization of bispecific ScFv antibodies (VH1/VH2 against antigens A and B and VK1/VK2 against antigens C and D)
该实施例证明，可以通过将针对ScFv载体中各自抗原而选择的Vκ和VH单域结合起来，产生双特异性ScFv抗体(针对抗原A和B的VH1/VH2以及针对抗原C和D的VK1/VK2)。This example demonstrates that bispecific ScFv antibodies (VH1/VH2 against antigens A and B and VK1 against antigens C and D can be generated by combining Vκ andVH single domains selected against the respective antigens in a ScFv vector /VK2).
为了产生双特异性抗体VH1/VH2，经NcoI/XhoI消化，从可变区载体1上切下VH1单域(图7)并连接到经NcoI/XhoI消化的可变区载体2上(图7)，产生VH1/可变区载体2。通过PCR，使用引物，从可变区载体1扩增VH2单区，将SalI限制位点引入5′端并将NotI限制位点引入3′端。再将PCR产物用SalI/NotI消化并连接到经SalI/NotI消化的VH1/可变区载体2上，产生VH1/VH2/可变区载体2。To generate the bispecific antibody VH1/VH2, VH1 single domain was excised from variable region vector 1 (Fig. 7) digested with NcoI/XhoI and ligated into
VK1/VK2/可变区载体2以类似方式产生。按照前述可溶性ScFvELISA的方法(参见实施例6，PCT/GB02/003014)，测定所产生的VH1/VH2 ScFV和VK1/VK2 ScFv的双特异性。按照前述方法(参见实施例8，PCT/GB02/003014)，进行竞争性ELISA。VK1/VK2/
可能结果：Possible outcomes:
-VH1/VH2 ScFv能同时抗原A和B-VH1/VH2 ScFv can simultaneously antigen A and B
-VK1/VK2 ScFv能同时抗原C和D-VK1/VK2 ScFv can simultaneously antigen C and D
-VH1/VH2 ScFV结合是竞争性的(当结合抗原A时，VH1/VH2ScFv不能结合抗原B)- VH1/VH2 ScFv binding is competitive (when binding antigen A, VH1/VH2 ScFv cannot bind antigen B)
-VK1/VK2 ScFv结合是竞争性的(当结合抗原C时，VK1/VK2ScFv不能结合抗原D)。- VK1/VK2 ScFv binding is competitive (when binding antigen C, VK1/VK2 ScFv cannot bind antigen D).
实施例5.双特异性VH1/VH2 Fab和VK1/VK2 Fab的构建及其结合特性的分析Example 5. Construction of bispecific VH1/VH2 Fab and VK1/VK2 Fab and analysis of their binding properties
为了产生VH1/VH2 Fab，将VH1单域连接到经NcoI/XhoI消化的CH载体上(图8)，产生VH1/CH，再将VH2单域连接到经SalI/NotI消化的CK载体上(图9)，产生VH2/CK。按照前述方法(参见实施例8，PCT/GB02/003014)，用来自VH1/CH和VH2/CK的质粒DNA用于共转化感受态大肠杆菌细胞。To generate VH1/VH2 Fab, VH1 single domain was ligated into NcoI/XhoI digested CH vector (Fig. 8) to generate VH1/CH, and then VH2 single domain was ligated into SalI/NotI digested CK vector (Fig. 9), produce VH2/CK. Plasmid DNA from VH1/CH and VH2/CK was used to co-transform competent E. coli cells as described previously (see Example 8, PCT/GB02/003014).
按照前述方法(参见实施例8，PCT/GB02/003014)，含有VH1/CH和VH2/CK质粒的克隆再用IPTG诱导，产生可溶性VH1/VH2 Fab。According to the aforementioned method (see Example 8, PCT/GB02/003014), clones containing VH1/CH and VH2/CK plasmids were then induced with IPTG to produce soluble VH1/VH2 Fab.
VK1/VK2 Fab按类似方法产生。VK1/VK2 Fabs were generated similarly.
按照前述的竞争性ELISA(参见实施例8，PCT/GB02/003014)，测定所产生的Fab的结合特性。The binding properties of the Fabs produced were determined following the competition ELISA described above (see Example 8, PCT/GB02/003014).
可能结果：Possible outcomes:
-VH1/VH2 Fab能同时抗原A和B-VH1/VH2 Fab can simultaneously antigen A and B
-VK1/VK2 Fab能同时抗原C和D-VK1/VK2 Fab can simultaneously antigen C and D
-VH1/VH2 Fab结合是竞争性的(当结合抗原A时，VH1/VH2 Fab不能结合抗原B)- VH1/VH2 Fab binding is competitive (when binding antigen A, VH1/VH2 Fab cannot bind antigen B)
-VK1/VK2 Fab结合是竞争性的(当结合抗原C时，VK1/VK2 Fab不能结合抗原D)。- VK1/VK2 Fab binding is competitive (when binding antigen C, VK1/VK2 Fab cannot bind antigen D).
实施例6.使dAb二聚体螯合Example 6. Sequestration of dAb dimers
概述overview
用柔性多肽接头，产生dAb-接头-dAb形式的VH和VK同型二聚体。产生dAb接头-dAb形式的载体，其含有不同长度的甘氨酸-丝氨酸接头3U：(Gly4Ser)3(SEQ ID NO：199)、5U：(Gly4Ser)5(SEQ ID NO：629)、7U：(Gly4Ser)7(SEQ TD NO：630)。用接头的定向dAb上游和接头之后相应第二dAb的文库，产生二聚体文库，所述接头为：TAR1-5(VK)、TAR1-27(VK)、TAR2-5(VH)或TAR2-6(VK)。采用这一方法，选择新的二聚体dAb。通过ELISA和BIAcore研究，在细胞中和及受体结合测定中，检测二聚化对抗原结合的影响。TAR1-5和TAR1-27的二聚化导致结合亲和力及中和水平得到改善。Using a flexible polypeptide linker, VH and VK homodimers are generated in the form of dAb-linker-dAb. Generation of dAb linker-dAb format vectors containing glycine-serine linkers of
1.0方法1.0 method
1.1文库的产生1.1 Generation of library
1.1.1载体1.1.1 Carrier
消化pEDA3U、pEDA5U和pEDA7U载体，引入与dAb-接头-dAb形式相容的不同接头长度。对于pEDA3U，使有义和反义的73个碱基对寡聚接头退火，采用缓慢退火程序(95℃-5分钟，80℃-10分钟，70℃-15分钟，56℃-15分钟，42℃直到使用)，在含有0.1M NaCl、10mMTris-HCl(pH7.4)的缓冲液中，用XhoI和NotI限制位点进行克隆。接头包含3(Gly4Ser)单元和填充区，其位于SalI和NotI克隆位点之间(方案1)。为了降低单体dAb被噬菌体展示选择的可能性，设计的填充区包含3个终止密码子、SacI限制位点和移码突变，以便将该区放在读框之外，当第二dAb不存在时。对于pEDA5U和pEDA7U，因为所需接头长度，消化重叠寡聚接头，用于各载体，退火并用Klenow延伸。然后纯化片段，用合适酶消化，再用XhoI和NotI限制位点克隆。The pEDA3U, pEDA5U and pEDA7U vectors were digested to introduce different linker lengths compatible with the dAb-linker-dAb format. For pEDA3U, the sense and
方案1
1.1.2文库的制备1.1.2 Library preparation
用NcoI和XhoI限制位点，将对应于定向dAb的N-端V基因克隆到接头上游。VH基因具有现成的相容性位点，然而，克隆VK基因需要引入合适的限制位点。通过使用修饰的PCR引物(VK-DLIBF：5′cggccatggcgtcaacggacat(SEQ ID NO：377)；VK XholR：5′atgtgcgctcgagcgtttgattt 3′(SEQ ID NO：378))，在30次循环的PCR扩增中，使用SuperTaq(HTBiotechnology Ltd)和pfu turbo(Stratagene)的2∶1混合物，来达到这一点。这在5′端保留NcoI位点，同时破坏相邻SalI位点并在3′端引入XhoI位点。将5个定向dAb克隆到3个二聚体载体的每个中：TAR1-5(VK)、TAR1-27(VK)、TAR2-5(VH)、TAR2-6(VK)和TAR2-7(VK)。所有构建体都经序列分析得以证实。The N-terminal V gene corresponding to the targeted dAb was cloned upstream of the linker using NcoI and XhoI restriction sites. VH genes have readily available compatibility sites, however, cloning VK genes requires the introduction of appropriate restriction sites. By using modified PCR primers (VK-DLIBF: 5' cggccatggcgtcaacggacat (SEQ ID NO: 377); VK XholR: 5' atgtgcgctcgagcgtttgattt 3' (SEQ ID NO: 378)), in 30 cycles of PCR amplification, use A 2:1 mixture of SuperTaq (HTBiotechnology Ltd) and pfu turbo (Stratagene) was used to achieve this. This preserves the NcoI site at the 5' end, while destroying the adjacent SalI site and introducing an XhoI site at the 3' end. Five targeted dAbs were cloned into each of three dimeric vectors: TAR1-5(VK), TAR1-27(VK), TAR2-5(VH), TAR2-6(VK) and TAR2-7( VK). All constructs were confirmed by sequence analysis.
每个载体((pEDA3U、pEDA 5U和pEDA 7U)：TAR1-5(VK)、TAR1-27(VK)、TAR2-5(VH)或TAR2-6(VK))都具有接头上游的克隆定向dAb，将相应的第二dAb的文库克隆到接头之后。为了达到这一点，经PCR，从经过第一轮选择而回收的噬菌体扩增互补(complimentary)dAb文库，所述噬菌体可以是当TAR1-5或TAR1-27是定向dAb时、针对人TNFα的VK文库的第一轮选择而回收的噬菌体(在第一轮后约1×106多样性)，或者是当TAR2-5或TAR2-6分别是定向dAb时、针对人p55 TNF受体的VH或VK文库的第一轮选择而回收的噬菌体(在第一轮后都约为1×105多样性)。对于VK文库，使用引物，在30次循环的PCR扩增中，使用SuperTaq和pfu turbo的2∶1混合物，进行PCR扩增。使用引物，对VH文库进行PCR扩增，以便在基因的5′端引入SalI限制位点。dAb文库PCR用合适限制酶消化，连接到相应载体接头下游，使用SalI/NotI限制位点，并经电穿孔进入新鲜制备的感受态TG1细胞中。Each vector ((pEDA3U,
各文库所得效价如下：The titers obtained for each library are as follows:
TAR1-5：pEDA3U＝4×108，pEDA5U＝8×107，pEDA7U＝1×108TAR1-5: pEDA3U=4×108 , pEDA5U=8×107 , pEDA7U=1×108
TAR1-27：pEDA3U＝6.2×108，pEDA5U＝1×108，pEDA7U＝1×109TAR1-27: pEDA3U=6.2×108 , pEDA5U=1×108 , pEDA7U=1×109
TAR2h-5：pEDA3U＝4×107，pEDA5U＝2×108，pEDA7U＝8×107TAR2h-5: pEDA3U=4×107 , pEDA5U=2×108 , pEDA7U=8×107
TAR2h-6：pEDA3U＝7.4×108，pEDA5U＝1.2×108，pEDA7U＝2.2×108TAR2h-6: pEDA3U=7.4×108 , pEDA5U=1.2×108 , pEDA7U=2.2×108
1.2选择1.2 Selection
1.2.1 TNFα1.2.1 TNFα
用被动包被在免疫管上的人TNFα，进行选择。简而言之，免疫管用1-4ml所需抗原包被过夜。免疫管再用PBS洗涤3次，用2％奶粉/PBS封闭1-2小时，然后再用PBS洗涤3次。噬菌体溶液用2％奶粉/PBS稀释，在室温下孵育2小时。这些管再用PBS洗涤，噬菌体用1mg/ml胰蛋白酶-PBS洗脱。对于TAR1-5二聚体文库，研究了3种选择策略。第一轮选择在免疫管中进行，用1μg/ml或20μg/ml的人TNFα包被，再用PBS 0.1％吐温洗涤20次。TG1细胞用经过洗脱的噬菌体感染，测定效价(例如Marks等，J.Mol.Biol.，1991年12月5日；222(3)：581-97，Richmann等，Biochemistry，1993年8月31日；32(34)：8848-55)。Selection was performed with human TNF[alpha] passively coated on immunotubes. Briefly, immunotubes were coated overnight with 1-4ml of the desired antigen. The immunotube was washed 3 times with PBS, blocked with 2% milk powder/PBS for 1-2 hours, and then washed 3 times with PBS. The phage solution was diluted with 2% milk powder/PBS and incubated at room temperature for 2 hours. The tubes were washed again with PBS and the phage were eluted with 1 mg/ml trypsin-PBS. For the TAR1-5 dimer library, 3 selection strategies were investigated. The first round of selection was carried out in immunotubes, coated with 1 μg/ml or 20 μg/ml human TNFα, and then washed 20 times with PBS 0.1% Tween. TG1 cells were infected with eluted phages, and the titer was determined (for example, Marks et al., J.Mol.Biol., December 5, 1991; 222(3):581-97, Richmann et al., Biochemistry, August 1993 31;32(34):8848-55).
回收的效价为：The recovered potency is:
pEDA3U＝2.8×107(1μg/ml TNF)，1.5×108(20μg/ml TNF)，pEDA3U=2.8×107 (1 μg/ml TNF), 1.5×108 (20 μg/ml TNF),
pEDA5U＝1.8×107(1μg/ml TNF)，1.6×108(20μg/ml TNF)，pEDA5U=1.8×107 (1 μg/ml TNF), 1.6×108 (20 μg/ml TNF),
pEDA7U＝8×106(1μg/ml TNF)，7×107(20μg/ml TNF)。pEDA7U=8×106 (1 μg/ml TNF), 7×107 (20 μg/ml TNF).
用下面三种不同的方法进行第二轮选择。The second round of selection was performed using the following three different methods.
1.在免疫管中，20次洗涤并过夜孵育，接着再是10次洗涤。1. In immunotubes, 20 washes and overnight incubation followed by 10 washes.
2.在免疫管中，20次洗涤并在室温下、在洗涤缓冲液中与(1μg/mlTNFα)孵育1小时，接着再是10次洗涤。2. In immunotubes, 20 washes and incubation with (1 μg/ml TNFα) in wash buffer for 1 hour at room temperature, followed by 10 more washes.
3.在链霉抗生物素珠上，使用33pmole生物素化人TNFα进行选择(Henderikx等，2002，Selection of antibodies against biotinylatedantigens.Antibody Phage Display：Methods and protocols，O′Brien和Atkin(主编)，Humana Press)。挑取来自第二轮选择的单个克隆，接种到96孔板中，以2ml 96孔板的形式制备粗上清液制备物。3. On streptavidin beads, use 33 pmole biotinylated human TNFα for selection (Henderikx et al., 2002, Selection of antibodies against biotinylated antigens. Antibody Phage Display: Methods and protocols, O'Brien and Atkin (editors), Humana Press). Single clones from the second round of selection were picked and seeded into 96-well plates, and crude supernatant preparations were prepared in the form of 2 ml 96-well plates.
表1.Table 1.
对于TAR1-27，按照前述方法并经以下改进，进行选择。在用1μg/ml或20μg/ml人TNFα包被、并用PBS 0.1％吐温洗涤20次的免疫管中，进行第一轮选择。在20次洗涤、过夜孵育并再次20次洗涤的免疫管中，进行第二轮选择。挑取来自第二轮选择的单个克隆，接种到96孔板，以2ml 96孔板的形式制备粗上清液制备物。For TAR1-27, select according to the aforementioned method with the following modifications. The first round of selection was performed in immunotubes coated with 1 μg/ml or 20 μg/ml human TNFα and washed 20 times with PBS 0.1% Tween. In immunotubes with 20 washes, overnight incubation, and another 20 washes, a second round of selection was performed. Single clones from the second round of selection were picked, seeded into 96-well plates, and crude supernatant preparations were prepared in the form of 2 ml 96-well plates.
TAR1-27效价如下：TAR1-27 potency is as follows:
表2.Table 2.
1.2.2 TNF受体1(p55受体；TAR2)1.2.2 TNF receptor 1 (p55 receptor; TAR2)
仅对于TAR2h-5文库，按照前述方法进行选择。在用1μg/ml人p55 TNF受体或10μg/ml人p55 TNF受体、并用PBS 0.1％吐温洗涤20次、过夜孵育并再次20次洗涤的免疫管中，进行第三轮选择。挑取来自第二轮和第三轮选择的单个克隆，接种到96孔板，以2ml 96孔板的形式制备粗上清液制备物。For the TAR2h-5 library only, selection was performed as described above. A third round of selection was performed in immunotubes with 1 μg/ml human p55 TNF receptor or 10 μg/ml human p55 TNF receptor, washed 20 times with PBS 0.1% Tween, incubated overnight and washed 20 times again. Single clones from the second and third rounds of selection were picked and seeded into 96-well plates, and crude supernatant preparations were prepared in the form of 2 ml 96-well plates.
TAR2h-5效价如下：TAR2h-5 titers are as follows:
表3.table 3.
1.3 筛选1.3 Screening
从每个3U、5U和7U文库中，适当时用不同选择方法挑取来自第二轮或第三轮选择的单个克隆。在37℃，将克隆在含有100μg/ml氨苄青霉素和1％葡萄糖的2xTY培养过夜。该培养物的1/100稀释液接种到2ml含有100μg/ml氨苄青霉素和0.1％葡萄糖的2xTY的2ml96孔板形式中，在37℃振荡培养直到OD600达到约0.9。然后，培养物用1mM IPTG在30℃诱导过夜。在sorval板式离心机中以4000rpm离心15分钟，使上清液澄清。上清液制备物用于初步筛选。From each of the 3U, 5U and 7U libraries, individual clones from the second or third round of selection were picked using different selection methods as appropriate. Colonies were grown overnight at 37°C in 2xTY containing 100 μg/ml ampicillin and 1% glucose. A 1/100 dilution of this culture was inoculated into a 2ml 96-well plate format in 2ml 2xTY containing 100 μg/ml ampicillin and 0.1% glucose and grown with shaking at 37°C until theOD600 reached about 0.9. Then, cultures were induced overnight at 30°C with 1 mM IPTG. The supernatant was clarified by centrifugation at 4000 rpm for 15 minutes in a Sorval plate centrifuge. Supernatant preparations were used for primary screening.
1.3.1 ELISA1.3.1 ELISA
通过蛋白A/L ELISA或抗原ELISA，对二聚体重组蛋白质与单体的结合活性进行比较。简而言之，96孔板用抗原或蛋白A/L在4℃包被过夜。该板用0.05％吐温-PBS洗涤，用2％吐温-PBS封闭2小时。将样品加到该板上，在室温下孵育1小时。洗涤该板，与第二试剂在室温下一起孵育1小时。洗涤该板，用TMB底物显色。蛋白A/L-HRP或India-HRP用作第二试剂。对于抗原ELISA，所用抗原浓度为1μg/ml的PBS，用于人TNFα和人THF受体1。因为大多数情况下存在定向dAb，二聚体给出阳性ELISA信号，因此解离速率经BIAcore检测。Compare the binding activity of the dimer recombinant protein to the monomer by protein A/L ELISA or antigen ELISA. Briefly, 96-well plates were coated overnight at 4°C with antigen or protein A/L. The plate was washed with 0.05% Tween-PBS and blocked with 2% Tween-PBS for 2 hours. Samples were added to the plate and incubated for 1 hour at room temperature. The plate was washed and incubated with the second reagent for 1 hour at room temperature. The plate was washed and developed with TMB substrate. Protein A/L-HRP or India-HRP was used as the second reagent. For antigen ELISA, the antigen concentration used was 1 [mu]g/ml in PBS for human TNF[alpha] and
1.3.2 BIAcore1.3.2 BIAcore
对TAR1-5和TAR2h-5克隆进行BIAcore分析。对于筛选，人TNFα以高密度(约10000RU)偶联到CM5芯片上。50μl人TNFα(50μg/ml)以5μl分钟的乙酸盐缓冲液(pH5.5)偶联到芯片上。分析后的芯片不能用标准方法再生，因为人TNFα不稳定，因此，每次分析样品之后，芯片用缓冲液洗涤10分钟。对于TAR1-5，用BIAcore筛选来自第二轮选择的克隆上清液。BIAcore analysis was performed on TAR1-5 and TAR2h-5 clones. For screening, human TNFα was coupled to a CM5 chip at high density (approximately 10000 RU). 50 μl of human TNFα (50 μg/ml) was coupled to the chip in 5 μl min of acetate buffer (pH 5.5). The analyzed chips could not be regenerated by standard methods because of the instability of human TNF[alpha], therefore, the chips were washed with buffer for 10 min after each analysis of samples. For TAR1-5, clone supernatants from the second round of selection were screened with BIAcore.
采用以下选择方法，从所得每个3U、5U和7U文库中筛选出48个克隆：48 clones were screened from each of the resulting 3U, 5U and 7U libraries using the following selection method:
R1：1μg/ml人TNFα免疫管，R2：1μg/ml人TNFα免疫管，过夜洗涤。R1: 1 μg/ml human TNFα immune tube, R2: 1 μg/ml human TNFα immune tube, washed overnight.
R1：20μg/ml人TNFα免疫管，R2：20/xg/ml人TNFα免疫管，过夜洗涤。R1: 20μg/ml human TNFα immune tube, R2: 20/xg/ml human TNFα immune tube, washed overnight.
R1：1μg/ml人TNFα免疫管，R2：33pmoles生物素化人TNFα在珠子上。R1: 1 μg/ml human TNFα immunotube, R2: 33 pmoles of biotinylated human TNFα on beads.
R1：20μg/ml人TNFa免疫管，R2：33pmoles生物素化人TNFα在珠子上。R1: 20 μg/ml human TNFα immunotube, R2: 33 pmoles biotinylated human TNFα on beads.
对于筛选，人p55 TNF受体以高密度(约4000RU)偶联到CM5芯片上。100μl人p55 TNF受体(10μg/ml)以5μl/分钟的乙酸盐缓冲液(pH5.5)偶联到芯片上。检查标准再生条件(甘氨酸pH2或pH3)，但是在每种情况下，抗原与TNFα一起从芯片表面除去，因此每次分析样品之后，芯片用缓冲液洗涤10分钟。For screening, the human p55 TNF receptor was coupled to a CM5 chip at high density (approximately 4000RU). 100 μl of human p55 TNF receptor (10 μg/ml) was coupled to the chip at 5 μl/min in acetate buffer (pH 5.5). Standard regeneration conditions (
对于TAR2-5，筛选了来自第二轮选择的克隆上清液。For TAR2-5, clonal supernatants from the second round of selection were screened.
采用以下选择方法，从每个3U、5U和7U文库中筛选出48个克隆：48 clones were screened from each of the 3U, 5U and 7U libraries using the following selection method:
R1：1μg/ml人p55 TNF受体免疫管，R2：1μg/ml人p55 TNF受体免疫管，过夜洗涤。R1: 1 μg/ml human p55 TNF receptor immune tube, R2: 1 μg/ml human p55 TNF receptor immune tube, washed overnight.
R1：10μg/ml人p55 TNF受体免疫管，R2：10μg/ml人p55 TNF受体免疫管，过夜洗涤。R1: 10 μg/ml human p55 TNF receptor immune tube, R2: 10 μg/ml human p55 TNF receptor immune tube, washed overnight.
1.3.3 受体和细胞测定1.3.3 Receptor and cellular assays
在受体测定中，二聚体中和能力的测定如下进行：In the receptor assay, the determination of the neutralization capacity of the dimer is performed as follows:
受体结合receptor binding
测定抗TNF dAb抑制TNF与重组TNF受体1(p55)结合的能力。简而言之，Maxisorp板与30mg/ml抗人Fc小鼠单克隆抗体(Zymed，SanFrancisco，USA)一起孵育过夜。各孔用含0.05％吐温-20的磷酸缓冲盐溶液(PBS)洗涤，再用1％BSA/PBS封闭，然后与100ng/ml TNF受体1Fc融合蛋白(R&D系统，Minneapolis，USA)一起孵育。抗TNFdAb与TNF(其加入到洗涤孔中，终浓度为10ng/ml)混合。TNF结合的测定如下：用0.2mg/ml生物素化抗TNF抗体(HyCult biotechnology，Uben，Netherlands)，接着用1∶500稀释的辣根过氧化物酶标记的链霉抗生物素(Amersham Biosciences，UK)，再与TMB底物(KPL，Gaithersburg，USA)一起孵育。加入HCl终止反应，在450nm读出吸光度。抗TNF dAb活性导致TNF结合的降低，因此，与仅用TNF对照相比，导致吸光度下降。The ability of anti-TNF dAbs to inhibit the binding of TNF to recombinant TNF receptor 1 (p55) was determined. Briefly, Maxisorp plates were incubated overnight with 30 mg/ml anti-human Fc mouse monoclonal antibody (Zymed, San Francisco, USA). Each well was washed with phosphate-buffered saline (PBS) containing 0.05% Tween-20, then blocked with 1% BSA/PBS, and then incubated with 100ng/ml TNF receptor 1Fc fusion protein (R&D Systems, Minneapolis, USA) . Anti-TNFdAb was mixed with TNF which was added to the washed wells at a final concentration of 10 ng/ml. TNF binding was determined as follows: 0.2 mg/ml biotinylated anti-TNF antibody (HyCult biotechnology, Uben, Netherlands), followed by 1:500 dilution of horseradish peroxidase-labeled streptavidin (Amersham Biosciences, UK), and then incubated with TMB substrate (KPL, Gaithersburg, USA). The reaction was terminated by adding HCl, and the absorbance was read at 450 nm. Anti-TNF dAb activity results in a decrease in TNF binding and, therefore, in a decrease in absorbance compared to TNF-only controls.
L929 细胞毒性测定L929 Cytotoxicity Assay
在小鼠L929成纤维细胞上，也测定了抗TNF dAb中和TNF细胞毒活性的能力(Evans，T.(2000)Molecular Biotechnology 15，243-248)。简而言之，将接种在微量滴定板上的L929细胞与抗TNF dAb、100pg/ml TNF和1mg/ml放线菌素D(Sigma，Poole，UK)一起孵育过夜。细胞存活率通过在490nm读出吸光度而测定，接着与[3-(4，5-二甲基噻唑-2-基)-5-(3-羧基(carbboxy)甲氧基苯基)-2-(4-磺基苯基)-2H-四唑(Promega，Madison，USA)一起孵育。抗TNF dAb活性导致TNF细胞毒性的降低，因此，与仅用TNF对照相比，导致吸光度增加。The ability of anti-TNF dAbs to neutralize the cytotoxic activity of TNF was also determined on mouse L929 fibroblasts (Evans, T. (2000)
在初步筛选中，如上所述，为BIAcore分析而制备的上清液也可用于受体测定。在受体和细胞测定中，使用纯化蛋白质，也可进行所选二聚体的进一步分析。In primary screening, supernatants prepared for BIAcore analysis were also used for receptor assays, as described above. Further analysis of selected dimers can also be performed using purified proteins in receptor and cellular assays.
HeLa IL-8测定HeLa IL-8 assay
在HeLa细胞中，测定抗TNFR1或抗TNFα dAb中和TNF对IL-8分泌的诱导作用(采用Akeson，L.等(1996)Journal of BiologicalChemistry 271，30517-30523所述方法的改进方法，所述文献介绍了在HUVEC中IL-1对IL-8的诱导作用；在此，我们观察了人TNFα的诱导作用，而且我们采用HeLa细胞替代HUVEC细胞系)。简而言之，接种在微量滴定板上的HeLa细胞与dAb和300pg/ml TNF一起孵育过夜。孵育后，吸出上清液，通过夹心ELISA(R&D系统)测定细胞和IL-8浓度。与仅用TNF对照相比，抗TNFR1 dAb活性导致上清液中IL-8分泌降低。In HeLa cells, the anti-TNFR1 or anti-TNFα dAb was measured to neutralize the induction of TNF on IL-8 secretion (using Akeson, L. et al. (1996) Journal of
L929测定用于以下实验；然而，优选采用HeLa IL-8测定以检测抗TNF受体1(p55)配体；在L929测定中，小鼠p55的存在对其使用具有某些限制。The L929 assay was used in the following experiments; however, the HeLa IL-8 assay was preferably employed to detect anti-TNF receptor 1 (p55) ligand; the presence of mouse p55 in the L929 assay has certain limitations to its use.
1.4序列分析1.4 Sequence analysis
对于在BIAcore和受体测定筛选中证明具有目标特性的二聚体进行测序。序列详见序列表。Sequencing was performed for dimers that demonstrated target properties in BIAcore and receptor assay screens. See sequence listing for details.
1.5结构形成(formatting)1.5 Formatting
1.5.1 TAR1-5-19二聚体1.5.1 TAR1-5-19 dimer
在细胞和受体测定中，重新精制并分析具有良好中和特性的TAR1-5二聚体。TAR1-5定向dab被亲和力成熟克隆TAR1-5-19取代。为了达到这一点，将该TAR1-5从单个二聚体对中克隆掉(cloneout)，并用经PCR扩增的TAR1-5-19取代。另外，在3U、5U和TU载体中构建TAR1-5-19同型二聚体。基因的N端拷贝经PCR扩增并如上所述进行克隆，用现有的SalI和NotI限制位点克隆C-端基因片段。TAR1-5 dimers with good neutralizing properties were re-purified and analyzed in cellular and receptor assays. The TAR1-5-directed dab was replaced by the affinity matured clone TAR1-5-19. To achieve this, the TAR1-5 was cloned out of a single dimer pair and replaced with PCR amplified TAR1-5-19. Additionally, TAR1-5-19 homodimers were constructed in 3U, 5U and TU vectors. The N-terminal copy of the gene was PCR amplified and cloned as described above, and the C-terminal gene fragment was cloned using the existing SalI and NotI restriction sites.
1.5.2诱变1.5.2 Mutagenesis
通过定点诱变，将dAb2中存在的琥珀终止密码子(其中一个在TAR1-5二聚体对C端的dAb)突变成谷氨酰胺。The amber stop codon present in dAb2 (one of the dAbs C-terminal to the TAR1-5 dimer pair) was mutated to glutamine by site-directed mutagenesis.
1.5.3 Fab1.5.3 Fabs
将含有TAR1-5或TAR1-5-19的二聚体重新精制到Fab表达载体中。用SfiI和NotI限制位点，将dAb克隆到含有CK基因或CH基因的表达载体中，通过序列分析而得以证实。CK载体来源于基于pUC的氨苄青霉素抗性载体，CH载体来源于pACYC氯霉素抗性载体。对于Fab表达，将dAb-CH和dAb-CK构建体共转化HB2151细胞，在含0.1％葡萄糖、100μg/ml氨苄青霉素和10μg/ml氯霉素的2xTY中培养。Dimers containing TAR1-5 or TAR1-5-19 were re-purified into Fab expression vectors. Using SfiI and NotI restriction sites, the dAbs were cloned into expression vectors containing the CK gene or the CH gene and confirmed by sequence analysis. The CK vector is derived from the pUC-based ampicillin-resistant vector, and the CH vector is derived from the pACYC chloramphenicol-resistant vector. For Fab expression, dAb-CH and dAb-CK constructs were co-transformed into HB2151 cells and cultured in 2xTY containing 0.1% glucose, 100 μg/ml ampicillin and 10 μg/ml chloramphenicol.
1.5.3铰链二聚化1.5.3 Hinge dimerization
检查了通过形成胱氨酸键的dAb二聚化。将氨基酸EPKSGDKTHTCPPCP(SEQ ID NO：379)的短序列(一种修饰形式的人IgGCl铰链)，经过改造连接在dAb的C端区。如上所述，合成编码该序列的寡聚接头并退火。将接头克隆到含有TAR1-5-19的pEDA载体中，用XhoI和NotI限制位点。二聚化在周质中原位发生。Dimerization of dAbs via formation of cystine bonds was examined. A short sequence of amino acids EPKSGDKTHTCPPCP (SEQ ID NO: 379) (a modified form of human IgGCl hinge) was engineered and linked to the C-terminal region of the dAb. Oligolinkers encoding this sequence were synthesized and annealed as described above. The linker was cloned into the pEDA vector containing TAR1-5-19, with XhoI and NotI restriction sites. Dimerization occurs in situ in the periplasm.
1.6表达和纯化1.6 Expression and purification
1.6.1表达1.6.1 Expression
如上所述，以2ml，96孔板形式制备上清液，用于初步筛选。初步筛选过程后，对所选二聚体进行进一步分析。二聚体构建体在TOP10F′或HB2151细胞上清液中表达。简而言之，将来自新鲜划线平板的单菌落在37℃、在含100μg/ml氨苄青霉素和1％葡萄糖的2xTY中培养过夜。将1/100稀释的该培养物接种到含有100μg/ml氨苄青霉素和0.1％葡萄糖的2xTY中，在37℃振荡培养，直到OD600达到约0.9。培养物再用1mM IPTG在30℃诱导过夜。离心除去细胞，上清液用蛋白A或L琼脂糖纯化。Supernatants were prepared in 2 ml, 96-well plate format for primary screening as described above. After the initial screening process, selected dimers were subjected to further analysis. Dimeric constructs were expressed in TOP10F' or HB2151 cell supernatants. Briefly, single colonies from freshly streaked plates were grown overnight at 37°C in 2xTY containing 100 μg/ml ampicillin and 1% glucose. This 1/100 diluted culture was inoculated into 2xTY containing 100 μg/ml ampicillin and 0.1% glucose, and cultured with shaking at 37°C until OD600 reached about 0.9. The cultures were further induced with 1 mM IPTG overnight at 30°C. Cells were removed by centrifugation and the supernatant purified with protein A or L agarose.
在HB2152细胞中，Fab和半胱氨酸铰链二聚体表达为周质蛋白。将1/100稀释的过夜培养物接种到含有0.1％葡萄糖和合适抗生素的2xTY中，在30℃振荡培养，直到OD600达到约0.9。培养物再用1mMIPTG在25℃诱导3-4小时。离心收获细胞，沉淀重悬于周质制备缓冲液(30mM Tris-HCl(pH8.0)、1mM EDTA、20％蔗糖)中。离心后，保留上清液，将沉淀重悬于5mM MgSO4。再次通过离心收获上清液，合并，然后纯化。In HB2152 cells, Fab and cysteine hinge dimers are expressed as periplasmic proteins.
1.6.2蛋白A/L纯化1.6.2 Purification of protein A/L
从蛋白L琼脂糖(Affitech，Norway)或蛋白A琼脂糖(Sigma，UK)中优化纯化二聚体蛋白，对其进行检测。蛋白质分批洗脱，或者用蠕动泵通过柱洗脱。检查3种缓冲液：0.1M磷酸-柠檬酸缓冲液(pH2.6)、0.2M甘氨酸(pH2.5)和0.1M甘氨酸(pH2.5)。确定蠕动泵条件下的优化条件，采用0.1M甘氨酸pH2.5，超过10倍柱体积。从蛋白A纯化，蠕动泵条件采用0.1M甘氨酸pH2.5。Dimeric proteins were detected by optimal purification from protein L agarose (Affitech, Norway) or protein A agarose (Sigma, UK). Proteins are eluted batchwise or through the column with a peristaltic pump. Three buffers were examined: 0.1M Phosphate-citrate buffer (pH2.6), 0.2M Glycine (pH2.5) and 0.1M Glycine (pH2.5). Determine the optimal conditions under the peristaltic pump conditions, using 0.1M glycine pH2.5, more than 10 times the column volume. Purification from protein A, peristaltic pump conditions using 0.1M glycine pH 2.5.
1.6.3 FPLC纯化1.6.3 FPLC purification
通过FPLC分析，在AKTA Explorer 100系统(AmershamBiosciences Ltd)上进行进一步纯化。TAR1-5和TAR1-5-19二聚体的分离是通过阳离子交换色谱(1ml Resource S-Amersham BiosciencesLtd)，用0-1M NaCl梯度/50mM乙酸盐缓冲液(pH4)洗脱。铰链二聚体的纯化是通过离子交换(1ml Resource Q Amersham BiosciencesLtd)，用0-1M NaCl梯度/25mM Tris HCl(pH8.0)洗脱。Fab的纯化是通过大小排阻层析，用superose 12(Amersham Biosciences Ltd)柱，流速0.5ml/分钟的PBS(含0.05％吐温)。纯化后，采用vivaspin 5K截止浓缩器(Vivascience Ltd)浓缩样品。Further purification was performed on an
2.0 结果2.0 result
2.1 TAR1-5二聚体2.1 TAR1-5 dimer
从第二轮选择中挑取6×96个克隆，包括所有文库和选择条件。制备上清液制备物，用抗原和蛋白L ELISA、BIAcore和受体测定来分析。在ELISA中，从各选择方法中鉴定出阳性结合克隆，它们分布在3U、5U和7U文库中。然而，因为定向dAb总是存在，所以通过该方法不能区别高和低亲和力结合物，因此进行BIAcore分析。6 × 96 clones were picked from the second round of selection, including all libraries and selection conditions. Supernatant preparations were prepared and analyzed by antigen and protein L ELISA, BIAcore and receptor assays. In ELISA, positive binding clones were identified from each selection method, which were distributed in 3U, 5U and 7U libraries. However, high and low affinity binders could not be discriminated by this method because targeted dAbs were always present, therefore BIAcore analysis was performed.
用2ml上清液进行BIAcore分析。BIAcore分析表明，与单体TAR1-5相比，二聚体K解离速率有很大改进。单体K解离速率范围为10-1M，二聚体K解离速率范围为10-3-10-4M。选择表现出解离速度很慢的16个克隆，它们来自3U、5U和TU文库，并对其进行测序。此外，在受体测定中，分析上清液中和人TNFα的能力。2 ml of supernatant was used for BIAcore analysis. BIAcore analysis revealed a large improvement in the dissociation rate of dimeric K compared to monomeric TAR1-5. The dissociation rate of monomer K is in the range of 10-1 M, and that of dimer K is in the range of 10-3 -10-4 M. Sixteen clones showing very slow dissociation rates from 3U, 5U and TU libraries were selected and sequenced. Additionally, in the receptor assay, supernatants were analyzed for their ability to neutralize human TNF[alpha].
对在这些测定中具有中和能力的6个先导克隆(以下称d1-d6)进行测序。结果显示所得6个克隆中仅3个具有不同第二dAb(dAb1、dAb2和dAb3)，然而，其中发现第二dAb不止一次连接不同长度的接头。Six lead clones (hereinafter referred to as d1-d6) with neutralizing ability in these assays were sequenced. The results showed that only 3 of the 6 clones obtained had different second dAbs (dAb1 , dAb2 and dAb3), however, among them the second dAbs were found to be attached to linkers of different lengths more than once.
TAR1-5d1：3U接头2nd dAb＝dAb1-1μg/ml Ag免疫管，过夜洗涤TAR1-5d1: 3U adapter2nd dAb=dAb1-1μg/ml Ag immunotube, washed overnight
TAR1-5d2：3U接头2nd dAb＝dAb2-1μg/ml Ag免疫管，过夜洗涤TAR1-5d2: 3U adapter2nd dAb=dAb2-1 μg/ml Ag immunotube, washed overnight
TAR1-5d3：5U接头2nd dAb＝dAb2-1μg/ml Ag免疫管，过夜洗涤TAR1-5d3: 5U adapter2nd dAb=dAb2-1 μg/ml Ag immunotube, washed overnight
TAR1-5d4：5U接头2nd dAb＝dAb3-20μg/ml Ag免疫管，过夜洗涤TAR1-5d4: 5U adapter2nd dAb=dAb3-20μg/ml Ag immunotube, washed overnight
TAR1-5d5：5U接头2nd dAb＝dAb1-20μg/ml Ag免疫管，过夜洗涤TAR1-5d5: 5U adapter2nd dAb=dAb1-20μg/ml Ag immunotube, washed overnight
TAR1-5d6：7U接头2nd dAb＝dAb1-R1：1μg/ml Ag免疫管，过夜洗涤TAR1-5d6: 7U adapter2nd dAb = dAb1-R1: 1 μg/ml Ag immunotube, washed overnight
R2：珠R2: beads
进一步检测6个先导克隆。蛋白质从周质和上清液产生，用蛋白L琼脂糖纯化，并在细胞和受体测定中进行检测。中和水平不同(表1)。检测了蛋白质制备的优化条件。从HB2151细胞上清液中得到的蛋白质得到最高收率(约10mg/L培养物)。将上清液与蛋白L琼脂糖一起在室温下孵育2小时或在4℃孵育过夜。珠子用PBS/NaCl洗涤，用蠕动泵装入FPLC柱。珠子用10倍体积的PBS/NaCl洗涤，用0.1M甘氨酸(pH2.5)洗脱。一般而言，二聚体蛋白在单体之后洗脱下来。Six lead clones were further tested. Proteins were produced from the periplasm and supernatant, purified with protein L agarose, and tested in cellular and receptor assays. Neutralization levels varied (Table 1). Optimal conditions for protein preparation were examined. The protein was obtained in the highest yield (approximately 10 mg/L culture) from HB2151 cell supernatant. Incubate the supernatant with protein L agarose for 2 h at room temperature or overnight at 4 °C. The beads were washed with PBS/NaCl and loaded into the FPLC column with a peristaltic pump. The beads were washed with 10 volumes of PBS/NaCl and eluted with 0.1 M glycine (pH 2.5). In general, dimeric proteins elute after monomers.
TAR1-5d1-6二聚体用FPLC纯化。FPLC纯化后得到3种，经SDSPAGE鉴定。一种对应于单体，而其它两种对应于不同大小的二聚体。两种中较大的一种可能是因为存在C端标记。在受体测定中检测这些蛋白质。数据见表1，代表了得自两种二聚体的优化结果(图11)TAR1-5d1-6 dimer was purified by FPLC. Three species were obtained after FPLC purification and identified by SDS PAGE. One corresponds to a monomer, while the other two correspond to dimers of different sizes. The larger of the two is likely due to the presence of a C-terminal tag. These proteins were detected in receptor assays. The data are presented in Table 1 and represent the optimization results obtained from the two dimers (Figure 11)
将来自二聚体对(即dAb1、dAb2和dAb3)的3种第二dAb，作为单体进行克隆，并通过ELISA以及在细胞和受体测定中进行检测。所有3种dAb都特异性结合TNF(经抗原ELISA)，而且不与塑料或BSA发生交叉反应。作为单体，在细胞或受体测定中，dAb都不中和。The three second dAbs from the dimer pair (ie dAb1 , dAb2 and dAb3) were cloned as monomers and tested by ELISA and in cellular and receptor assays. All 3 dAbs specifically bound TNF (by antigen ELISA) and did not cross-react with plastic or BSA. As monomers, neither dAb neutralized in either cellular or receptor assays.
2.1.2 TAR1-5-19二聚体2.1.2 TAR1-5-19 dimer
在6个先导克隆中，用TAR1-5-19代替TAR1-5。在细胞和受体测定中，对所有TAR1-5-19二聚体进行分析，使用总蛋白(仅纯化蛋白L)，除非另有说明(表2)。在细胞测定中，TAR1-5-19d4和TAR1-5-19d3具有最佳ND50(～5nM)，这与受体测定结果是一致的，并且相比TAR1-5-19单体(ND50～30nM)来说得到了改善。尽管纯化TAR1-5二聚体在受体和细胞测定中得到不同结果，但是TAR1-5-19二聚体更一致。在蛋白质纯化期间，当使用不同洗脱缓冲液时会有变动。用0.1M磷酸-柠檬酸缓冲液(pH2.6)或0.2M甘氨酸(pH2.5)洗脱，尽管在大多数情况下从蛋白L琼脂糖中除去所有蛋白质，这使它功能更少。In the 6 lead clones, TAR1-5 was replaced by TAR1-5-19. In cell and receptor assays, all TAR1-5-19 dimers were analyzed using total protein (purified protein L only), unless otherwise stated (Table 2). In cell assays, TAR1-5-19d4 and TAR1-5-19d3 had the best ND50 (~5nM), which was consistent with the receptor assay results, and compared to TAR1-5-19 monomer (ND50 ~ 30nM) was improved. While purified TAR1-5 dimers gave different results in receptor and cellular assays, TAR1-5-19 dimers were more consistent. During protein purification there are variations when using different elution buffers. Elution with 0.1 M phosphate-citrate buffer (pH 2.6) or 0.2 M glycine (pH 2.5) although in most cases removes all protein from protein L agarose makes it less functional.
TAR1-5-19d4在发酵罐中表达，然后在阳离子交换FPLC中纯化，得到十分纯的二聚体。与TAR1-5d4一起，通过FPLC纯化得到3种对应于单体和两种二聚体。对该二聚体进行氨基酸测序。然后，在受体测定中检测TAR1-5-19单体和TAR1-5-19d4，单体所得IC50为30nM，二聚体为8nM。与TAR1-5-19单体、TAR1-5-19d4和TAR1-5d4相比的受体测定结果见图10。TAR1-5-19d4 was expressed in fermentors and then purified in cation-exchange FPLC to give a very pure dimer. Together with TAR1-5d4, 3 corresponding monomers and two dimers were obtained by FPLC purification. Amino acid sequencing was performed on the dimer. TAR1-5-19 monomer and TAR1-5-19d4 were then tested in a receptor assay, giving anIC50 of 30 nM for the monomer and 8 nM for the dimer. The results of the receptor assay compared to TAR1-5-19 monomer, TAR1-5-19d4 and TAR1-5d4 are shown in Figure 10.
在3U、5U和7U载体中制备TAR1-5-19同型二聚体，表达并在蛋白L上纯化。蛋白质在细胞和受体测定中进行检验，测定所得IC50(对于受体测定)和ND50(对于细胞测定)(表3，图12)。TAR1-5-19 homodimers were prepared in 3U, 5U and 7U vectors, expressed and purified on protein L. The proteins were tested in cellular and receptor assays and the resultingIC50 (for receptor assay) andND50 (for cellular assay) were determined (Table 3, Figure 12).
2.2 Fab2.2 Fab
TAR1-5和TAR1-5-19二聚体克隆到Fab形式中，表达并在蛋白L琼脂糖上纯化。Fab在受体测定中进行评价(表4)。结果表明，TAR1-5-19和TAR1-5二聚体的中和水平都类似于其来源的原Gly4Ser接头二聚体。表达TAR1-5-19 Fab(其中TAR1-5-19展示在CH和CK上)，经蛋白L纯化，在受体测定中进行评价。所得IC50约为1nM。TAR1-5 and TAR1-5-19 dimers were cloned into Fab format, expressed and purified on protein L agarose. Fabs were evaluated in receptor assays (Table 4). The results showed that both TAR1-5-19 and TAR1-5 dimers had neutralization levels similar to their original Gly4 Ser adapter dimers from which they were derived. TAR1-5-19 Fab (where TAR1-5-19 is displayed on CH and CK) was expressed, protein L purified, and evaluated in receptor assays. The resultingIC50 was approximately 1 nM.
2.3 TAR1-27二聚体2.3 TAR1-27 dimer
从第二轮选择中挑取3×96个克隆，包括所有文库和选择条件。制备2ml上清液制备物，用于在ELISA和生物测定中进行分析。抗原ELISA得到71个阳性克隆。粗上清液的受体测定得到42个具有抑制特性的克隆(TNF结合0-60％)。在大多数情况下，抑制特性与强烈ELISA信号相关。对42个克隆进行测序，其中有39个具有独特的第二dAb序列。对具有最佳抑制特性的12个二聚体进行进一步分析。3 × 96 clones were picked from the second round of selection, including all libraries and selection conditions. A 2 ml supernatant prep was prepared for analysis in ELISA and bioassays. Antigen ELISA obtained 71 positive clones. Receptor assays of crude supernatants yielded 42 clones with inhibitory properties (TNF binding 0-60%). In most cases, inhibitory properties correlate with a strong ELISA signal. Of the 42 clones sequenced, 39 had unique second dAb sequences. The 12 dimers with the best inhibitory properties were further analyzed.
12个中和克隆在200ml上清液制备物中表达，再在蛋白L上纯化。它们通过蛋白L和抗原ELISA、BIAcore和在受体测定中进行评价。在所有情况下都得到强烈阳性ELISA信号。BIAcore分析表明，所有克隆具有快的缔合和解离速度。与单体TAR1-27相比，改进了解离速率，然而，与先前检测的TAR1-5二聚体的速度(K解离范围约为10-3M～10-4M)相比，TAR1-27二聚体的解离速度更快(K解离范围约为10-1M～10-2M)。纯化二聚体的稳定性是个问题，因此为了改进稳定性，在2 TAR1-27二聚体(d2和d16)的纯化中加入5％甘油、0.5％Triton X100或0.5％NP40(Sigma)。加入NP40或Triton X100TM提高纯化产物的收率约2倍。在受体测定中评价这两种二聚体。在所有纯化条件下TAR1-27d2的IC50都约为30nM。当纯化没有使用稳定剂时，TAR1-27d16显示出没有中和效应，但当纯化在稳定条件下进行时，得到IC50约为50nM。没有进行进一步分析。Twelve neutralizing clones were expressed in 200 ml supernatant preparations and purified on protein L. They were evaluated by protein L and antigen ELISA, BIAcore and in receptor assays. Strong positive ELISA signals were obtained in all cases. BIAcore analysis indicated that all clones had fast association and dissociation rates. Compared with monomeric TAR1-27, the dissociation rate was improved, however, compared with the previously detected speed of TAR1-5 dimer (K dissociation range is about 10-3 M ~ 10-4 M), TAR1- The dissociation rate of 27 dimer is faster (K dissociation range is about 10-1 M ~ 10-2 M). Stability of purified dimers was an issue, so to improve stability, 5% glycerol, 0.5% Triton X100 or 0.5% NP40 (Sigma) were added to the purification of 2 TAR1-27 dimers (d2 and d16). Adding NP40 or Triton X100TM increased the yield of the purified product about 2-fold. Both dimers were evaluated in receptor assays. TheIC50 of TAR1-27d2 was approximately 30 nM under all purification conditions. TAR1-27d16 showed no neutralizing effect when purified without a stabilizer, but gave anIC50 of approximately 50 nM when purified under stable conditions. No further analysis was performed.
2.4 TAR2-5二聚体2.4 TAR2-5 dimer
从第二轮选择中挑取3×96个克隆，包括所有文库和选择条件。制备2ml上清液制备物，用于分析。对各板进行蛋白A和抗原ELISA。通过BIAcore鉴定出30个目标克隆，具有良好解离速率(K解离范围介于10-2-10-3M)。对克隆进行测序，通过序列分析鉴定了13个独特的二聚体。3 × 96 clones were picked from the second round of selection, including all libraries and selection conditions. A 2ml supernatant prep was prepared for analysis. Protein A and antigen ELISAs were performed on each plate. Thirty target clones were identified by BIAcore with good off-rates (K off-range ranging from 10−2 to 10−3 M). The clones were sequenced and 13 unique dimers were identified by sequence analysis.
表4： TAR1-5二聚体Table 4: TAR1-5 dimers
*注释：二聚体2和二聚体3具有相同的第二dAb(称为dAb2)，但却具有不同接头长度(d2＝(Gly4Ser)3、d3＝(Gly4Ser)3)。dAb1是二聚体1、5和6的配偶体dAb。dAb3是二聚体4的配偶体dAb。配偶体dAb都不能单独中和。除非另有说明，否则FPLC纯化是通过阳离子交换。在这些测定中，对经FPLC所得的每种二聚体，测定优化二聚体种类。* Note:
表5：TAR1-5-19二聚体Table 5: TAR1-5-19 dimers
表6：TAR1-5-19同型二聚体Table 6: TAR1-5-19 homodimers
表7：TAR1-5/TAR1-5-19 FabTable 7: TAR1-5/TAR1-5-19 Fab
实施例7.通过末端半胱氨酸键的dAb二聚化Example 7. dAb dimerization via terminal cysteine bonds
概述overview
对于dAb二聚化，将游离半胱氨酸拼接到蛋白质的C-端。当表达时，该蛋白形成二聚体，所述二聚体可通过两个步骤的纯化方法而纯化。For dAb dimerization, a free cysteine is spliced to the C-terminus of the protein. When expressed, the protein forms dimers that can be purified by a two-step purification method.
TAR1-5-19CYS二聚体的PCR构建PCR Construction of TAR1-5-19CYS Dimer
按照实施例8描述dAb三聚体的方法。三聚体方案得到单体、二聚体和三聚体的混合物。The procedure for dAb trimers was described as in Example 8. The trimer scheme yields a mixture of monomers, dimers, and trimers.
TAR1-5-19CYS二聚体的表达和纯化Expression and purification of TAR1-5-19CYS dimer
按照实施例8概述的方法，从培养物上清液中，通过蛋白L琼脂糖捕获而纯化二聚体。Dimers were purified from culture supernatants by protein L agarose capture following the procedure outlined in Example 8.
TAR1-5-19CYS单体与TAR1-5-19CYS二聚体的分离Separation of TAR1-5-19CYS monomer and TAR1-5-19CYS dimer
在阳离子交换分离之前，按照制造商的使用说明书，用PD-10柱(Amersham Pharmacia)将单体/二聚体混合样品的缓冲液更换成50mM乙酸钠缓冲液(pH4.0)。再将样品用于预先用50mM乙酸钠(pH4.0)平衡的1mL Resource S阳离子交换柱(Amersham Pharmacia)。用以下盐梯度的50mM乙酸钠(pH4.0)分离出单体和二聚体：Prior to cation-exchange separation, the buffer exchange of monomer/dimer mixed samples into 50 mM sodium acetate buffer (pH 4.0) was performed using a PD-10 column (Amersham Pharmacia) according to the manufacturer's instructions. Samples were then applied to 1 mL Resource S cation exchange columns (Amersham Pharmacia) pre-equilibrated with 50 mM sodium acetate (pH 4.0). Monomers and dimers were separated using the following salt gradient of 50 mM sodium acetate, pH 4.0:
150-200mM氯化钠超过15倍柱体积150-200mM NaCl over 15 column volumes
200-450mM氯化钠超过10倍柱体积200-450mM NaCl over 10 column volumes
450-1000mM氯化钠超过15倍柱体积450-1000mM NaCl over 15 column volumes
仅含二聚体的部分用SDS-PAGE鉴定后合并，加入1/5体积的1MTris(pH8.0)将pH升至8。Fractions containing only dimers were identified by SDS-PAGE and pooled, and the pH was raised to 8 by adding 1/5 volume of 1MTris (pH 8.0).
体外功能性结合测定：TNF受体测定和细胞测定。In vitro functional binding assays: TNF receptor assay and cellular assay.
采用TNF受体和细胞测定，测定二聚体对人TNFα的亲和力。受体测定中的IC50约为0.3-0.8nM；细胞测定中的ND50约为3-8nM。其它可能的TAR1-5-19CYS二聚体形式The affinity of the dimers for human TNF[alpha] was determined using TNF receptor and cellular assays. TheIC50 in the receptor assay is about 0.3-0.8nM; theND50 in the cell assay is about 3-8nM. Other possible dimer forms of TAR1-5-19CYS
PEG二聚体和委托合成的马来酰亚胺二聚体PEG dimer and commissioned maleimide dimer
Nektar(Shearwater)提供一系列双马来酰亚胺PEG[mPEG2-(MAL)2或mPEG-(MAL)2]，它们可让单体成为二聚体，其中用小接头隔开dAb，这两者都连接有PEG(大小范围为5-40kDa)。已经知道，5kDa mPEG-(MAL)2(即[TAR1-5-19]-Cys-马来酰亚胺-PEG×2，其中马来酰亚胺一起连接在二聚体中)在TNF受体测定中的亲和力为～1-3nM。另外，也可用TMEA(三[2-马来酰亚胺乙基]胺)(PierceBiotechnology)或其它双功能接头产生二聚体。Nektar (Shearwater) offers a range of bismaleimide PEGs [mPEG2-(MAL)2 or mPEG-(MAL)2] that allow monomers to dimerize with a small linker separating the dAb, the two Both have PEG attached (size range 5-40 kDa). It is known that 5kDa mPEG-(MAL)2 (i.e. [TAR1-5-19]-Cys-maleimide-PEG×2, in which the maleimides are linked together in a dimer) in the TNF receptor Affinities in the assay were ~1-3 nM. Alternatively, TMEA (tris[2-maleimidoethyl]amine) (Pierce Biotechnology) or other bifunctional linkers can be used to generate dimers.
通过化学偶联方法，采用2，2′-二硫双吡啶(Sigma Aldrich)和还原单体，也可产生二硫化物二聚体。将多肽接头或铰链加到dAb的C-端。或者(Gly4Ser)n(其中n＝1-10，例如1、2、3、4、5、6或7)、或者免疫球蛋白(例如IgG铰链区或随机肽序列(例如选自随机肽序列文库)等小接头，可以添加在dAb和末端半胱氨酸残基之间。这可用于制备如上所述的二聚体。Disulfide dimers were also produced by chemical coupling methods using 2,2'-dithiobispyridine (Sigma Aldrich) and reducing monomers. A polypeptide linker or hinge is added to the C-terminus of the dAb. or (Gly4 Ser)n (where n = 1-10, such as 1, 2, 3, 4, 5, 6 or 7), or an immunoglobulin (such as an IgG hinge region or a random peptide sequence (such as selected from random peptide A small linker, such as a sequence library), can be added between the dAb and the terminal cysteine residue. This can be used to make dimers as described above.
实施例8.dAb三聚化Example 8. dAb trimerization
概述overview
对于dAb三聚化，蛋白质C-端需要游离半胱氨酸。半胱氨酸残基，一旦还原得到游离巯基，则可用于将蛋白质特异性偶联到三聚体马来酰亚胺分子上，例如TMEA(三[2-马来酰亚胺乙基]胺)。For dAb trimerization, a free cysteine is required at the C-terminus of the protein. Cysteine residues, once reduced to a free sulfhydryl group, can be used to specifically couple proteins to trimeric maleimide molecules such as TMEA (tris[2-maleimidoethyl]amine ).
TAR1-5-19CYS的PCR构建PCR construction of TAR1-5-19CYS
下列寡核苷酸专门用于具有SalI和BamHI位点的PCR TAR1-5-19，用于克隆并引入C-端半胱氨酸残基：The following oligonucleotides were specific for PCR TAR1-5-19 with SalI and BamHI sites for cloning and introduction of the C-terminal cysteine residue:
SalISalI
～～～～～～～~~~~~~~
Trp Ser Ala Ser Thr Aap* Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValTrp Ser Ala Ser Thr Aap* Ile Gln Met Thr Gln Ser Pro Ser Ser Ser Leu Ser Ala Ser Val
1 TGG AGC GCG TCG ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA1 TGG AGC GCG TCG ACG GAC ATC CAG ATG ACC CAG TCT CCA TCC TCT CTG TCT GCA TCT GTA
ACC TCG CGC AGC TGC CTG TAG GTC TAC TGG GTC AGA GGT AGG AGA GAC AGA CGT AGA CATACC TCG CGC AGC TGC CTG TAG GTC TAC TGG GTC AGA GGT AGG AGA GAC AGA CGT AGA CAT
Gly Asp Arg Val Thr Ils Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr Leu His TrpGly Asp Arg Val Thr Ils Thr Cys Arg Ala Ser Gln Ser Ile Asp Ser Tyr Leu His Trp
61 GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT TTA CAT TGG61 GGA GAC CGT GTC ACC ATC ACT TGC CGG GCA AGT CAG AGC ATT GAT AGT TAT TTA CAT TGG
CCT CTG GCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG TAA CTA TCA ATA AAT GTA ACCCCT CTG GCA CAG TGG TAG TGA ACG GCC CGT TCA GTC TCG TAA CTA TCA ATA AAT GTA ACC
Tyr Gln Gln Lys Pro Gly Lye Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Glu Leu GlnTyr Gln Gln Lys Pro Gly Lye Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Glu Leu Gln
121 TAC CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT AGT GCA TCC GAG TTG CAA121 TAC CAG CAG AAA CCA GGG AAA GCC CCT AAG CTC CTG ATC TAT AGT GCA TCC GAG TTG CAA
ATG GTC GTC TTT GGT CCC TTT CGG GGA TTC GAG GAC TAG ATA TCA CGT AGG CTC AAC GTTATG GTC GTC TTT GGT CCC TTT CGG GGA TTC GAG GAC TAG ATA TCA CGT AGG CTC AAC GTT
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr IleSer Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
181 AGT GGG GTC CCA TCA CGT TTC AGT GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC181 AGT GGG GTC CCA TCA CGT TTC AGT GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC
TCA CCC CAG GGT AGT GCA AAG TCA CCG TCA CCT AGA CCC TGT CTA AAG TGA GAG TGG TAGTCA CCC CAG GGT AGT GCA AAG TCA CCG TCA CCT AGA CCC TGT CTA AAG TGA GAG TGG TAG
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg ProSer Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Val Trp Arg Pro
241 AGC AGT CTG CAA CCT GAA GAT TTT GCT ACG TAC TAC TGT CAA CAG GTT GTG TGG CGT CCT241 AGC AGT CTG CAA CCT GAA GAT TTT GCT ACG TAC TAC TGT CAA CAG GTT GTG TGG CGT CCT
TCG TCA GAC GTT GGA CTT CTA AAA CGA TGC ATG ATG ACA GTT GTC CAA CAC ACC GCA GGATCG TCA GAC GTT GGA CTT CTA AAA CGA TGC ATG ATG ACA GTT GTC CAA CAC ACC GCA GGA
BamHIBamHI
～～～～～～～～~~~~~~~~
Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Cys *** *** Gly Ser GlyPhe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Cys *** *** Gly Ser Gly
301 TTT ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG TGC TAA TAA GGA TCC GGC301 TTT ACG TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA CGG TGC TAA TAA GGA TCC GGC
AAA TGC AAG CCG GTT CCC TGG TTC CAC CTT TAG TTT GCC ACG ATT ATT CCT AGG CCGAAA TGC AAG CCG GTT CCC TGG TTC CAC CTT TAG TTT GCC ACG ATT ATT CCT AGG CCG
(*TAR1-5-19CYS序列的开始；TAR1-5-19CYS氨基酸序列(SEQID NO：293；TAR1-5-19CYS核苷酸序列(SEQ ID NO：294，编码链；SEQ ID NO：295，非编码链))(* Start of TAR1-5-19CYS sequence; TAR1-5-19CYS amino acid sequence (SEQ ID NO: 293; TAR1-5-19CYS nucleotide sequence (SEQ ID NO: 294, coding strand; SEQ ID NO: 295, non coding chain))
正向引物forward primer
5’-TGGAGCGCGTCGACGGACATCCAGATGACCCAGTCTCCA-3’(SEQ ID NO：296)5'-TGGAGCGCGTCGACGGACATCCAGATGACCCAGTCTCCA-3' (SEQ ID NO: 296)
反向引物reverse primer
5’-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3’(SEQ ID NO：297)5'-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3' (SEQ ID NO: 297)
如下进行PCR反应(50μL体积)：200μM dNTP、0.4μM各引物、5μL 10x Pfu Turbo缓冲液(Stratagene)、100ng模板质粒(编码TAR1-5-19)、1μL Pfu Turbo酶(Stratagene)并用无菌水将体积调至50μL。采用以下PCR条件：起始变性步骤94℃2分钟，再是25次循环(94℃30秒，64℃30秒，72℃30秒)。最终延伸步骤也包括72℃5分钟。纯化PCR产物并用SalI和BamHI消化，再连接到用相同限制酶切割的载体上。正确克隆经DNA测序得以证实。Perform a PCR reaction (50 μL volume) as follows: 200 μM dNTPs, 0.4 μM each primer, 5 μL 10x Pfu Turbo buffer (Stratagene), 100 ng template plasmid (encoding TAR1-5-19), 1 μL Pfu Turbo enzyme (Stratagene) and rinse with sterile water Adjust the volume to 50 μL. The following PCR conditions were used: an initial denaturation step at 94°C for 2 minutes, followed by 25 cycles (94°C for 30 seconds, 64°C for 30 seconds, 72°C for 30 seconds). The final extension step also included 5 minutes at 72°C. The PCR product was purified and digested with SalI and BamHI, and ligated into a vector cut with the same restriction enzymes. Correct cloning was confirmed by DNA sequencing.
TAR1-5-19CYS的表达和纯化Expression and purification of TAR1-5-19CYS
按照制造商的方案，将TAR1-5-19CYS载体转化BL21(DE3)pLysS化学感受态细胞(Novagen)中。用100μg/mL羧苄青霉素和37μg/mL氯霉素选择携带dAb质粒的细胞。在含有500mL terrific肉汤(Sigma-Aldrich)、100μg/mL羧苄青霉素和37μg/mL氯霉素的2L带挡板的瓶子中建立培养。培养物在30℃以200rpm振荡培养，直到O.D.600为1-1.5，然后用1mM IPTG(异丙基-β-D-硫代吡喃半乳糖苷，Melford Laboratories)诱导。让dAb的表达在30℃持续12-16小时。发现大多数dAb存在于培养基中。因此，通过离心(8,000xg 30分钟)，从培养基中分离出细胞，上清液用于纯化dAb。每升上清液中加入30mL蛋白L琼脂糖(Affitech)，让dAb分批结合，同时搅拌2小时。然后让树脂在重力下沉降1小时，再吸去上清液。将琼脂糖装入XK 50柱(Amersham Phamacia)中，用10倍体积的PBS洗涤。结合dAb用100mM甘氨酸(pH2.0)洗脱，含蛋白部分通过加入1/5体积的1M Tris(pH8.0)而中和。每升培养上清液中分离到20mg纯蛋白，其中含有50∶50比例的单体与二聚体。The TAR1-5-19CYS vector was transformed into BL21(DE3)pLysS chemically competent cells (Novagen) following the manufacturer's protocol. Cells carrying dAb plasmids were selected with 100 μg/mL carbenicillin and 37 μg/mL chloramphenicol. Cultures were established in 2 L baffled bottles containing 500 mL terrific broth (Sigma-Aldrich), 100 μg/mL carbenicillin, and 37 μg/mL chloramphenicol. The culture was grown at 30°C with shaking at 200 rpm until the O.D.600 was 1-1.5, and then induced with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside, Melford Laboratories). Expression of the dAb was allowed to continue for 12-16 hours at 30°C. Most dAbs were found to be present in the culture medium. Therefore, cells were separated from the culture medium by centrifugation (8,000xg for 30 min), and the supernatant was used for dAb purification. 30 mL of protein L agarose (Affitech) was added per liter of supernatant and the dAbs were allowed to bind in batches with stirring for 2 hours. The resin was then allowed to settle by gravity for 1 hour before the supernatant was aspirated. Agarose was loaded into an
TAR1-5-19CYS的三聚化Trimerization of TAR1-5-19CYS
将2.5ml 100μM TAR1-5-19CYS用5mM二硫苏糖醇还原，在室温下放置20分钟。用PD-10柱(Amersham Pharmacia)给样品更换缓冲液。该柱预先用5mM EDTA、50mM磷酸钠(pH6.5)平衡，按照制造商的指南，使用样品并洗脱。将样品放在冰上备用。TMEA(三[2-马来酰亚胺乙基]胺)购自Pierce Biotechnology。用100％DMSO(二甲基亚砜)配制20mM TMEA贮液。发现TMEA浓度大于3∶1(dAb∶TMEA的摩尔比)时，引起快速沉淀和蛋白质交联。当pH增加时也会增加沉淀速率和交联速率。因此，使用100μM还原TAR1-5-19CYS，加入25μM TMEA使蛋白质三聚化，让反应在室温下进行2小时。发现在偶联反应进行时，加入添加剂(例如甘油或乙二醇)至20％(v/v)，可显著减少三聚体沉淀。偶联后，SDS-PAGE分析表明，溶液中同时存在单体、二聚体和三聚体。2.5ml of 100μM TAR1-5-19CYS was reduced with 5mM dithiothreitol and left at room temperature for 20 minutes. Samples were buffer exchanged with PD-10 columns (Amersham Pharmacia). The column was pre-equilibrated with 5 mM EDTA, 50 mM sodium phosphate (pH 6.5), and samples were used and eluted according to the manufacturer's instructions. Keep samples on ice until ready to use. TMEA (tris[2-maleimidoethyl]amine) was purchased from Pierce Biotechnology. A 20 mM TMEA stock solution was prepared in 100% DMSO (dimethyl sulfoxide). It was found that concentrations of TMEA greater than 3:1 (molar ratio of dAb:TMEA) caused rapid precipitation and protein cross-linking. The rate of precipitation and the rate of crosslinking also increase as the pH increases. Therefore, use 100 μM reduced TAR1-5-19CYS, add 25 μM TMEA to trimerize the protein, and let the reaction proceed for 2 hours at room temperature. It was found that the addition of additives (such as glycerol or ethylene glycol) up to 20% (v/v) significantly reduced trimer precipitation while the coupling reaction was proceeding. After coupling, SDS-PAGE analysis showed that monomer, dimer and trimer existed simultaneously in the solution.
三聚体TAR1-5-19CYS的纯化Purification of Trimeric TAR1-5-19CYS
每mL TMEA-TAR1-5-19cys反应物中加入40μL 40％冰乙酸，将pH降至～4。再将样品加到1mL Resource S阳离子交换柱(AmershamPharmacia)，该柱预先用50mM乙酸钠(pH4.0)平衡。用340-450mM氯化钠的盐梯度、50mM乙酸钠(pH4.0)超过30倍柱体积，部分分离二聚体和三聚体。仅含三聚体的部分用SDS-PAGE鉴定后合并，加入1/5体积的1M Tris(pH8.0)将pH升至8。为了在浓缩步骤(采用5K截止Viva离心浓缩器；Vivascience)中防止三聚体沉淀，向样品中加入10％甘油。Add 40 μL of 40% glacial acetic acid per mL of TMEA-TAR1-5-19cys reaction to lower the pH to ~4. The sample was then applied to a 1 mL Resource S cation exchange column (AmershamPharmacia) pre-equilibrated with 50 mM sodium acetate (pH 4.0). Dimers and trimers were partially separated using a salt gradient of 340-450 mM sodium chloride, 50 mM sodium acetate, pH 4.0, over 30 column volumes. The fractions containing only trimers were identified by SDS-PAGE and combined, and 1/5 volume of 1M Tris (pH 8.0) was added to raise the pH to 8. To prevent trimer precipitation during the concentration step (5K cut-off Viva centrifugal concentrators were used; Vivascience), 10% glycerol was added to the samples.
体外功能性结合测定：TNF受体测定和细胞测定In vitro functional binding assays: TNF receptor assay and cellular assay
采用TNF受体和细胞测定，测定三聚体对人TNFα的亲和力。受体测定中的IC50约为0.3nM；细胞测定中的ND50约为3-10nM(例如3nM)。The affinity of the trimers for human TNFα was determined using TNF receptor and cellular assays. TheIC50 in the receptor assay is about 0.3 nM; theND50 in the cell assay is about 3-10 nM (eg, 3 nM).
其它可能的TAR1-5-19CYS三聚体形式Other possible trimeric forms of TAR1-5-19CYS
采用以下试剂，也可将TAR1-5-19CYS制成三聚体：TAR1-5-19CYS can also be made into trimers using the following reagents:
PEG三聚体和委托合成的马来酰亚胺三聚体PEG trimer and commissioned maleimide trimer
Nektar(Shearwater)提供一系列多臂PEG，它们在PEG末端可以被化学修饰。因此，采用在各臂末端具有马来酰亚胺官能团的PEG三聚体，使dAb三聚体化的方式类似于以上概述的用TMEA的方式。PEG也具有增加三聚体溶解度的优势，因而能防止聚集的问题。因此，可以产生dAb三聚体，其中每个dAb具有与马来酰亚胺官能团连接的C-末端半胱氨酸，所述马来酰亚胺官能团连接在PEG三聚体上。Nektar (Shearwater) offers a series of multi-armed PEGs which can be chemically modified at the PEG termini. Thus, using PEG trimers with maleimide functional groups at the ends of each arm, dAbs were trimerized in a manner similar to that outlined above with TMEA. PEG also has the advantage of increasing the solubility of the trimer, thereby preventing aggregation problems. Thus, dAb trimers can be generated wherein each dAb has a C-terminal cysteine linked to a maleimide functional group attached to the PEG trimer.
将多肽接头或铰链加到dAb的C-端。A polypeptide linker or hinge is added to the C-terminus of the dAb.
或者(Gly4Ser)n(其中n＝1-10，例如1、2、3、4、5、6或7)、或者免疫球蛋白(例如IgG铰链区或随机肽序列(例如选自随机肽序列文库)等小接头，可以添加在dAb和末端半胱氨酸残基之间。当用于制备多聚体(例如二聚体或三聚体)时，这又将引入更大程度的柔性和各个单体间的距离，这些会改善与靶标(例如多亚基靶标例如人TNFα)的结合特性。or (Gly4 Ser)n (where n = 1-10, such as 1, 2, 3, 4, 5, 6 or 7), or an immunoglobulin (such as an IgG hinge region or a random peptide sequence (such as selected from random peptide A small linker, such as a sequence library), can be added between the dAb and the terminal cysteine residue. This in turn introduces a greater degree of flexibility when used to make multimers such as dimers or trimers and the distance between individual monomers, these will improve the binding properties to the target (eg a multi-subunit target such as human TNFα).
实施例9.针对人血清白蛋白(HSA)和小鼠血清白蛋白(MSA)的一批单域抗体(dAb)的选择Example 9. Selection of a batch of single domain antibodies (dAbs) against human serum albumin (HSA) and mouse serum albumin (MSA)
该实施例说明了制备针对血清白蛋白的单域抗体(dAb)的方法。描述了针对小鼠血清白蛋白(MSA)和人血清白蛋白(HSA)的dAb的选择。该实验中使用了3个人噬菌体展示抗体文库，它们各自基于VH的单一人构架(参见图13：基于V3-23/DP47和JH4b的dummy VH序列)或Vκ(参见图15：基于o12/o2/DPK9和Jk1的dummy Vκ序列)，其由NNK密码子编码的侧链多样性结合到互补决定区(CDR1、CDR2和CDR3)中。This example illustrates a method for making single domain antibodies (dAbs) against serum albumin. A selection of dAbs against mouse serum albumin (MSA) and human serum albumin (HSA) is described. Three human phage display antibody libraries were used in this experiment, each based on a single humanVH framework (see Figure 13: dummy VH sequences based on V3-23/DP47 and JH4b) or Vκ (see Figure 15: based on o12/ o2/DPK9 and Jk1 dummy Vκ sequences), whose side chain diversity encoded by NNK codons is integrated into the complementarity determining regions (CDR1, CDR2 and CDR3).
文库1(VH)：Library 1 (VH ):
在以下位置的多样性：H30、H31、H33、H35、H50、H52、H52a、H53、H55、H56、H58、H95、H97、H98。Diversity at the following positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98.
文库大小：6.2×109Library size: 6.2×109
文库2(VH)：Library 2 (VH ):
在以下位置的多样性：H30、H31、H33、H35、H50、H52、H52a、H53、H55、H56、H58、H95、H97、H98、H99、H100、H100a、H100b。Diversity at the following positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a, H100b.
文库大小：4.3×109Library size: 4.3×109
文库3(Vκ)：Library 3 (Vκ):
在以下位置的多样性：L30、L31、L32、L34、L50、L53、L91、L92、L93、L94、L96Diversity at the following positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96
文库大小：2×109Library size: 2×109
预选VH和Vκ文库分别对蛋白A和蛋白L通用配体的结合性，使得在未选择文库中的大部分克隆是功能性的。以上所示的文库大小对应于预选后的大小。Preselection of theVH and VK libraries for protein A and protein L universal ligand binding, respectively, resulted in the majority of clones in the unselected libraries being functional. The library sizes shown above correspond to sizes after preselection.
分别用各文库，针对血清白蛋白进行两轮选择。对于每次选择，将浓度为100μg/ml的抗原/4ml PBS包被在免疫管(nunc)上。在第一轮选择中，3个文库中的每一个都分别针对HSA(Sigma)和MSA(Sigma)进行选择。在第二轮选择中，来自第一轮选择的6个噬菌体中的每一个都针对以下抗原进行选择：(i)再次用相同抗原(例如第一轮MSA，第二轮MSA)和(ii)针对互换抗原(例如第一轮MSA，第二轮HSA)，产生总共12个第二轮选择。在每种情况下，在第二轮选择后，测定48个克隆与HSA和MSA的结合。按照Harrison等，MethodsEnzymol.1996；267：83-109和标准ELISA方案(Hoogenboom等(1991)Nucleic Acids Res.，19：4133)所述，产生可溶性dAb片段，只是用2％吐温PBS作为封闭缓冲液，以及用蛋白L-HRP(Sigma)(用于Vκ)和蛋白A-HRP(Amersham Pharmacia Biotech)(用于VH)检测结合dAb。Using each library separately, two rounds of selection were performed against serum albumin. For each selection, immunotubes (nunc) were coated with antigen at a concentration of 100 μg/ml/4 ml PBS. In the first round of selection, each of the 3 libraries was selected against HSA (Sigma) and MSA (Sigma) respectively. In the second round of selection, each of the six phages from the first round of selection were selected against: (i) the same antigen again (e.g. first round MSA, second round MSA) and (ii) A total of 12 second round selections were generated against swapped antigens (eg first round MSA, second round HSA). In each case, 48 clones were assayed for binding to HSA and MSA after the second round of selection. Soluble dAb fragments were generated as described by Harrison et al., Methods Enzymol. 1996; 267:83-109 and the standard ELISA protocol (Hoogenboom et al. (1991) Nucleic Acids Res., 19:4133), except that 2% Tween PBS was used as blocking buffer solution, and bound dAbs were detected with protein L-HRP (Sigma) (for VK) and protein A-HRP (Amersham Pharmacia Biotech) (forVH ).
在ELISA中测定dAb，得到大于背景的信号，表明与MSA、HSA或这两者结合，不溶性形式仅结合塑料，但是所有都对血清白蛋白具有特异性。对克隆进行测序(参见下表)表明，鉴定出了21个独特dAb序列。所选Vκ dAb克隆之间的最小相似性(在氨基酸水平上)为86.25％((69/80)×100)；这是当所有多样化残基都不同(例如克隆24和34)时的结果。所选VH dAb克隆之间的最小相似性为94％((127/136)×100)。The dAb was assayed in an ELISA and gave a signal above background indicating binding to MSA, HSA or both, the insoluble form only bound plastic but all were specific for serum albumin. Sequencing of the clones (see table below) revealed that 21 unique dAb sequences were identified. The minimal similarity (at the amino acid level) between the selected Vκ dAb clones was 86.25% ((69/80) × 100); this is the result when all the diversifying residues differ (
然后，测定血清白蛋白结合dAb从溶液中捕获生物素化抗原的能力。按照ELISA方案(同上)，只是ELISA板用1μg/ml蛋白L(对于Vκ克隆)和1μg/ml蛋白A(对于VH克隆)包被。按照方案，从溶液中捕获可溶性dAb，再用生物素化MSA或HSA和链霉抗生物素HRP进行检测。生物素化MSA和HAS是按照制造商的使用说明书来制备，其目的是为了获得平均2个生物素/血清白蛋白分子。在ELISA中，鉴定出了24个克隆从溶液中捕获生物素化MSA。这其中有两个(以下克隆2和38)也捕获生物素化HSA。然后，测定dAb对CM5biacore芯片上包被的MSA的结合能力。发现8个克隆能结合biacore上的MSA。Serum albumin binding dAbs were then assayed for their ability to capture biotinylated antigen from solution. The ELISA protocol (supra) was followed except that ELISA plates were coated with 1 μg/ml protein L (for VK clones) and 1 μg/ml protein A (forVH clones). Following the protocol, soluble dAbs were captured from solution and detected with biotinylated MSA or HSA and streptavidin-HRP. Biotinylated MSA and HAS were prepared according to the manufacturer's instructions in order to obtain an average of 2 biotin/serum albumin molecules. In the ELISA, 24 clones were identified that captured biotinylated MSA from solution. Two of these (
表8.Table 8.
在所有情况下，构架与在相应dummy序列中的构架相同，其在CDR中的多样性见上表。In all cases, the framework was identical to that in the corresponding dummy sequence, whose diversity among the CDRs is given in the table above.
在结合biacore上的MSA的8个克隆中，选择在大肠杆菌中高度表达的2个克隆(克隆MSA16和MSA26)用于进一步研究(参见实施例10)。MSA16和MSA 26的完整核苷酸序列和氨基酸序列见图16。Among the 8 clones that bound MSA on biacore, 2 clones highly expressed in E. coli (clone MSA16 and MSA26) were selected for further study (see Example 10). The complete nucleotide and amino acid sequences of MSA16 and
实施例10.测定结合MSA的dAb MSA16和MSA26的小鼠亲和力和血清半衰期Example 10. Determination of mouse affinity and serum half-life of dAbs MSA16 and MSA26 that bind MSA
dAb MSA16和MSA26在大肠杆菌周质中表达，其纯化采用分批吸附到蛋白L-琼脂糖亲和树脂(Affitech，Norway)上，然后用甘氨酸(pH2.2)洗脱。然后，通过抑制biacore分析纯化的dAb，测定Kd。简而言之，对纯化MSA16和MS A26进行测试，以测定在高密度MSA包被的biacore CM5芯片上达到200RU响应所需的dAb浓度。一旦测出dAb所需浓度，将预期Kd左右的MSA抗原浓度范围与dAb预混合并孵育过夜。然后以30μl/分钟的高流速，测定各预混物中dAb与MSA包被的biacore芯片的结合。用所得曲线绘制Klotz曲线，根据曲线估计对于MSA16的Kd为200nM，而对于MSA26则为70nM(图17A和图17B)。dAbs MSA16 and MSA26 were expressed in E. coli periplasm and purified by batch adsorption onto protein L-Sepharose affinity resin (Affitech, Norway), followed by elution with glycine (pH 2.2). The purified dAbs were then analyzed by inhibition biacore to determine theKd . Briefly, purified MSA16 and MS A26 were tested to determine the concentration of dAb required to achieve a 200RU response on a high-density MSA-coated biacore CM5 chip. Once the desired concentration of dAb has been determined, the MSA antigen concentration range around the expectedKd is premixed with the dAb and incubated overnight. Binding of dAbs in each premix to MSA-coated biacore chips was then assayed at a high flow rate of 30 μl/min. Klotz curves were plotted from the obtained curves, from which the Kd was estimated to be 200 nM for MSA16 and 70 nM for MSA26 ( FIGS. 17A and 17B ).
然后，将克隆MSA16和MSA26克隆到表达载体中，所述载体带有HA标记(核酸序列：TATCCTTATGATGTTCCTGATTATGCA(SEQ ID NO：373)和氨基酸序列：YPYDVPDYA(SEQ ID NO：374))，2-10mg量在大肠杆菌中表达，用蛋白L-琼脂糖亲和树脂(Affitech，Norway)并用甘氨酸(pH2.2)洗脱而从上清液中纯化。测定dAb的小鼠血清半衰期。给CD1小鼠单次静脉注射约1.5mg/kg的MSA26和MSA16。通过山羊抗HA(Abcam，UK)捕获和用4％Marvel封闭的蛋白L-HRP(invitrogen)测定ELISA，分析血清水平。用0.05％吐温PBS洗涤。在1x小鼠血清存在下，绘制已知浓度的dAb的标准曲线，以保证与试验样品的可比性。用2区室模型建模，显示MSA26的tα为0.16小时，tβ为14.5小时，曲线下面积(AUC)为465小时.mg/ml(数据未显示)，MSA16的tα为0.98小时，tβ为36.5小时，AUC为913小时.mg/ml(图18)。与HEL4(一种抗鸡卵清溶菌酶dAb)相比，这两个抗MSA克隆具有相当长的半衰期，其tα为0.06小时，tβ为0.34小时。Then, the clones MSA16 and MSA26 are cloned into an expression vector with a HA marker (nucleic acid sequence: TATCCTTATGATGTTCCTGATTATGCA (SEQ ID NO: 373) and amino acid sequence: YPYDVPDYA (SEQ ID NO: 374)), 2-10 mg amount Expressed in E. coli, purified from the supernatant using protein L-sepharose affinity resin (Affitech, Norway) and eluted with glycine (pH 2.2). The mouse serum half-life of the dAbs was determined. MSA26 and MSA16 were given a single intravenous injection of about 1.5 mg/kg to CD1 mice. Serum levels were analyzed by goat anti-HA (Abeam, UK) capture and ELISA with 4% Marvel blocked protein L-HRP (invitrogen). Wash with 0.05% Tween PBS. In the presence of 1x mouse serum, a standard curve of known concentrations of dAb was prepared to ensure comparability with test samples. Modeling with a 2-compartment model showed that tα for MSA26 was 0.16 hours, tβ was 14.5 hours, and the area under the curve (AUC) was 465 hours.mg/ml (data not shown), and tα for MSA16 was 0.98 hours, tβ was 36.5 hours, and AUC was 913 hours.mg/ml (Figure 18). These two anti-MSA clones had considerably longer half-lives compared to HEL4, an anti-chicken egg white lysozyme dAb, with a tα of 0.06 hours and a tβ of 0.34 hours.
实施例11.VH-VH和Vκ-Vκ双特异性Fab样片段的产生Example 11. Generation ofVH -VH and Vκ-Vκ bispecific Fab-like fragments
该实施例描述了制备VH-VH和Vκ-Vκ双特异性抗体(作为Fab样片段)的方法。在构建各种所述Fab样片段之前，按照实施例9所述的类似方法，首先从dAb文库中选出能结合所选靶标的dAb。分离出一种能结合鸡卵溶菌酶(Sigma)的VH dAb即HEL4，也分离出能结合TNFα受体(R和D系统)的第二VH dAb(TAR2h-5)。这些序列在序列表中给出。通过选择和亲和力成熟，分离出能结合TNFα(TAR1-5-19)的Vκ dAb，其序列也在序列表中给出。实施例9中描述的第二Vκ dAb(MSA26)(其序列见图17B)也可用于这些实验。This example describes a method for making VH-VH and VK-VK bispecific antibodies (as Fab-like fragments) . Before constructing various Fab-like fragments, according to the similar method described in Example 9, dAbs capable of binding the selected target were first selected from the dAb library. OneVH dAb, HEL4, which binds chicken egg lysozyme (Sigma), was isolated as well as a secondVH dAb (TAR2h-5), which binds TNF[alpha] receptors (R and D systems). These sequences are given in the Sequence Listing. By selection and affinity maturation, a VK dAb capable of binding TNFa (TAR1-5-19) was isolated and its sequence is also given in the Sequence Listing. A second VK dAb (MSA26) described in Example 9 (see Figure 17B for its sequence) was also used in these experiments.
将来自含有上述4种dAb的表达载体的DNA用酶SalI和NotI消化，切下dAb的DNA编码区。消化物通过琼脂糖凝胶电泳，纯化出预期大小的条带(300-400bp)，切下该条带，再用Qiagen凝胶纯化试剂盒(Qiagen，UK)进行凝胶纯化。再将编码dAb的DNA插入到CH载体或Cκ载体中(图8和9)，如下表所示。The DNA from the expression vector containing the above four dAbs was digested with enzymes SalI and NotI, and the DNA coding regions of the dAbs were excised. The digest was subjected to agarose gel electrophoresis to purify a band of expected size (300-400 bp), which was excised and gel-purified with Qiagen Gel Purification Kit (Qiagen, UK). The dAb-encoding DNA was then inserted into the CH vector or CK vector (Figures 8 and 9), as shown in the table below.
表9.Table 9.
VH CH和VH Cκ构建体共转化HB2151细胞。另外，Vκ CH和Vκ Cκ构建体共转化HB2151细胞。将各共转化细胞系的培养物培养过夜(在含有5％葡萄糖、10μg/ml氯霉素和100μg/ml氨苄青霉素的2xTy中，以维持对CH质粒和Cκ质粒的抗生素选择)。用过夜培养物接种新鲜培养基(2xTy，10μg/ml氯霉素和100μg/ml氨苄青霉素)并培养至OD0.7-0.9，然后加入IPTG进行诱导，以表达它们的CH构建体和Cκ构建体。再通过蛋白A纯化(用于共转化的VH CH和VH Cκ)和MSA亲和树脂纯化(用于共转化的Vκ CH和Vκ Cκ)，对表达的Fab样片段进行纯化。TheVHCH andVH CK constructs co-transformed HB2151 cells. Additionally, the VκCH and Vκ CK constructs co-transformed HB2151 cells. Cultures of each co-transformed cell line were grown overnight (in 2xTy containing 5% glucose, 10 μg/ml chloramphenicol and 100 μg/ml ampicillin to maintain antibiotic selection for CH andCK plasmids). Overnight cultures were inoculated with fresh media (2xTy, 10 μg/ml chloramphenicol and 100 μg/ml ampicillin) and grown to OD 0.7-0.9 before induction with IPTG to express theirCH constructs and CK constructs body. The expressed Fab-like fragments were then purified by protein A purification (for co-transformedVHCH andVH CK) and MSA affinity resin purification (for co-transformed VκCH and Vκ CK).
VH-VH双特异性VH -VH bispecific
通过蛋白质凝胶电泳，测定VH CH和VH Cκ双特异性抗体的表达。将凝胶印迹后，通过myc标记和flag标记，在蛋白质印迹上检测Fab片段期望大小的条带，表明Fab样片段的VH CH和VH Cκ部分都存在。然后，为了确定双特异性抗体的两个半边是否存在于同一Fab样片段上，将ELISA板用100μl/孔3mg/ml鸡卵溶菌酶(HEL)的碳酸氢钠缓冲液，在4℃包被过夜。该板再用2％吐温PBS封闭(按照实施例1所述)，接着与VH CH/VH Cκ双特异性Fab样片段一起孵育。通过非关联链，用9e10(一种结合myc标记的单克隆抗体，Roche)和抗小鼠IgG-HRP(Amersham Pharmacia Biotech)，检测双特异性片段与HEL的结合。VH CH/VH Cκ双特异性Fab样片段的信号为0.154，相比之下，对于单独表达的VH Cκ链的背景信号为0.069。这证明Fab样片段对靶抗原具有结合特异性。The expression ofVHCH andVH CK bispecific antibodies was determined by protein gel electrophoresis. After the gel was blotted, a band of the expected size for the Fab fragment was detected on the Western blot by myc- and flag-tagging, indicating the presence of both theVHCH andVH CK moieties of the Fab-like fragment. Then, to determine whether the two halves of the bispecific antibody are present on the same Fab-like fragment, ELISA plates were coated with 100 μl/well of 3 mg/ml chicken egg lysozyme (HEL) in sodium bicarbonate buffer at 4°C overnight. The plate was then blocked with 2% Tween PBS (as described in Example 1), followed by incubation withtheVHCH /VHCK bispecific Fab-like fragment. Binding of the bispecific fragment to HEL was detected via the non-associated chain using 9e10 (a myc-tagged monoclonal antibody, Roche) and anti-mouse IgG-HRP (Amersham Pharmacia Biotech). TheVHCH /VH CK bispecific Fab-like fragment had a signal of 0.154 compared to a background signal of 0.069 for theVH CK chain expressed alone. This demonstrates that the Fab-like fragment has binding specificity for the target antigen.
Vκ-Vκ双特异性Vκ-Vκ bispecific
在MSA亲和树脂上纯化共转化的Vκ CH和Vκ Cκ双特异性Fab样片段之后，用所得蛋白质探测1μg/ml TNFα包被的ELISA板和10μg/ml MSA包被的ELISA板。不出所料，当在两种ELISA板上用蛋白L-HRP检测时，产生高于背景的信号(数据未显示)。这表明，能够结合MSA的蛋白质部分(并因此而在MSA亲和柱上纯化)也能在随后的ELISA中结合TNFα，这证实了抗体片段的双特异性。这样的蛋白质部分再用于随后的两个实验。第一个，1μg/ml TNFα包被的ELISA板用双特异性Vκ CH和Vκ Cκ Fab样片段进行探测，同时也用对照即结合TNFα的dAb进行探测，该对照的浓度为经计算在ELISA上得到相似信号的浓度。在2mg/ml MSA存在和不存在时，用双特异性dAb和对照dAb探测ELISA板。双特异性孔的信号降低超过50％，但dAb孔的信号却完全没有降低(参见图19a)。同一蛋白也用于受体测定(有和没有MSA)，也显示出与MSA的竞争性(参见图19c)。这证明MSA竞争性结合双特异性抗体和TNFα。After purification of co-transformed VκCH and Vκ CK bispecific Fab-like fragments on MSA affinity resin, 1 μg/ml TNFα-coated ELISA plates and 10 μg/ml MSA-coated ELISA plates were probed with the resulting protein. As expected, a signal above background was generated when detected with protein L-HRP on both ELISA plates (data not shown). This demonstrates that the portion of the protein capable of binding MSA (and thus purified on the MSA affinity column) also binds TNF[alpha] in the subsequent ELISA, confirming the bispecificity of the antibody fragment. Such protein fractions were reused for two subsequent experiments. In the first, 1 μg/ml TNFα-coated ELISA plates were probed with bispecific VκCH and Vκ Cκ Fab-like fragments, as well as a control, a TNFα-binding dAb, at a concentration calculated in the ELISA Concentrations at which similar signals were obtained. ELISA plates were probed with bispecific dAbs and control dAbs in the presence and absence of 2 mg/ml MSA. The signal was reduced by more than 50% for the bispecific wells but not at all for the dAb wells (see Figure 19a). The same protein was also used in receptor assays (with and without MSA) and also showed competition with MSA (see Figure 19c). This demonstrates that MSA competitively binds the bispecific antibody and TNFα.
实施例12.对小鼠血清白蛋白和TNFα具有特异性的Vκ_Vκ双特异性cys键合的双特异性抗体的产生Example 12. Generation of Vκ_Vκ bispecific cys-linked bispecific antibodies specific for mouse serum albumin and TNFα
该实施例描述了通过二硫键的化学偶联制备对小鼠血清白蛋白和TNFα都具有特异性的双特异性抗体片段的方法。MSA16(来自实施例1)和TAR1-5-19 dAb都再克隆到具有C端半胱氨酸和没有标记的基于pET的载体中。两种dAb表达水平均为4-10mg，用蛋白L-琼脂糖亲和树脂(Affitiech，Norway)从上清液中纯化出来。半胱氨酸标记的dAb再用二硫苏糖醇还原。TAR1-5-19 dAb再与二硫双吡啶偶联，封闭二硫键的再形成，从而形成PEP 1-5-19同型二聚体。再将两种不同dAb在pH6.5混合，促进形成二硫键并产生TAR1-5-19，MSA16 cys键合的异型二聚体。两种不同蛋白质缀合物的这种制备方法最初由King等(King TP，Li Y Kochoumian L Biochemistry，1978，第17卷：1499-506 Preparation of Protein Conjugates via intermoleculardisulfide bond formation)描述的。通过阳离子交换将异型二聚体与单体分开。通过SDS凝胶上存在预期大小的条带来证实分离。所得异型二聚体种类在TNF受体测定中进行测定，发现其中和TNF的IC50约为18nM。然后，用恒定浓度的异型二聚体(18nM)和系列稀释的MSA和HAS，重复受体测定。适当浓度范围(最多2mg/ml)的HAS的存在，并不引起二聚体抑制TNFα能力的下降。然而，加入MSA，以剂量依赖性方式引起二聚体抑制TNFα能力的下降(图20)。这证明MSA和TNFα竞争性结合cys键合的TAR1-5-19、MSA16二聚体。This example describes the preparation of bispecific antibody fragments specific for both mouse serum albumin and TNF[alpha] by chemical coupling of disulfide bonds. Both MSA16 (from Example 1) and TAR1-5-19 dAbs were recloned into pET-based vectors with a C-terminal cysteine and no tag. Both dAbs were expressed at 4-10 mg and were purified from the supernatant using protein L-Sepharose affinity resin (Affitiech, Norway). Cysteine-labeled dAbs were then reduced with dithiothreitol. The TAR1-5-19 dAb was then conjugated with dithiobispyridine to block the reformation of the disulfide bond, thereby forming a PEP 1-5-19 homodimer. The two different dAbs were then mixed at pH 6.5 to promote disulfide bond formation and generate TAR1-5-19, MSA16 cys-linked heterodimers. This preparation of conjugates of two different proteins was first described by King et al. (King TP, Li Y Kochoumian L Biochemistry, 1978, Vol. 17: 1499-506 Preparation of Protein Conjugates via inmoleculardisulfide bond formation). The heterodimer is separated from the monomer by cation exchange. Separation was confirmed by the presence of bands of the expected size on the SDS gel. The resulting heterodimeric species was assayed in a TNF receptor assay and found to neutralize TNF with anIC50 of approximately 18 nM. Receptor assays were then repeated with a constant concentration of heterodimer (18 nM) and serial dilutions of MSA and HAS. The presence of HAS in an appropriate concentration range (up to 2 mg/ml) did not cause a decrease in the ability of the dimer to inhibit TNFα. However, addition of MSA caused a decrease in the ability of the dimer to inhibit TNF[alpha] in a dose-dependent manner (Figure 20). This demonstrates that MSA and TNF[alpha] compete for binding to the cys-linked TAR1-5-19, MSA16 dimer.
数据汇总data summary
上述实施例的实验中所得数据的汇总见附录4。See
实施例13.抗小鼠TNFR1 dAb和抗人TNFR1 dAb的活性Example 13. Activity of anti-mouse TNFR1 dAb and anti-human TNFR1 dAb
表10.抗小鼠TNFR1 dAb的活性Table 10. Activity of anti-mouse TNFR1 dAbs
n/d，未检出n/d, not detected
表11.抗人TNFR1 dAb的活性Table 11. Activity of anti-human TNFR1 dAbs
n/d，未检出n/d, not detected
MRC-5 IL-8释放测定MRC-5 IL-8 Release Assay
在以下MRC-5细胞测定中，评价了某些能结合人TNFR1的dAb的活性。测定是基于在MRC-5细胞中TNF诱导的IL-8分泌，该测定方法是Alceson，L等，Journal of Biological Chemistry 271：30517-30523(1996)所述方法的改进方法，所述文献介绍了在HUVEC中IL-1对IL-8的诱导作用。用MRC-5细胞代替HUVEC细胞系，通过评价人TNFα对IL-8的诱导，测定了dAb的活性。简而言之，MRC-5细胞接种在微量滴定板上，将这些板与dAb和人TNFα(300pg/ml)一起孵育过夜。孵育后，吸出培养物上清液，通过夹心ELISA(R&D系统)测定上清液中的IL-8浓度。与仅与TNFα一起孵育的对照孔相比，抗TNFR1 dAb活性导致上清液中IL-8分泌降低。The activity of certain dAbs that bind human TNFRl was evaluated in the following MRC-5 cell assay. The assay is based on the TNF-induced IL-8 secretion in MRC-5 cells, which is an improved method of the method described in Alceson, L, etc., Journal of Biological Chemistry 271: 30517-30523 (1996), which describes Induction of IL-1 by IL-8 in HUVECs. The dAb activity was determined by evaluating the induction of IL-8 by human TNF[alpha] using MRC-5 cells instead of the HUVEC cell line. Briefly, MRC-5 cells were seeded on microtiter plates and these plates were incubated overnight with dAbs and human TNF[alpha] (300 pg/ml). After incubation, the culture supernatant was aspirated and the IL-8 concentration in the supernatant was determined by sandwich ELISA (R&D Systems). Anti-TNFR1 dAb activity resulted in decreased IL-8 secretion in the supernatant compared to control wells incubated with TNFα alone.
实施例14.小鼠脓毒性休克模型Example 14. Mouse Septic Shock Model
在良好建立的脓毒性休克综合征的实验模型中，评价抗TNFR1dAb的体内功效(Rothe等，Circulatory Shock 44：51-56，(1995))。在该模型中，LPS诱导的死亡取决于TNFR-1(p55)的活化。在该模型中，用D-半乳糖胺(D-GaIN)使小鼠对LPS毒性敏感。在该项研究中，对野生型动物致死的LPS剂量约为10ng。The in vivo efficacy of anti-TNFRl dAbs was evaluated in a well-established experimental model of septic shock syndrome (Rothe et al., Circulatory Shock 44:51-56, (1995)). In this model, LPS-induced death depends on the activation of TNFR-1 (p55). In this model, mice are sensitized to LPS toxicity with D-galactosamine (D-GaIN). In this study, the dose of LPS lethal to wild-type animals was approximately 10 ng.
腹膜内注射LPS(肠炎沙门氏菌(Salmonella enteritidis)，Sigma，USA)和D-半乳糖胺(D-GaIN，Sigma，USA)。D-GalN-致敏(10mg/小鼠)的对照小鼠在用LPS(10ng)攻击后18小时内死亡。在攻击后超过1天的周期中，记录非致敏小鼠的死亡率。LPS (Salmonella enteritidis, Sigma, USA) and D-galactosamine (D-GaIN, Sigma, USA) were injected intraperitoneally. D-GalN-sensitized (10 mg/mouse) control mice died within 18 hours after challenge with LPS (10 ng). Mortality of non-sensitized mice was recorded over a period of more than 1 day post-challenge.
给小鼠腹膜内注射双特异性配体(所述配体能结合小鼠TNFR1和小鼠血清白蛋白(TAR2m-21-23 3U TAR7m-16；TAR7m-16在本文中也称为MSA16))或ENBREL(entarecept；Immunex Corporation)，4小时后给予LPS。(参见表12)。在48小时内以4-6小时的间隔监控存活情况。用存活率证明抗小鼠TNFR1 dAb的功效。Mice were injected intraperitoneally with a dual-specific ligand (the ligand binds mouse TNFR1 and mouse serum albumin (TAR2m-21-23 3U TAR7m-16; TAR7m-16 is also referred to herein as MSA16)) or ENBREL(R) (entarecept; Immunex Corporation), followed by
表12.Table 12.
TAR2m-21-23 3U TAR7m-16是含有dAb并能结合小鼠TNFR1的双特异性配体，小鼠TNFR1通过肽接头与能结合小鼠血清白蛋白的dAb结合。编码TAR2m-21-23 3U TAR7m-16的核苷酸序列和双特异性配体的氨基酸序列分别见下列SEQ ID NO：375和SEQ ID NO：376。TAR2m-21-23 3U TAR7m-16 is a dual-specific ligand that contains a dAb and binds to mouse TNFR1, which binds to a dAb that binds to mouse serum albumin through a peptide linker. The nucleotide sequence encoding TAR2m-21-23 3U TAR7m-16 and the amino acid sequence of the dual-specific ligand are shown in the following SEQ ID NO: 375 and SEQ ID NO: 376, respectively.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCC
GCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGT
GGTACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCGGTACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTC
CAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTAT
ATTACTGTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGATTACTGTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAG
GGAACCCAGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGGGAACCCAGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG
CGGTGGCGGGTCGACGGACATCCAGATGACCCSGTCTCCATCCTCCCTGTCTGCATCGGTGGCGGGTCGACGGACATCCAGATGACCCSGTCTCCCATCCTCCCCTGTCTGCAT
CTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTATTAAGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTATTAAGCAT
TTAAAGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCTTAAAGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGC
ATCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGGACAGATCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGGACAG
ATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACGTACTACTGTATTTCACTTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACGTACTACTGT
CAACAGGGGGCTCGGTGGCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAACAACAGGGGGCTCGGTGGCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAA
ACGGGCGGCCGCAGAACAAAACTCATCTCAGAAGAGGATCTGAATACGGGCGGCCGCAGAACAAAACTCATCTCCAGAAGAGGATCTGAAT
(SEQ ID NO：375)(SEQ ID NO: 375)
EVQLLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGREVQLLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGR
GTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQ
GTQVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSIIKHGTQVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSIIKH
LKWYQQKPGKAPKLLIYGASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLKWYQQKPGKAPKLLIYGASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
QQGARWPQTFGQGTKVEIKRAAAEQKLISEEDLNQQGARWPQTFGQGTKVEIKRAAAEQKLISEEDLN
(SEQ ID NO：376)(SEQ ID NO: 376)
在TAR2m-21-23 3U TAR7m-16治疗组中有幸存者，这证明抗TNFR1 dAb对体内抑制受体活性是有效的，其结果证明效果是剂量依赖性的。此外，TAR2m-21-23 3U TAR7m-16的治疗功效比ENBREL(entarecept；Immunex Corporation)的功效更好。仅用TAR2m-21-23 3U TAR7m-16治疗的动物(第5组，没有LPS攻击)的存活率也证明，TAR2m-21-23 3U TAR7m-16无毒并且在体内通过受体交联而不激动受体。There were survivors in the TAR2m-21-23 3U TAR7m-16 treatment group, which demonstrated that the anti-TNFR1 dAb was effective in inhibiting receptor activity in vivo, and the results demonstrated that the effect was dose-dependent. Furthermore, the therapeutic efficacy of TAR2m-21-23 3U TAR7m-16 was better than that of ENBREL(R) (entarecept; Immunex Corporation). The survival rate of animals treated with only TAR2m-21-23 3U TAR7m-16 (
进一步研究证明，当TNFα不存在时，抗TNFR1 dAb不激动TNFR1(起到TNFR1激动剂的作用)。将L929细胞在分别含有以下成分之一的培养基中培养：不同浓度的TAR2m-21-23单体、与市售抗myc抗体(9E10)交联的TAR2m-21-23单体、TAR2m-21-23 3UTAR7m-16或TAR2m-21-23 40K PEG。就与抗myc抗体交联的TAR2m-21-23单体而言，将dAb和抗体按2∶1比例混合，在室温下预孵育1小时，刺激体内免疫交联效应，然后进行培养。浓度为3000nM的TAR2m-21-23单体与L929细胞一起孵育。TAR2m-21-23单体和抗Myc抗体与浓度为3000nM的dAb一起孵育。将浓度为25nM、83.3nM、250nM、833nM和2500nM的TAR2m-21-23 3U TAR7m-16与细胞一起孵育。将浓度为158.25nM、527.5nM、1582.5nM、5275nM和15825nM的TAR2m-21-23 40K PEG与细胞一起孵育。孵育过夜后，按照L929细胞的细胞毒性测定所述，评价细胞存活率。结果表明，与不同数量的dAb一起孵育，不导致培养物中无活力细胞数量的增加。将L929细胞与10nM、1nM和0.1nM市售抗TNFR1 IgG抗体一起孵育，导致以剂量依赖方式增加无活力细胞，因此证明了这些细胞对TNFR1介导的激动作用的敏感性(图26)。Further studies demonstrated that the anti-TNFR1 dAb does not agonize TNFR1 (acts as a TNFR1 agonist) in the absence of TNFα. L929 cells were cultured in media containing one of the following components: different concentrations of TAR2m-21-23 monomer, TAR2m-21-23 monomer cross-linked with commercially available anti-myc antibody (9E10), TAR2m-21 -23 3UTAR7m-16 or TAR2m-21-23 40K PEG. For TAR2m-21-23 monomer cross-linked with anti-myc antibody, dAb and antibody were mixed in a 2:1 ratio, pre-incubated for 1 hour at room temperature to stimulate the immune cross-linking effect in vivo, and then incubated. TAR2m-21-23 monomer at a concentration of 3000 nM was incubated with L929 cells. TAR2m-21-23 monomer and anti-Myc antibody were incubated with dAb at a concentration of 3000 nM. TAR2m-21-23 3U TAR7m-16 at concentrations of 25nM, 83.3nM, 250nM, 833nM and 2500nM were incubated with the cells. TAR2m-21-23 40K PEG at concentrations of 158.25nM, 527.5nM, 1582.5nM, 5275nM and 15825nM were incubated with the cells. After overnight incubation, cell viability was assessed as described for the cytotoxicity assay of L929 cells. The results showed that incubation with different amounts of dAb did not lead to an increase in the number of non-viable cells in the culture. Incubation of L929 cells with 10 nM, 1 nM and 0.1 nM of a commercially available anti-TNFR1 IgG antibody resulted in a dose-dependent increase in non-viable cells, thus demonstrating the sensitivity of these cells to TNFR1-mediated agonism ( FIG. 26 ).
实施例15.慢性炎性疾病的模型Example 15. Models of Chronic Inflammatory Diseases
A.小鼠胶原诱发的关节炎模型A. Mouse collagen-induced arthritis model
给DBA/1小鼠注射一次Arthrogen-CIA佐剂和Arthrogen-CIA胶原(MD-biosciences)的乳剂。在第21天，从研究中除去具有高关节炎评分的动物，剩下的动物分成10组，雄性和雌性动物数量相等。在第21天，治疗开始用腹膜内注射盐水、ENBREL(entarecept；Immunex Corporation)或TAR2m-21-23 40k PEG，并持续28天。对动物四肢的每肢都进行临床关节炎评分，分值为0-4，0分代表正常肢，4分代表涉及多关节的最严重的发炎肢。DBA/1 mice were injected once with an emulsion of Arthrogen-CIA adjuvant and Arthrogen-CIA collagen (MD-biosciences). On
在该模型中，四肢关节炎评分总和从最大的16降至(a)14-15，(b)12-15，(c)10-15，(d)9-15，(e)7-15，(f)5-15，(g)3-15，或(h)1-15是有益结果。可产生的有益结果是四肢关节炎评分总和为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。与未处理的对照组相比，关节炎发作延迟也是有益结果。In this model, the sum of arthritis scores in the extremities decreased from a maximum of 16 to (a) 14-15, (b) 12-15, (c) 10-15, (d) 9-15, (e) 7-15 , (f) 5-15, (g) 3-15, or (h) 1-15 are beneficial results. Beneficial results can be produced with a sum of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 arthritis scores in the extremities. Delayed onset of arthritis was also a beneficial outcome compared to untreated controls.
临床评分清楚表明，与盐水对照相比，用TAR2m-21-23 40k PEG进行治疗，对抑制关节炎发展具有非常好的作用，而且TAR2m-21-2340k PEG治疗比用ENBREL(entarecept；Immunex Corporation)更好。这首次证明了抑制TNFR1对治疗慢性炎性疾病模型是有效的。Clinical scores clearly demonstrated that treatment with TAR2m-21-23 40k PEG had a very good effect on inhibiting the development of arthritis compared to saline controls, and TAR2m-21-23 40k PEG treatment was more effective than ENBREL(R) (entarecept; Immunex Corporation )better. This is the first demonstration that inhibition of TNFR1 is effective in treating models of chronic inflammatory disease.
B.小鼠IBD和关节炎的ΔARE模型B. ΔARE model of mouse IBD and arthritis
在TNF mRNA的3′富含AU的元件(ARE)中带有定向缺失的小鼠(称为TnfΔARE小鼠)过量产生TNF并发生炎性肠病，该病与节段性回肠炎在组织病理学上相似。(Kontoyiannis等，J.Exp.Med.196：1563-74(2002))。在这些小鼠中，节段性回肠炎样疾病在4-8周龄时发生，并且这些动物还出现了类风湿性关节炎的临床症状。Mice with a targeted deletion in the 3' AU-rich element (ARE) of TNF mRNA (termed TnfΔARE mice) overproduce TNF and develop inflammatory bowel disease, which is associated with Crohn's disease in tissue Pathologically similar. (Kontoyiannis et al., J. Exp. Med. 196:1563-74 (2002)). In these mice, Crohn's disease-like disease developed at 4-8 weeks of age, and the animals also developed clinical signs of rheumatoid arthritis.
在TnfΔARE小鼠，评价能结合小鼠TNFR1的dAb抑制节段性回肠炎样病理和关节炎的功效。ENBREL(entarecept；ImmunexCorporation)用作阳性对照。按照表13所示的给药和剂量方案，将这些药物通过腹膜内注射给予。In TnfΔARE mice, the efficacy of dAbs that bind mouse TNFRl to inhibit Crohn's-like pathology and arthritis was evaluated. ENBREL(R) (entarecept; Immunex Corporation) was used as a positive control. These drugs were administered by intraperitoneal injection according to the dosing and dosage schedule shown in Table 13.
所研究的dAb包括：The dAbs studied included:
1)具有一个40kD PEG部分的TAR2m-21-23 PEG化；1) PEGylation of TAR2m-21-23 with a 40kD PEG moiety;
2)能结合小鼠TNFR-1和小鼠血清白蛋白的双特异性TAR2m-21-23 3U TAR7m-16。2) Bispecific TAR2m-21-23 3U TAR7m-16 that can bind mouse TNFR-1 and mouse serum albumin.
表13.Table 13.
在给药周期结束时，处死小鼠，切下回肠末端和近侧结肠进行分析。At the end of the dosing period, mice were sacrificed and the terminal ileum and proximal colon were excised for analysis.
对于组织学分析，将组织样品切片并用苏木精和伊红染色，用半定量评分系统对急性和慢性炎症进行评分。评分规定如下：急性炎症评分0＝0-1多形核(PMN)细胞/高倍视野(PMN/hpf)；1＝2-10PMN/hpf粘膜内；2＝11-20 PMN/hpf粘膜内；3＝21-30 PMN/hpf粘膜内或11-20 PMN/hpf延伸到粘膜肌层下；4＝＞30 PMN/hpf粘膜内或＞20 PMN/hpf延伸到粘膜肌层下；慢性炎症评分0＝0-10单核白细胞(ML)/hpf(ML/hpf)粘膜内；1＝11-20 ML/hpf粘膜内；2＝21-30ML/hpf粘膜内或11-20 ML/hpf延伸到粘膜肌层下；3＝31-40 ML/hpf粘膜内或滤泡增生；和4＝＞40 ML/hpf粘膜内或＞30 ML/hpf延伸到粘膜肌层下或滤泡增生。For histological analysis, tissue samples were sectioned and stained with hematoxylin and eosin, and acute and chronic inflammation were scored using a semiquantitative scoring system. Scores are defined as follows:
按照以下系统，每周对关节炎的宏观表型体征进行评分：0＝没有关节炎(正常外观和屈曲)；1＝轻度关节炎(关节变形)；2＝中度关节炎(肿胀，关节变形)；3＝重度关节炎(严重受损的运动)。Macroscopic phenotypic signs of arthritis were scored weekly according to the following system: 0 = no arthritis (normal appearance and flexion); 1 = mild arthritis (joint deformation); 2 = moderate arthritis (swelling, joint deformation); 3 = severe arthritis (severely impaired movement).
在小鼠关节炎ΔARE模型中，TAR2m-21-23 dAb证明有良好的体内功效，与40kD PEG化单体(TAR2m21-23 40kD PEG)和双特异性抗TNFR1/抗SA形式(TAR2m-21-23 3U TAR7m-16)一样。在第9周，TAR2m-21-23和TAR2m-21-23 3U TAR7m-16治疗组的平均关节炎评分小于0.4。相比之下，盐水对照组的中度至严重关节炎的平均关节炎评分＞1.0。与TAR2m-21-23和TAR2m-21-23 3U TAR7m-16(每周用药2次)相比，用ENBREL(entarecept；Immunex Corporation)治疗组(每周用药3次)的平均评分为0.5-1.0。这些结果表明，用能结合TNFR1的dAb形式的疗法，是高度有效的抗关节炎疗法，所研究的PEG化dAb和双特异性dAb形式都是对慢性炎性疾病十分有效的药物。而且，这些结果还证明，能结合TNFR1的dAb仅以拮抗方式结合受体。In the mouse arthritis ΔARE model, the TAR2m-21-23 dAb demonstrated good in vivo efficacy, comparable to the 40kD PEGylated monomer (TAR2m21-23 40kD PEG) and the bispecific anti-TNFR1/anti-SA format (TAR2m-21- 23 3U TAR7m-16). At
C.小鼠IBD DSS模型C. Mouse IBD DSS Model
给予小鼠溶于饮用水的葡聚糖硫酸钠(DSS)可诱发IBD。(参见例如Okayasu I.等，Gastroenterology 98：694-702(1990)；Podolsky K.，J.Gasteroenterol.38 supp1 XV：63-66(2003))。无幽门螺杆菌(H.pylori)的成年BDF1小鼠笼养2周，稳定它们的昼夜节律。所有小鼠都单独放在带有专门的无病原体(SPF)屏障单元的通风笼子里，在12小时光-暗周期下。让动物随意进食饮水。Administration of dextran sodium sulfate (DSS) dissolved in drinking water to mice induces IBD. (See, eg, Okayasu I. et al., Gastroenterology 98:694-702 (1990); Podolsky K., J. Gasteroenterol. 38 suppl XV:63-66 (2003)). H. pylori-free adult BDF1 mice were housed for 2 weeks to stabilize their circadian rhythm. All mice were housed individually in ventilated cages with specialized pathogen-free (SPF) barrier units under a 12-h light-dark cycle. Animals were given food and water ad libitum.
研究进行7天。饮用水含有5％DSS，用于整个研究阶段中。所有动物在1-7天的早晨(0900-1000)和傍晚(1600-1700)进行治疗。(参见表14)。第1天相当于单次预防剂量。所有动物每天称重，任何腹泻发生率都记录下来。在最后一次治疗后24小时，处死所有动物，在处死前40分钟给予溴脱氧尿苷脉冲。从动物身上切下远侧大肠。将远侧大肠的小样品放在“RNAlater”中，剩余部分在Carnoy氏固定剂中固定，石蜡包埋，切片(非连续切片/每玻片)并用苏木精和伊红染色。切片用目测评价IBD严重程度并给予严重程度评分。进行组织学分析，测定平均损害面积(溃疡面积)、平均上皮面积和平均壁内炎症面积。The study was conducted for 7 days. Drinking water containing 5% DSS was used throughout the study period. All animals were treated in the morning (0900-1000) and evening (1600-1700) on days 1-7. (See Table 14).
采用Zeiss Axiohome显微镜，大肠H&E横切片可用于记录一系列组织尺寸，这可准确量化面积。对于每个横切片，可以测量上皮加上固有层的面积和结缔组织面积。然后可单独计算出上皮面积，差就是固有层面积。在正常组织中，该组织对面积的相对贡献约为10％，但是它随炎症的增加而上升。因此上皮∶固有层的相对比例发生改变。Using a Zeiss Axiohome microscope, H&E cross sections of the large intestine can be used to document a range of tissue dimensions, which allows accurate quantification of area. For each transverse section, the area of epithelium plus lamina propria and connective tissue area can be measured. The epithelial area can then be calculated separately, and the difference is the lamina propria area. In normal tissue, the relative contribution of this tissue to area is about 10%, but it rises with increasing inflammation. The relative ratio of epithelium:lamina propria is thus altered.
随着严重程度增加，该面积的深度变狭窄(由于溃疡)且结肠长度变短。这些现象共同引起内腔横切面面积增加。因此，这一参数可用于确定疾病的严重程度。With increasing severity, the depth of this area narrows (due to ulceration) and the length of the colon becomes shorter. Together, these phenomena lead to an increase in the cross-sectional area of the lumen. Therefore, this parameter can be used to determine the severity of the disease.
组织样品可通过显微镜观察并进行严重程度评分，其中0＝无炎症；1＝隐窝底部周围轻度炎症；2＝大量炎症浸润，粘膜结构被破坏；3＝大量炎症浸润，粘膜结构被破坏以及溃疡。Tissue samples can be viewed microscopically and scored for severity, where 0 = no inflammation; 1 = mild inflammation around the base of the crypt; 2 = massive inflammatory infiltrate with disrupted mucosal structure; 3 = massive inflammatory infiltrate with disrupted mucosal structure and ulcer.
在该模型中，相对于盐水对照组严重程度评分而言，当治疗导致严重程度评分下降，就表明有效。例如治疗组严重程度评分可以降低0.1至约1、1至约2、或2至约3。评分约2以下、1至约2、或1以下，表明有效。In this model, treatment is indicated to be effective when it results in a decrease in severity scores relative to the severity scores of the saline control group. For example, the treatment group severity score can be reduced by 0.1 to about 1, 1 to about 2, or 2 to about 3. A score of about 2 or less, 1 to about 2, or less than 1 indicates efficacy.
9组(每组6只)动物如下进行治疗：9 groups (6 animals in each group) were treated as follows:
表14.Table 14.
经口管饲将给予ZANTAC(盐酸雷尼替丁；GlaxoSmithKline)。ZANTAC(R) (ranitidine hydrochloride; GlaxoSmithKline) will be administered by oral gavage.
D.小鼠慢性阻塞性肺病(COPD)模型D. Mouse Chronic Obstructive Pulmonary Disease (COPD) Model
对抗TNFR1 dAb在小鼠亚慢性吸烟(TS)模型中疾病发展的功效进行评价。(参见例如Wright JL和Churg A.，Chest 122：306S-309S(2002))。抗小鼠TNFR1 dAb经腹膜内注射给予，每48小时一次(在第一次暴露给TS之前24小时开始)并且以血清半衰期延长形式(例如包含抗SA dAb的PEG化、双特异性配体)给予。The efficacy of anti-TNFR1 dAbs on disease development in a mouse model of subchronic smoking (TS) was evaluated. (See eg Wright JL and Churg A., Chest 122:306S-309S (2002)). Anti-mouse TNFR1 dAb given by intraperitoneal injection every 48 hours (starting 24 hours before first exposure to TS) and in a serum half-life-extended form (e.g., PEGylated, dual-specific ligand containing anti-SA dAb) give.
或者，抗TNFR1 dAb将通过鼻内给药方式给予，每24小时一次(在第一次暴露给TS之前4小时开始)并且以单体dAb给予。用ENBREL(entarecept；Immunex Corporation)作为阳性对照。每天都接触TS，研究持续1-2周。(参见例如Vitalis等，Eru.Respir.J.，11：664-669(1998))。最后一次TS暴露之后，分析支气管肺泡灌洗的总细胞计数和细胞分类计数，包括嗜中性粒细胞、嗜酸性粒细胞、巨噬细胞和T淋巴细胞亚类。将肺叶在10％缓冲福尔马林中固定，分析组织切片中肺泡和肺泡管的增大、小气管壁增厚情况，并进行细胞计数，包括嗜中性粒细胞、嗜酸性粒细胞、巨噬细胞和T淋巴细胞亚类。因ES暴露而升高的嗜中性粒细胞、嗜酸性粒细胞、巨噬细胞和T-淋巴细胞亚类数目的下降，以及TS诱导的肺泡和肺泡管的增大和小气管壁增厚的降低，证明了功效。Alternatively, the anti-TNFR1 dAb will be administered intranasally every 24 hours (starting 4 hours before the first exposure to TS) and as a monomeric dAb. ENBREL(R) (entarecept; Immunex Corporation) was used as a positive control. TS was exposed daily and the study lasted 1-2 weeks. (See eg Vitalis et al., Eru. Respir. J., 11:664-669 (1998)). After the last TS exposure, bronchoalveolar lavage was analyzed for total and differential cell counts, including neutrophils, eosinophils, macrophages, and T lymphocyte subsets. The lung lobes were fixed in 10% buffered formalin, and the tissue sections were analyzed for enlargement of alveoli and alveolar ducts, thickening of small airway walls, and cell counts, including neutrophils, eosinophils, macrophages and T lymphocyte subsets. Decreased numbers of neutrophils, eosinophils, macrophages, and T-lymphocyte subsets, which were elevated by ES exposure, and decreased TS-induced enlargement of alveoli and alveolar ducts and small airway wall thickening , demonstrating the efficacy.
实施例16.重组嵌合TNFR1分子的构建和表达Example 16. Construction and expression of recombinant chimeric TNFR1 molecules
该实施例说明了由不同鼠TNFR1域和人TNFR1域组成的分子的制备方法(Banner DW等，Cell，75(3)：431-45(1993))，使得该分子含有4种已定义的TNFR1胞外区，但是这些在小鼠和人TNFR1蛋白间的衍生差异是不同的。根据不同结构域的作用和功能，所产生的嵌合受体共享人TNFR1和小鼠TNFR1的特性。该分子提供对结合人或小鼠TNFR1的dAb、抗体及其抗原结合片段和其它分子(例如有机化合物、NCE；或蛋白质域，例如亲和体、LDL受体域或EGF域)的结构域特异性的评价方法。This example illustrates the preparation of molecules consisting of different murine TNFR1 domains and human TNFR1 domains (Banner DW et al., Cell, 75(3):431-45 (1993)) such that the molecule contains the four defined TNFR1 extracellular domains, but these differ in their derivation between mouse and human TNFR1 proteins. According to the roles and functions of different domains, the generated chimeric receptors shared the properties of human TNFR1 and mouse TNFR1. This molecule provides domain specificity for dAbs, antibodies and antigen-binding fragments thereof, and other molecules (e.g., organic compounds, NCE; or protein domains, such as Affibodies, LDL receptor domains, or EGF domains) that bind human or mouse TNFR1 sexual evaluation method.
方法method
将人和小鼠TNFR1序列先通过EcoRI和NotI限制性内切核酸酶位点克隆到毕赤酵母表达载体pPicZoα(Invitrogen)中。模板小鼠TNFR1 DNA(因此嵌合受体构建体结束于鼠域4)含有3′6x组氨酸标记。人TNFR1(因此嵌合受体构建体结束于人域4)在序列的3′端同时含有Myc和6x组氨酸标记。Human and mouse TNFR1 sequences were first cloned into Pichia pastoris expression vector pPicZoα (Invitrogen) via EcoRI and NotI restriction endonuclease sites. The template mouse TNFR1 DNA (and thus the chimeric receptor construct ending in murine domain 4) contained a 3' 6x histidine tag. Human TNFR1 (and thus the chimeric receptor construct ending in human domain 4) contains both a Myc and a 6x histidine tag at the 3' end of the sequence.
按照标准PCR条件，采用RubyTaq DNA聚合酶(USB Corporation，Cleveland，Ohio)、100ng模板DNA(包含全长mTNFR1或hTNFR1DNA相关DNA小量制备物模板)，进行起始PCR。Initial PCR was performed using RubyTaq DNA polymerase (USB Corporation, Cleveland, Ohio) and 100 ng of template DNA (including full-length mTNFR1 or hTNFR1 DNA-related DNA miniprep template) according to standard PCR conditions.
典型的PCR反应如下进行：含聚合酶的25μl 10X RubyTaq PCR缓冲液；2μl第一引物(来自10μM贮液)；2μl第二引物(来自10μl贮液)；1μl(100ng)全长TNFR1模板DNA；20μl dH2O(至终体积50μl)。反应在薄壁管中进行，并放在热循环仪中，其中按照以下参数进行反应。A typical PCR reaction is performed as follows: 25 μl 10X RubyTaq PCR buffer containing polymerase; 2 μl primary primer (from 10 μM stock); 2 μl second primer (from 10 μl stock); 1 μl (100 ng) full-length TNFR1 template DNA; 20 μl dH2 O (to a final volume of 50 μl). Reactions were carried out in thin-walled tubes and placed in a thermal cycler where the reactions were carried out according to the following parameters.
用于产生嵌合构建体的起始PCR反应的概述Overview of the initial PCR reactions used to generate chimeric constructs
*注释：H＝人结构域；M＝小鼠结构域；例如MHHH＝小鼠结构域1，人结构域2-4。* Note: H = human domain; M = mouse domain; eg MHHH =
将这些起始PCR所产生的PCR产物从1％琼脂糖凝胶上切下来，用凝胶纯化试剂盒(Qiagen)进行纯化，然后洗脱到50μl dH2O中。PCR products from these initial PCRs were excised from 1% agarose gels, purified using a gel purification kit (Qiagen) and eluted into 50 [mu]ldH2O .
用于产生嵌合TNFR1构建体的引物Primers used to generate chimeric TNFR1 constructs
SOE PCRSOE PCR
装配PCR(也称为′pull-through′或剪接重叠延伸(SOE)，参见Gene，15：77(1)：61-8(1989))使初次PCR产物无需消化或连接而联合在一起，使用初次PCR产物互补末端。在该过程中，初次产物联合在一起并变性，接着在Taq DNA聚合酶和dNTP存在下，让它们的互补末端一起退火。重复退火和延伸多次循环，导致互补链补平，产生全长模板。添加邻接全长构建体盒的引物，进行常规PCR，扩增装配产物。进行SOE PCR，使来自上述起始PCR的不同TNFR1域一起退火并扩增。装配SOE PCR如下进行：含MgCl2的40μl 10×PCR缓冲液；～2μl(100ng)起始PCR1的纯化产物；～2μl(100ng)起始PCR2的纯化产物；36μl dH2O(至终体积80μl)。在以下装配步骤后加入SOE引物混合物：2μl 5′侧翼引物(引物1)；2μl 3′侧翼引物(引物2)；10μl 10×PCR缓冲液；6μl dH2O(至终体积20μl)。Assembly PCR (also known as 'pull-through' or splicing overlap extension (SOE), see Gene, 15:77(1):61-8 (1989)) combines primary PCR products without digestion or ligation, using Complementary ends of primary PCR products. In this process, primary products are brought together and denatured, followed by annealing together of their complementary ends in the presence of Taq DNA polymerase and dNTPs. Repeated multiple cycles of annealing and extension result in complementary strand fill-in, yielding a full-length template. A primer is added adjacent to the full-length construct cassette and conventional PCR is performed to amplify the assembled product. SOE PCR was performed to anneal together and amplify the different TNFR1 domains from the initial PCR described above. Assembly SOE PCR was performed as follows: 40 μl of 10×PCR buffer containingMgCl2 ; ~2 μl (100 ng) of the purified product of the starting PCR1; ~2 μl (100 ng) of the purified product of the starting PCR2; 36 μl ofdH2O (to a final volume of 80 μl ). The SOE primer mix was added after the following assembly steps: 2 μl 5' flanking primer (primer 1); 2 μl 3' flanking primer (primer 2); 10
使用下述程序进行PCR反应。起始装配循环需要约45分钟，然后将热循环仪设置停顿在94℃。20μl引物混合物加入到各反应物中并混合。PCR reactions were performed using the procedure described below. The initial assembly cycle takes approximately 45 minutes, after which the thermal cycler is set to dwell at 94°C. 20 μl of primer mix was added to each reaction and mixed.
步骤1装配
步骤2扩增(停顿在94℃，然后加入引物混合物)
将3-5μl各反应物在1％琼脂糖凝胶上电泳，检查PCR产物。PCR products were checked by running 3-5 μl of each reaction on a 1% agarose gel.
3)将装配TNFR1嵌合体克隆到毕赤酵母表达载体中3) Cloning the assembled TNFR1 chimera into the expression vector of Pichia pastoris
pPicZα载体(Invitrogen)依次用EcoRI和NotI酶消化，再用Chromaspin TE-1000凝胶过滤柱(Clontech，Mountain View，CA)纯化。The pPicZα vector (Invitrogen) was sequentially digested with EcoRI and NotI enzymes, and then purified with a Chromaspin TE-1000 gel filtration column (Clontech, Mountain View, CA).
4)将TNFR1嵌合构建体转化到大肠杆菌中4) Transformation of the TNFR1 chimeric construct into E. coli
连接后的嵌合构建体转化到HB2151电感受态(electrocompetent)大肠杆菌细胞中，在低盐LB培养基中回收1小时，然后接种在含有0.25μg/ml零霉素(ZEOCIN)、含腐草霉素D的抗生素制剂(Cayla，Toulouse，法国)的低盐LB琼脂上，在37℃培养24小时。单菌落经序列证实，以保证表达载体内嵌合构建体有正确序列，大规模Maxiprep质粒制备物由各嵌合构建体载体组成。The ligated chimeric construct was transformed into HB2151 electrocompetent (electrocompetent) Escherichia coli cells, recovered in low-salt LB medium for 1 hour, and then inoculated in a medium containing 0.25 μg/ml zeocin (ZEOCIN), containing saprophyllum Mycin D antibiotic preparation (Cayla, Toulouse, France) was cultured on low-salt LB agar at 37°C for 24 hours. Single colonies were sequence-confirmed to ensure the correct sequence of the chimeric constructs within the expression vectors, and large-scale Maxiprep plasmid preparations consisted of each chimeric construct vector.
所制备的嵌合构建体的核苷酸序列见下文。嵌合构建体根据其结构域来源而命名(从左到右是域1至域4)。例如，HMMM含有人域1和小鼠域2-4。含有小鼠域4的嵌合蛋白仅具有His标记并且缺乏跨膜区和域4之间的间隔区。The nucleotide sequences of the prepared chimeric constructs are given below. Chimeric constructs are named according to their domain origin (
HMMMHMMM
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTAGTGTGTGTCCCCAAGGAAAATATCCACCTCAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGCCTCAGTTGCGGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGCCTCAGTTGC
AAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTGACAAGGACAAAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCCTCTCCTTGCCAAGCTGACAAGGACA
CGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTGCGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTG
CGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAACCGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAAC
ACCGTGTGTAACTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCCACCGTGTGTAACTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCC
ACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAGCGCTCATCATCATCATCATCAACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAAGCGCTCATCATCATCATCATCA
TTAATGA(SEQ ID NO：619)TTAATGA (SEQ ID NO: 619)
HHHMHHHM
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTAGTGTGTGTCCCCAAGGAAAATATCCACCTCAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC
TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACATCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTTCTTGCACAGTGGACCGGGACA
CCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCAGTG
CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAACCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC
ACCGTGTGCACCTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCCACCGTGTGCACCTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCC
ACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAGCGCTCATCATCATCATCATCAACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAAGCGCTCATCATCATCATCATCA
TTAATGA(SEQ ID NO：620)TTAATGA (SEQ ID NO: 620)
HHMHHHMH
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTAGTGTGTGTCCCCAAGGAAAATATCCACCTCAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC
TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACATCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTTCTTGCACAGTGGACCGGGACA
CCGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTGCCGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTG
CGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAACCGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAAC
ACCGTGTGTAACTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACCGTGTGTAACTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA
ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG
CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAACACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCCAGAA
GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA(SEQ ID NO：621)GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA (SEQ ID NO: 621)
HMHHHMHH
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTAGTGTGTGTCCCCAAGGAAAATATCCACCTCAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAGTTGCGGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAGTTGC
AAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTGACAAGGACAAAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCCTCTCCTTGCCAAGCTGACAAGGACA
CGGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCGGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG
CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAACCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC
ACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA
ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG
CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAACACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCCAGAA
GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA(SEQ ID NO：622)GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA (SEQ ID NO: 622)
MHHHMHHH
AGCTTGTGTCCCCAAGGAAAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGTAGCTTGTGTCCCCAAGGAAAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGT
GCCACAAAGGAACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAGGCCACAAAGGAACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAG
GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC
TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACATCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTTCTTGCACAGTGGACCGGGACA
CCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCAGTG
CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAACCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC
ACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA
ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCIACCCCAGATTGAGAATGTTAAGGGACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCIACCCCAGATTGAGAATGTTAAGGG
CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAACACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCCAGAA
GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA(SEQ ID NO：623)GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA (SEQ ID NO: 623)
5)TNFR1嵌合构建体的制备及转化到巴斯德毕赤酵母中5) Preparation of TNFR1 chimeric construct and transformation into Pichia pastoris
将通过各大量制备(maxiprep)产生的质粒DNA用稀有切割限制内切核酸酶PmeI消化，以便使DNA线性化，然后进行毕赤酵母转化。线性化DNA再通过苯酚/氯仿抽提和乙醇沉淀而纯化，然后重悬于30μl dH2O。10μl线性化DNA溶液与80μl电感受态(electro-competent)KM71H毕赤酵母细胞混合5分钟，然后进行电穿孔(1.5kV，200Ω，25μF)。细胞立即用YPDS恢复并在30℃孵育2小时，然后接种在在含有100μg/ml零霉素(ZEOCIN)、含腐草霉素D的抗生素制剂(Cayla，Toulouse，法国)的YPDS琼脂板上培养2天。Plasmid DNA produced by each maxiprep was digested with the rare-cutting restriction endonuclease PmeI to linearize the DNA prior to Pichia transformation. The linearized DNA was then purified by phenol/chloroform extraction and ethanol precipitation, then resuspended in 30 μldH2O . 10 μl of linearized DNA solution was mixed with 80 μl of electro-competent KM71H Pichia cells for 5 minutes before electroporation (1.5 kV, 200Ω, 25 μF). Cells were immediately recovered with YPDS and incubated at 30°C for 2 hours, and then seeded on YPDS agar plates containing 100 μg/ml zeocin (ZEOCIN), an antibiotic preparation containing phleomycin D (Cayla, Toulouse, France). 2 days.
6)构建体在毕赤酵母中的表达6) Expression of the construct in Pichia pastoris
挑取各构建体的单个转化菌落，接种到5ml BMGY(作为起子培养物)中，在30℃培养24小时。用该培养物接种500ml BMGY培养基，在30℃培养24小时，然后在室温下以1500-3000g离心5分钟收获细胞。将细胞重悬于100ml BMMY中，培养4天，同时交错增加甲醇浓度(第1天0.5％，第2天1％，第3天1.5％，第4天2％)。表达后，以3300g离心15分钟，回收上清液。A single transformed colony of each construct was picked, inoculated into 5 ml of BMGY (as a starter culture) and grown at 30°C for 24 hours. Use this culture to inoculate 500ml of BMGY medium, incubate at 30°C for 24 hours, and then harvest the cells by centrifugation at 1500-3000g for 5 minutes at room temperature. Cells were resuspended in 100 ml BMMY and cultured for 4 days with staggered increases in methanol concentration (0.5% on
7)用Nickel树脂纯化TNFR1嵌合构建体7) Purification of TNFR1 chimeric constructs with Nickel resin
培养上清液先加入10mM终浓度的咪唑和2×PBS来缓冲。在室温下加入Nickel-NT A树脂，分批吸附His-标记的蛋白质达4小时(振荡)。上清液/树脂混合物再倒入poly-prep柱(Biorad)。树脂再用10倍体积的2×PBS洗涤，用250mM咪唑1×PBS洗脱。更换缓冲液后，用EndoH脱糖基化酶使嵌合构建体表达脱糖基化，再通过SDS-PAGE得以证实。The culture supernatant was first buffered by adding 10 mM imidazole and 2×PBS at a final concentration. Nickel-NTA resin was added at room temperature, and the His-tagged protein was adsorbed in batches for 4 hours (shaking). The supernatant/resin mixture was then poured onto a poly-prep column (Biorad). The resin was then washed with 10 times the volume of 2×PBS, and eluted with 250
PCR过程中所用的模板DNA序列Template DNA sequence used during PCR
人(Homo sapiens)TNFR1(胞外区Genbank检索号33991418)Human (Homo sapiens) TNFR1 (Extracellular region Genbank accession number 33991418)
CTGGTCCCTCACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGCTGGTCCCTCACCCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGG
AAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAAGGAAAATATATCCACCTCAAAAATAATTCGATTTGCTGTACCAAGTGCCACAAAGG
AACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGG
AGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCA
GCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAG
TGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGTGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGG
AGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGAGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTG
CACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTCCACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTC
TTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAGTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAG
TGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGCACTGAGGACTCATGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGCACTGAGGACTCA
GGCACCACA(SEQ ID N0：624)GGCACCACA (SEQ ID NO: 624)
所编码的人TNFR1胞外区具有下列氨基酸序列。The encoded extracellular domain of human TNFR1 has the following amino acid sequence.
LVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECELVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPPGPGQDTDCRECE
SGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFSGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLF
QCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFRENECVSCSNCKKSLECTKLCLP
QIENVKGTEDSGTT(SEQ ID N0：603)QIENVKGTEDSGTT (SEQ ID N0:603)
鼠(小家鼠(Mus musculus))TNFR1(胞外区Genbank检索号31560798)Mouse (Mus musculus) TNFR1 (Extracellular Region Genbank Accession No. 31560798)
CTAGTCCCTTCTCTTGGTGACCGGGAGAAGAGGGATAGCTTGTGTCCCCAAGGACTAGTCCCTTCTCTTGGTGACCGGGAGAAGAGGGATAGCTTGTGTCCCCAAGGA
AAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGTGCCACAAAGGAAAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGTGCCACAAAGGA
ACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAGGGAACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAGGGA
GTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAGGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAG
TTGCAAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTTGCAAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCCTCTCCTTGCCAAGC
TGACAAGGACACGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGATGACAAGGACACGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGA
GTGAGACACACTTCCAGTGCGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGAGTGAGACACACTTCCAGTGCGTGGACTGCAGCCCCCTGCTTCAACGGCACCGTGA
CAATCCCCTGTAAGGAGACTCAGAACACCGTGTGTAACTGCCATGCAGGGTTCTCAATCCCCCTGTAAGGAGACTCAGAACACCGTGTGTAACTGCCATGCAGGGTTCT
TTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCCACTGCAAGAAAAATGAGGAGTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCCACTGCAAGAAAAAATGAGGAG
TGTATGAAGTTGTGCCTACCTCCTCCGCTTGCAAATGTCACAAACCCCCAGGACTGTATGAAGTTGTGCCTACCTCCTCCGCTTGCAAATGTCACAAACCCCCAGGAC
TCAGGTACTGCG(SEQ ID NO：625)TCAGGTACTGCG (SEQ ID NO: 625)
所编码的鼠(小家鼠(Mus musculus))TNFR1胞外区具有下列氨基酸序列。The encoded extracellular domain of murine (Mus musculus) TNFR1 has the following amino acid sequence.
LVPSLGDREKRDSLCPQGKYVHSKNNSICCTKCHKGTYLVSDCPSPGRDTVCRECELVPSLGDREKRDSLCPQGKYVHSKNNSICCTKCHKGTYLVSDCPSPGRDTTVCRECE
KGTFTASQNYLRQCLSCKTCRKEMSQVEISPCQADKDTVCGCKENQFQRYLSETHFKGTFTASQNYLRQCLSCKTCRKEMSQVEISPCQADKDTVCGCKENQFQRYLSETHF
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实施例17.抗TNFR1 dAb的结构域特异性Example 17. Domain specificity of anti-TNFR1 dAbs
该实施例描述了用于确定结合TNFR1的dAb的结构域特异性的方法。该方法采用表面等离子共振(SPR)(Detection of immuno-complex formation via surface plasmon resonance on gold-coateddiffraction，gratings.′Biosensors.1987-88；3(4)：211-25.)，以确定抗体与固定在SPR芯片表面的完全人或小鼠生物素化TNFR1结合的能力，当抗体孵育并与过量的实施例16所述的嵌合分子平衡之后。在该测定中，抗TNFR1 dAb流过TNFR1表面，产生SPR信号，表示与固定在SPR芯片上的TNFR1结合的dAb量。如果dAb经预孵育并用包含TNFR1域(该结构域尤其可与dAb结合)的嵌合分子平衡，那么与单独的dAb相比，该混合物流过TNFR1表面，将会产生更弱的SPR信号。然而，如果dAb经预孵育并用不包含TNFR1域(该结构域尤其可与dAb结合)的嵌合分子平衡，那么与单独的dAb所得到的信号相比，该混合物流过TNFR1表面，将会产生大致相同的SPR信号。This example describes methods for determining the domain specificity of dAbs that bind TNFR1. The method adopts surface plasmon resonance (SPR) (Detection of immuno-complex formation via surface plasmon resonance on gold-coated diffraction, gratings.'Biosensors.1987-88; 3(4):211-25.) to determine the relationship between antibody and immobilization. Ability to bind fully human or mouse biotinylated TNFR1 on the surface of an SPR chip after antibody incubation and equilibration with an excess of the chimeric molecule described in Example 16. In this assay, anti-TNFR1 dAbs flow across the TNFR1 surface, generating an SPR signal that represents the amount of dAb bound to TNFR1 immobilized on the SPR chip. If the dAb is pre-incubated and equilibrated with a chimeric molecule containing a TNFR1 domain that specifically binds the dAb, flow of this mixture across the TNFR1 surface will produce a weaker SPR signal compared to the dAb alone. However, if the dAb is pre-incubated and equilibrated with a chimeric molecule that does not contain the TNFR1 domain that specifically binds the dAb, flow of this mixture across the TNFR1 surface will produce roughly the same SPR signal.
方法method
1)SPR芯片TNFR1表面的产生1) Generation of SPR chip TNFR1 surface
TNFR1表面的选择由待测抗TNFR1dAb的物种特异性来决定。因此，用人TNFR1包被的表面来评价抗人TNFR1 dAb，并且用小鼠TNFR1包被的芯片来评价抗小鼠TNFR1 dAb。The choice of TNFR1 surface is determined by the species specificity of the anti-TNFR1 dAb to be tested. Therefore, human TNFR1-coated surfaces were used to evaluate anti-human TNFR1 dAbs, and mouse TNFR1-coated chips were used to evaluate anti-mouse TNFR1 dAbs.
生物素化TNFR1在合适SPR缓冲液中稀释，并在BIACORE 3000SPR仪器(Biacore International AB，Uppsala，Sweden)中通过链霉抗生物素(SA)传感器芯片。低流速(5-10μl/分钟)用于使生物素化TNFR1和链霉抗生物素表面之间的接触时间最大化。持续流速，直到链霉抗生物素表面被生物素化材料饱和，以产生具有最大TNFR1表面的芯片。芯片通常结合几百个到几千个生物素化材料的SPR响应单元。Biotinylated TNFR1 was diluted in appropriate SPR buffer and passed over a streptavidin (SA) sensor chip in a BIACORE 3000 SPR instrument (Biacore International AB, Uppsala, Sweden). A low flow rate (5-10 μl/min) was used to maximize the contact time between the biotinylated TNFR1 and the streptavidin surface. Continue the flow rate until the streptavidin surface is saturated with biotinylated material to produce a chip with the largest TNFR1 surface. Chips typically incorporate hundreds to thousands of SPR-responsive elements of biotinylated material.
2)SPR芯片上抗TNFR1响应的效价2) Titer of anti-TNFR1 response on SPR chip
一个成功的竞争实验需要抗TNFR1 dAb的起始最佳浓度，使得最少量的dAb流过其表面，给出明显的SPR信号。在某些浓度范围内，dAb可以剂量依赖性方式结合表面，使得dAb结合的RU数能反映流过芯片表面的dAb浓度。A successful competition experiment requires an optimal initial concentration of anti-TNFR1 dAb such that the least amount of dAb flows across its surface, giving a clear SPR signal. Within certain concentration ranges, dAbs can bind to the surface in a dose-dependent manner such that the number of RUs bound by dAbs reflects the concentration of dAbs flowing across the surface of the chip.
为了确定这样的剂量依赖性浓度范围，在10倍系列稀释的Biacore缓冲液(范围从1∶10稀释到1∶1,000,000稀释)中滴定抗TNFR1dAb。然后将稀释液单独且序贯注射到TNFR1芯片表面，从最稀的样品开始。测定各稀释液所得的最大RU数。必要时，每次注射后，TNFR1表面用合适的SPR再生缓冲液进行再生，以除去结合的抗TNFR1 dAb。采用该方法，测出产生约100RU信号所需的抗TNFR1dAb的最小浓度。To determine such a dose-dependent concentration range, anti-TNFRl dAbs were titrated in 10-fold serial dilutions of Biacore buffer (ranging from 1:10 dilution to 1:1,000,000 dilution). The dilutions were then injected onto the TNFR1 chip surface individually and sequentially, starting with the most dilute sample. Determine the maximum number of RU obtained for each dilution. When necessary, after each injection, the TNFR1 surface was regenerated with an appropriate SPR regeneration buffer to remove bound anti-TNFR1 dAb. Using this method, the minimum concentration of anti-TNFRldAb required to produce a signal of approximately 100 RU was determined.
3)抗TNFR1 dAb/嵌合体的预平衡3) Pre-equilibration of anti-TNFR1 dAb/chimera
一旦确定了抗TNFR1 dAb的最佳浓度，就可建立抗TNFR1 dAb/嵌合TNFR1混合物。建立混合物，使得抗TNFR1 dAb的终浓度与先前测定的最佳浓度相同。反应通常在100μl体积(含有50微升2x浓缩的抗TNFR1 dAb、40微升Biacore缓冲液和10微升纯化嵌合蛋白)中进行。最终混合物通常的浓度为约10-100μM嵌合蛋白和约10-100nM抗TNFR1 dAb。在室温下让混合物平衡30分钟。Once the optimal concentration of anti-TNFR1 dAb has been determined, an anti-TNFR1 dAb/chimeric TNFR1 mixture can be established. Set up the mixture so that the final concentration of anti-TNFR1 dAb is the same as the previously determined optimal concentration. Reactions were typically performed in 100 μl volumes (containing 50
4)竞争性Biacore实验4) Competitive Biacore experiment
平衡后，各抗TNFR1 dAb/嵌合TNFR1混合物序贯通过TNFR1SPR表面并测定响应单元数目。各混合物注射后，表面经再生以除去结合的抗TNFR1 dAb，然后注射下一种混合物。用不同嵌合体所产生的不同响应，可以测出结合特定dAb的TNFR1域。After equilibration, each anti-TNFR1 dAb/chimeric TNFR1 mixture was sequentially passed over the TNFR1 SPR surface and the number of responsive units was determined. After each mixture was injected, the surface was regenerated to remove bound anti-TNFRl dAbs before the next mixture was injected. Using the different responses produced by the different chimeras, the domain of TNFR1 that binds a particular dAb can be determined.
这些研究表明，TAR2m-21-23结合小鼠TNFR1的域1，TAR2h-205结合人TNFR1的域1，TAR2h-10-27、TAR2h-131-8、TAR2h-15-8、TAR2h-35-4、TAR2h-154-7、TAR2h-154-10和TAR2h-185-25结合人TNFR1的域3。These studies showed that TAR2m-21-23
实施例18.筛选方法Example 18. Screening method
实施例16中描述的这些嵌合受体蛋白质可用于测定或筛选，以分离出能结合TNFR1内特定结构域的物质(例如抗体、dAb、化合物)。简而言之，这些方法描述了将嵌合蛋白加入到粗抗体制备物中，然后通过ELISA或表面等离子共振筛选TNFR1结合。另外，它们描述了在表面(例如ELISA板或SPR芯片)包被的嵌合蛋白的用途并通过测定它们与该表面上嵌合蛋白的结合而筛选抗体。The chimeric receptor proteins described in Example 16 can be used in assays or screens to isolate agents (eg, antibodies, dAbs, compounds) that bind specific domains within TNFRl. Briefly, these methods describe the addition of chimeric proteins to crude antibody preparations, followed by screening for TNFR1 binding by ELISA or surface plasmon resonance. In addition, they describe the use of chimeric proteins coated on surfaces (eg ELISA plates or SPR chips) and screening of antibodies by assaying their binding to the chimeric proteins on the surface.
1)可溶性ELISA筛选1) Soluble ELISA screening
该方法可用于从庞大的未知特异性的抗体或抗体片段库中，快速分离出能结合TNFR1特定结构域的抗体或抗体片段(例如dAb)。This method can be used to quickly isolate antibodies or antibody fragments (such as dAbs) that can bind to a specific domain of TNFR1 from a large library of antibodies or antibody fragments of unknown specificity.
96孔测定板用100μl/孔的嵌合TNFR1在4℃包被过夜。各孔用0.1％TPBS(含有浓度0.1％的吐温-20的磷酸缓冲盐溶液)洗涤3次。加入200μl/孔的1％TPBS封闭该板并将该板在室温下孵育1-2小时。各孔用PBS洗涤3次，然后加入含有可溶性抗体或抗体片段(其含有c-Myc表位标记)/50μl 0.2％TPBS的50μl细菌上清液或periprep。将该板在室温下孵育1小时。然后，将该板用0.1％TPBS(0.1％吐温-20/PBS)洗涤5次，再向各孔中加入100μl第一抗c-Myc小鼠单克隆抗体/0.1％TPBS，将该板在室温下孵育1小时。弃去这样的第一抗体溶液，再将该板用0.1％TPBS洗涤5次。然后加入来自山羊的100μl预稀释的抗小鼠IgG(Fc特异性)HRP缀合物(Sigma产品目录号：A0168)，将该板在室温下孵育1小时。弃去第二抗体，将该板用0.1％TPBS洗涤6次，再用PBS洗涤2次。向各孔中加入50μl TMB过氧化物酶溶液，将该板在室温下放置2-60分钟。加入50μl 1M盐酸，终止反应。在加酸后的30分钟之内，在96孔板阅读器上读出该板在450nm处的OD值。粗制细菌上清液或peripreps中存在的、能结合嵌合蛋白内存在的TNFR1域的这些抗体，将会给出较强烈的ELISA信号，而那些不能结合的则不能给出信号。A 96-well assay plate was coated with 100 μl/well of chimeric TNFR1 overnight at 4°C. Each well was washed 3 times with 0.1% TPBS (phosphate-buffered saline solution containing 0.1% Tween-20). The plate was blocked by adding 200 μl/well of 1% TPBS and the plate was incubated at room temperature for 1-2 hours. The wells were washed 3 times with PBS before addition of 50 μl bacterial supernatant or periprep containing soluble antibody or antibody fragment (which contains the c-Myc epitope tag)/50 μl 0.2% TPBS. The plate was incubated for 1 hour at room temperature. Then, the plate was washed 5 times with 0.1% TPBS (0.1% Tween-20/PBS), and 100 μl of primary anti-c-Myc mouse monoclonal antibody/0.1% TPBS was added to each well, and the plate was placed in Incubate for 1 hour at room temperature. Such a primary antibody solution was discarded, and the plate was washed 5 times with 0.1% TPBS. 100 [mu]l of pre-diluted anti-mouse IgG (Fc-specific) HRP conjugate from goat (Sigma Cat#: A0168) was then added and the plate was incubated for 1 hour at room temperature. The secondary antibody was discarded and the plate was washed 6 times with 0.1% TPBS and 2 times with PBS. 50 μl of TMB peroxidase solution was added to each well and the plate was left at room temperature for 2-60 minutes. Add 50 μl of 1M hydrochloric acid to stop the reaction. Read the OD of the plate at 450 nm on a 96-well plate reader within 30 minutes after acid addition. Those antibodies present in crude bacterial supernatants or peripreps that bind the TNFR1 domain present within the chimeric protein will give a stronger ELISA signal, whereas those that do not bind will not give a signal.
2)竞争性ELISA筛选2) Competitive ELISA screening
该方法可以用于快速筛选一系列能结合TNFR1的不同粗抗体或抗体片段制备物，以测定它们的结构域结合特异性。This method can be used to quickly screen a series of different crude antibody or antibody fragment preparations that can bind TNFR1 to determine their domain binding specificity.
96孔测定板用100μl/孔的鼠或人TNFR1(人或小鼠)在4℃包被过夜。各孔用0.1％TPBS(含有浓度0.1％的吐温-20的磷酸缓冲盐溶液)洗涤3次。加入200μl/孔的1％TPBS(1％吐温-20/PBS)封闭该板，然后将该板在室温下孵育1-2小时。各孔用PBS洗涤3次。同时，细菌上清液或peripreps用预优化浓度的嵌合TNFR1蛋白/溶液预平衡。将含有可溶性抗体或抗体片段的50μl该粗制细菌制备物/嵌合蛋白混合物加入到ELISA板中。将该板在室温下孵育1小时。然后，将该板用0.1％TPBS(0.1％吐温-20/PBS)洗涤5次，再向各孔中加入100μl第一检测抗体(或蛋白A-HRP或蛋白L HRP)的0.1％TPBS，将该板在室温下孵育1小时。弃去这样的第一抗体溶液，再将该板用0.1％TPBS洗涤5次。如有必要，加入来自山羊的100μl预稀释的第二抗体-HRP缀合物，将该板在室温下孵育1小时。然后弃去第二抗体，将该板用0.1％TPBS洗涤6次，再用PBS洗涤2次。向各孔中加入50μl TMB过氧化物酶溶液，将该板在室温下放置2-60分钟。加入50μl1M盐酸，终止反应。在加酸后的30分钟之内，在96孔板阅读器上读出该板在450nm处的OD值。ELISA信号减少，表明抗体与板上包被的嵌合TNFR1域、而不是完整TNFR1结合，因此，该抗体能结合嵌合蛋白内的一个结构域。96-well assay plates were coated with 100 μl/well of murine or human TNFR1 (human or mouse) overnight at 4°C. Each well was washed 3 times with 0.1% TPBS (phosphate-buffered saline solution containing 0.1% Tween-20). The plate was blocked by adding 200 μl/well of 1% TPBS (1% Tween-20/PBS), and then the plate was incubated at room temperature for 1-2 hours. Each well was washed 3 times with PBS. At the same time, bacterial supernatants or peripreps were pre-equilibrated with pre-optimized concentrations of chimeric TNFR1 protein/solution. 50 [mu]l of this crude bacterial preparation/chimeric protein mixture containing soluble antibody or antibody fragment was added to the ELISA plate. The plate was incubated for 1 hour at room temperature. Then, the plate was washed 5 times with 0.1% TPBS (0.1% Tween-20/PBS), and then 100 μl of 0.1% TPBS of the primary detection antibody (or protein A-HRP or protein L HRP) was added to each well, The plate was incubated for 1 hour at room temperature. Such a primary antibody solution was discarded, and the plate was washed 5 times with 0.1% TPBS. If necessary, 100 μl of pre-diluted secondary antibody-HRP conjugate from goat was added and the plate was incubated for 1 hour at room temperature. The secondary antibody was then discarded and the plate was washed 6 times with 0.1% TPBS and 2 times with PBS. 50 μl of TMB peroxidase solution was added to each well and the plate was left at room temperature for 2-60 minutes. Add 50 μl of 1M hydrochloric acid to terminate the reaction. Read the OD of the plate at 450 nm on a 96-well plate reader within 30 minutes after acid addition. The ELISA signal was reduced, indicating that the antibody bound to the chimeric TNFR1 domain coated on the plate, rather than intact TNFR1, thus, the antibody binds to a domain within the chimeric protein.
3)对与参考抗体或抗体片段竞争性结合TNFR1的抗体和抗体片段的竞争性ELISA筛选3) Competitive ELISA screening for antibodies and antibody fragments that competitively bind to TNFR1 with reference antibodies or antibody fragments
该方法可以用于快速筛选一系列能结合TNFR1的不同粗抗体或抗体片段制备物，所述抗体或抗体片段与参考抗体或抗体片段(例如TAR2m-21-23)竞争性结合TNFR1或者结合TNFR1的所需结构域(例如域1)。该方法采用含有不同可检测标记(表位标记)的参考抗体或抗体片段和试验抗体或抗体片段(例如有待筛选的抗体群体)。This method can be used to rapidly screen a series of preparations of different crude antibodies or antibody fragments that bind to TNFR1 that compete with a reference antibody or antibody fragment (e.g. TAR2m-21-23) for binding to TNFR1 or that bind to TNFR1 Desired domain (eg domain 1). The method employs a reference antibody or antibody fragment and a test antibody or antibody fragment (eg, a population of antibodies to be screened) that contain different detectable labels (epitope tags).
96孔测定板用100μl/孔的鼠或人TNFR1在4℃包被过夜。各孔用0.1％TPBS(含有浓度0.1％的吐温-20的磷酸缓冲盐溶液)洗涤3次。加入200μl/孔的1％TPBS(1％吐温-20/PBS)封闭该板，将该板在室温下孵育1-2小时。各孔用PBS洗涤3次。同时，待测粗抗体制备物与预优化浓度的参考抗体或抗体片段(例如域1-结合抗体；TAR2m-21-23)/溶液混合。正如所述，重要的是该抗体不包含与有待筛选结构域结合特异性的抗体相同的检测标记。将50μl该粗制抗体/参考抗体混合物加入到ELISA板中。将该板在室温下孵育1小时。然后，将该板用0.1％TPBS洗涤5次，向各孔中加入100μl第一检测抗体(其结合仅存于待筛选的抗体群体中的标记)的0.1％TPBS，将该板在室温下孵育1小时。弃去这样的第一抗体溶液，再将该板用0.1％TPBS洗涤5次。然后，加入能识别第一检测抗体的100μl预稀释的第二抗体-HRP缀合物，将该板在室温下孵育1小时。弃去第二抗体溶液，将该板用0.1％TPBS洗涤6次，再用PBS洗涤2次。向各孔中加入50μl TMB过氧化物酶溶液，将该板在室温下放置2-60分钟。加入50μl 1M盐酸，终止反应。在加酸后的30分钟之内，在96孔板阅读器上读出该板在450nm处的OD值。应该平行地进行一个单独而平行的ELISA，采用该方法，但不加参考抗体或抗体片段。与用相同抗体制备物、而没有竞争性参考抗体或抗体片段所得到的ELISA信号相比，在参考抗体或抗体片段存在时ELISA信号减少，表明特定抗体或抗体片段与参考抗体或抗体片段竞争性结合TNFR1域，并且与参考抗体或抗体片段一样结合同一TNFR1域。96-well assay plates were coated with 100 μl/well of mouse or human TNFR1 overnight at 4°C. Each well was washed 3 times with 0.1% TPBS (phosphate-buffered saline solution containing 0.1% Tween-20). The plate was blocked by adding 200 μl/well of 1% TPBS (1% Tween-20/PBS), and the plate was incubated at room temperature for 1-2 hours. Each well was washed 3 times with PBS. Simultaneously, the crude antibody preparation to be tested is mixed with a pre-optimized concentration of a reference antibody or antibody fragment (eg domain 1 -binding antibody; TAR2m-21-23)/solution. As stated, it is important that the antibody does not contain the same detectable label as the antibody to be screened for domain binding specificity. 50 μl of this crude antibody/reference antibody mixture was added to the ELISA plate. The plate was incubated for 1 hour at room temperature. Then, the plate was washed 5 times with 0.1% TPBS, 100 μl of primary detection antibody (which binds to the marker present only in the antibody population to be screened) in 0.1% TPBS was added to each well, and the plate was incubated at
4)SPR筛选4) SPR screening
可以容易地修改上述ELISA方法，以便采用表面等离子共振，例如用BIACORE 3000 SPR仪器(Biacore International AB，Uppsala，Sweden)。通常，嵌合蛋白可固定在SPR芯片上，或者嵌合蛋白可用含有抗TNFR1抗体或抗体片段的粗制细菌上清液平衡，所得混合物流过用全长人TNFR1或鼠TNFR1包被的SPR芯片。The ELISA method described above can be easily modified to employ surface plasmon resonance, for example with a BIACORE 3000 SPR instrument (Biacore International AB, Uppsala, Sweden). Typically, the chimeric protein can be immobilized on an SPR chip, or the chimeric protein can be equilibrated with crude bacterial supernatant containing an anti-TNFR1 antibody or antibody fragment, and the resulting mixture flowed over an SPR chip coated with full-length human TNFR1 or murine TNFR1 .
实施例19.TAR2m21-23二聚体是高度亲合力的TNFR1拮抗剂Example 19. TAR2m21-23 dimer is a high affinity TNFR1 antagonist
采用实施例8描述的方法，通过制备在羧基端含有cys残基的TAR2m21-23形式，制备TAR2m21-23二聚体。蛋白质(TAR2m21-23CYS)在毕赤酵母中表达，并用Streamline蛋白A进行纯化。非还原性SDS-PAGE分析表明，～40-50％的蛋白质以二聚体形式存在于溶液中。二聚体用凝胶过滤色谱进一步纯化。The TAR2m21-23 dimer was prepared using the method described in Example 8 by preparing a form of TAR2m21-23 containing a cys residue at the carboxy terminus. The protein (TAR2m21-23CYS) was expressed in Pichia pastoris and purified using Streamline protein A. Non-reducing SDS-PAGE analysis indicated that -40-50% of the protein existed as dimers in solution. The dimer was further purified by gel filtration chromatography.
2mg蛋白质浓缩至约250μl并上样到Superdex 75 HR凝胶过滤柱(Amersham Bioscience)上，该柱先前已用PBS进行平衡。该柱运行的流速为0.5ml/分钟，收集0.5ml流分。在280nm处监控从该柱上洗脱的蛋白质，含有二聚体的流分经非还原性SDS-PAGE得以证实。合并含有二聚体、但不含单体TAR2m-21-23 CYS的流分。浓缩合并的流分，在L929 TNF细胞的细胞毒性测定(实施例6)中测定二聚体dAb的效力。2 mg of protein was concentrated to approximately 250 μl and loaded onto a
在L929细胞毒性测定中，将TAR2m21-23二聚体的生物学效力与单体TAR2m-21-23进行比较。在该测定中，测定了TAR2m21-23单体和TAR2m21-23二聚体对由TNF诱导的小鼠L929细胞的细胞毒性的抑制，其结果表示为在测定中抑制50％细胞毒性的dAb单体或二聚体的浓度(中和剂量50，ND50)。The biological potency of TAR2m21-23 dimers was compared to monomeric TAR2m-21-23 in the L929 cytotoxicity assay. In this assay, the inhibition of TAR2m21-23 monomers and TAR2m21-23 dimers on TNF-induced cytotoxicity in mouse L929 cells was determined and the results are expressed as dAb monomers that inhibited 50% of the cytotoxicity in the assay or dimer concentration (neutralizing
在测定中，单体dAb的ND50约为600pM。在测定中，二聚体dAb(TAR2m21-23二聚体)的ND50约低10倍(ND50约60-70pM)。这些结果表明，与dAb单体相比，TAR2m21-23二聚体对细胞表面TNFR1的亲和力具有显著提高。结果表明，TAR2m21-23二聚体同时结合细胞表面上的两个单独的TNFR1分子，针对多聚体的所得亲合力效应导致对TNFR1抑制的改善。TheND50 of the monomeric dAb was approximately 600 pM in the assay. TheND50 of the dimeric dAb (TAR2m21-23 dimer) was approximately 10-fold lower in the assay (ND50 approximately 60-70 pM). These results demonstrate that the TAR2m21-23 dimer has a significantly increased affinity for cell surface TNFR1 compared to the dAb monomer. The results demonstrate that the TAR2m21-23 dimer binds simultaneously to two separate TNFR1 molecules on the cell surface and that the resulting avidity effect for the multimer leads to improved inhibition of TNFR1.
然后测定二聚体形式(TAR2m21-23二聚体)，以观察它是否表现出TNFR1激动的任何征兆，所述TNFR1激动意即引起TNFR1交联并促进胞内信号转导和细胞死亡的能力。采用改进L929细胞的细胞毒性测定，可达到这一点，其中未加TNF-α，而是检测抗TNFR1抗体或dAb形式，以观察它们是否激动TNFR1并诱导细胞死亡。在测定中，测定了作为已知TNFR1激动剂的抗TNFR1抗体AF-425-PB(R&D系统)，以及作为已报道的TNFR1、TAR2m-21-23和TAR2m21-23二聚体的拮抗剂的抗TNFR1抗体MAB430(R&D系统)。The dimer form (TAR2m21-23 dimer) was then assayed to see if it exhibited any signs of TNFRl agonism, ie the ability to cause TNFRl to cross-link and promote intracellular signaling and cell death. This was achieved using a modified cytotoxicity assay in L929 cells in which TNF-[alpha] was not added and instead anti-TNFRl antibody or dAb formats were tested to see if they agonized TNFRl and induced cell death. In the assay, the anti-TNFR1 antibody AF-425-PB (R&D Systems), which is a known TNFR1 agonist, and the anti-TNFR1 antibody, which is a reported antagonist of TNFR1, TAR2m-21-23 and TAR2m21-23 dimers, were tested. TNFR1 antibody MAB430 (R&D Systems).
在测定中，抗体AF-425-PB激活TNFR1并诱导细胞毒性，其ND50约为100pM。在测定中，甚至已报道的拮抗剂抗体MAB430也引起受体交联和细胞杀伤，其ND50约为10nM。相比之下，在测定中，TAR2m-21-23二聚体却不引起任何细胞死亡，甚至当以极高浓度存在时(＞1μM)。这些结果表明，TAR2m21-23二聚体不是TNFR1激动剂。In the assay, antibody AF-425-PB activates TNFR1 and induces cytotoxicity with anND50 of approximately 100 pM. Even the reported antagonist antibody MAB430 caused receptor crosslinking and cell killing withan ND50 of approximately 10 nM in the assay. In contrast, TAR2m-21-23 dimers did not cause any cell death in the assay, even when present at very high concentrations (>1 [mu]M). These results suggest that TAR2m21-23 dimers are not TNFR1 agonists.
结果表明，结合TNFR1的dAb的二聚体、三聚体或其它多聚体对细胞表面表达的TNFR1具有高亲合力，而且是有效TNFR1拮抗剂。此外，该研究结果表明，结合TNFR1的域1的dAb多聚体(例如TAR2m21-23二聚体)，能结合两个TNFR1分子(如同测定中作为激动剂的抗体所能结合的一样)，而且与TNFR1的结构域或表位靶(域1)的结合，阻止受体链的紧密结合，而这正是起始TNFR1信号转导所必需的。该特性对二价分子来说是独特的，所述分子能交联细胞表面的TNFR1，但不引起TNFR1信号转导和细胞死亡。The results indicate that dimers, trimers or other multimers of dAbs that bind TNFR1 have high affinity for TNFR1 expressed on the cell surface and are potent TNFR1 antagonists. In addition, the results of this study demonstrate that dAb multimers that bind
实施例20.结合人TNFR1和小鼠TNFR1的dAb的分离Example 20. Isolation of dAbs that bind human TNFR1 and mouse TNFR1
已知序列的dAb在大肠杆菌中表达，并用蛋白A层流(streamline)树脂纯化。洗脱到Tris-甘氨酸中，然后dAb流过SPR芯片，其上已经固定有生物素化人TNFR1(流动池2)和生物素化鼠TNFR1(流动池4)。(该SPR芯片用相同密度的人TNFR1和鼠TNFR1包被)。流动池1和3为空白，作为无抗原参考表面，用于检测并减去非特异性结合。dAbs of known sequence were expressed in E. coli and purified using protein A streamline resin. After elution into Tris-glycine, the dAbs were then flowed through an SPR chip on which biotinylated human TNFRl (flow cell 2) and biotinylated murine TNFRl (flow cell 4) had been immobilized. (The SPR chip was coated with the same density of human TNFR1 and murine TNFR1).
dAb流过连续的4个流动池(即依次为流动室1、2、3和4)，测定流动室2与流动室1之间的响应差异，也测定流动室4与流动室3之间的响应差异。前者测定的是与人TNFR1的结合，而后者是与鼠TNPR1的结合。记录用于结合人TNFR1和鼠TNFR1的特异性结合曲线，在曲线中注意到，与鼠TNFR1相比，人TNFR1的缔合速率更快。解离速率也大致相似。通过每次结合事件所得到的最大响应单位(RU)数来评价交叉反应。在该实施例中，人生物素化TNFR1表面在流动室2上包含约900RU的TNFR1，而在流动室4包含约1400RU的鼠TNFR1。作为对照，浓度为2微摩尔TAR2h-154-7(一种人特异性dAb)结合人表面的最大响应为385RU，而在小鼠表面的响应仅为4.5RU。2微摩尔TAR2h-205在人表面得到的响应为435RU，而在小鼠表面为266RU。The dAb was flowed through four consecutive flow cells (i.e.,
本申请书提到的所有出版物以及所述出版物中引用的参考文献，全都通过引用结合到本文中。本领域技术人员显而易见的是，只要不偏离本发明的范围和精神，可以对本发明描述的方法和系统进行各种的修改和变动。尽管用具体的优选实施方案描述了本发明，但是应该理解，要求保护的发明并不限于这些具体的实施方案。当然，对用于实施本发明的所述方式的各种修改，对于分子生物学或相关领域技术人员来说是显而易见的，这样的修改落入所附权利要求书范围之内。All publications mentioned in this application, as well as references cited in said publications, are hereby incorporated by reference in their entirety. It will be apparent to those skilled in the art that various modifications and changes can be made to the methods and systems described herein without departing from the scope and spirit of the invention. Although the invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed is not limited to such specific embodiments. Naturally, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
附录1：延长体内半衰期的多肽Appendix 1: Peptides that extend half-life in vivo
α-1糖蛋白(血清类粘蛋白(Orosomucoid))(AAG)Alpha-1 Glycoprotein (Orosomucoid) (AAG)
α-1抗胰凝乳蛋白酶(ACT)Alpha-1 Antichymotrypsin (ACT)
α-1抗胰蛋白酶(AAT)Alpha-1 Antitrypsin (AAT)
α-1微球蛋白(蛋白HC)(AIM)Alpha-1 Microglobulin (Protein HC) (AIM)
α-2巨球蛋白(A2M)Alpha-2 Macroglobulin (A2M)
抗凝血酶III(AT III)Antithrombin III (AT III)
载脂蛋白A-1(Apo A-1)Apolipoprotein A-1 (Apo A-1)
载脂蛋白B(Apo B)Apolipoprotein B (Apo B)
β-2-微球蛋白(B2M)β-2-microglobulin (B2M)
血浆铜蓝蛋白(Cp)Ceruloplasmin (Cp)
补体成分(C3)Complement component (C3)
补体成分(C4)Complement component (C4)
C1酯酶抑制剂(C1 INH)C1 Esterase Inhibitor (C1 INH)
C-反应蛋白(CRP)C-reactive protein (CRP)
半胱氨酸蛋白酶抑制剂C(Cys C)Cysteine Protease Inhibitor C (Cys C)
铁蛋白(FER)Ferritin (FER)
血纤蛋白原(FIB)Fibrinogen (FIB)
纤连蛋白(FN)Fibronectin (FN)
触珠蛋白(Hp)Haptoglobin (Hp)
血红素结合蛋白(HPX)Hemopexin (HPX)
免疫球蛋白A(IgA)Immunoglobulin A (IgA)
免疫球蛋白D(IgD)Immunoglobulin D (IgD)
免疫球蛋白E(IgE)Immunoglobulin E (IgE)
免疫球蛋白G(IgG)Immunoglobulin G (IgG)
免疫球蛋白M(IgM)Immunoglobulin M (IgM)
免疫球蛋白轻链(κ/λ)Immunoglobulin light chain (κ/λ)
脂蛋白(a)[Lp(a)]Lipoprotein(a) [Lp(a)]
甘露糖结合蛋白(MBP)Mannose-binding protein (MBP)
肌红蛋白(Myo)Myoglobin (Myo)
血纤蛋白溶解酶原(PSM)Plasminogen (PSM)
前白蛋白(运甲状腺素蛋白(Transthyretin))(PAL)Prealbumin (Transthyretin) (PAL)
视黄醇结合蛋白(RBP)Retinol binding protein (RBP)
类风湿因子(RF)Rheumatoid factor (RF)
血清淀粉样蛋白A(SAA)Serum amyloid A (SAA)
可溶性转铁蛋白受体(sTfR)Soluble transferrin receptor (sTfR)
转铁蛋白(Tf)Transferrin (Tf)
附录2
附录3：肿瘤学组合Appendix 3: Oncology Portfolio
附录4
数据汇总data summary
Claims (241)
Translated fromChineseApplications Claiming Priority (3)
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| GBPCT/GB04/04253 | 2004-10-08 | ||
| PCT/GB2004/004253WO2005035572A2 (en) | 2003-10-08 | 2004-10-08 | Antibody compositions and methods |
| US10/985,847 | 2004-11-10 |
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| CN200910246166ADivisionCN101724071A (en) | 2004-10-08 | 2005-10-07 | Single domain antibodies against TNFRl and methods of use therefor |
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| CN102405236A (en)* | 2009-02-19 | 2012-04-04 | 葛兰素集团有限公司 | Improved anti-tnfr1 polypeptides, antibody variable domains & antagonists |
| CN102686239A (en)* | 2009-10-27 | 2012-09-19 | 葛兰素集团有限公司 | Stable anti-TNFR1 polypeptides, antibody variable domains & antagonists |
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| CN109476745B (en)* | 2016-05-13 | 2023-11-24 | 综合医院公司 | Antagonistic anti-tumor necrosis factor receptor superfamily antibodies |
| CN106279413A (en)* | 2016-08-30 | 2017-01-04 | 苏州普罗达生物科技有限公司 | D2E7 polypeptide and application |
| CN110573529A (en)* | 2017-04-05 | 2019-12-13 | 韩国生命工学研究院 | Fusion protein for activating NK cells, NK cells and pharmaceutical compositions containing same |
| CN108926720A (en)* | 2018-07-16 | 2018-12-04 | 中山大学附属第三医院（中山大学肝脏病医院） | TNFR1 gene and its application for encoding albumen |
| CN113302205A (en)* | 2018-11-15 | 2021-08-24 | 综合医院公司 | agonistic tumor necrosis factor receptor superfamily polypeptide |
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