CN109310755A

CN109310755A - The bispecific binding protein of PD-L1 and KDR

Info

Publication number: CN109310755A
Application number: CN201780021304.8A
Authority: CN
Inventors: Z.朱; D.陆
Original assignee: Kadmon Corp LLC
Current assignee: Kadmon Corp LLC
Priority date: 2016-02-02
Filing date: 2017-02-02
Publication date: 2019-02-05
Also published as: EP3411068A4; JP2019506863A; WO2017136562A2; WO2017136562A3; EP3411068A2; EA201891732A1

Abstract

Provide bispecific binding protein, such as the bispecific antibody in conjunction with human PD-L 1 and people KDR.The bispecific binding protein can be used for treating the disease and symptom that feature is generated as with immunosupress and/or excessive blood vessel.

Description

The bispecific binding protein of PD-L1 and KDR

The cross reference of related application

This application claims the priority of 2 months U.S. Provisional Application No. submitted for 2nd 62/290,350 in 2016.This application Content be incorporated herein by reference in their entirety.

Invention field

There is provided herein bispecific binding proteins, it includes the first combined area for combining human PD-L 1 and combine people KDR's Second combined area.It additionally provides the method for preparing bispecific binding protein and needs to subtract using bispecific binding protein treatment Less or inhibits immunosupress or reduction or inhibit the method for the disease or symptom of angiogenesis.

Background of invention

Programmed death receptor 1 (PD-1) is the member of CD28 receptor family, the family include CD28, CTLA-4, PD-1, (Freeman etc. (2000) J Exp Med 192:1027-34 of ICOS and BTLA；Latchman etc. (2001) Nat Immunol 2:261-8).During PD-1 is immunopathogenesis status progression, the immune suppression of the induction type mainly raised on activating T cell and B cell Receptor processed.The interaction of PD-1 and its ligand PD-L1 leads to the suppression generated to the TCR and BCR proliferation mediated and cell factor It makes and induction is conducted by the negative signal of the endogenous PD-1 inhibition motif (ITIM) based on immunity receptor tyrosine mediated T cells with antigenic specificity apoptosis (Agata etc. (1996) Int.Immunol.8:765, Unkeless and Jin. (1997) (2002) such as Curr.Opin.Immunol.9:338-343, Okzaki etc. (2001) PNAS98:13866-71, Dong Nat.Med.8:793-800).PD-L1 be cell surface glycoprotein and be PD-1 major ligand.After cell activation, PD-L1 also can induce in lymphoid tissue and non-lymph peripheral tissues.In addition to immunocyte, PD-L1 is in a variety of impacted cells It raises in type, including cancer cell and stroma cell, and has a positive effect in immunosupress during disease progression (Iwai etc. (2002) PNAS 99:12293-7, Ohigashi etc. (2005) Clin Cancer Res 11:2947-53).Incited somebody to action PD-L1 up-regulation and the bad clinical effectiveness of kinds cancer and virus infection connect (Hofmeyer etc. (2011) J.BioMed.Biotech.2011:1-9, McDermott and Atkins. (2013) Cancer Med.2:662-73).In clinic In preceding and clinical setting, antibody promotes the infiltration of CD8 T cell, CTL activity and existing Th1 thin the blocking of PD-1 or PD-L1 Intracellular cytokine IFN-γ increases ((2007) Clin Cancer such as Zhou etc. (2010) J.Immunol.185:5082-92, Nomi Res.13:2152-7, Flies etc. (2011) YaleJ.Bio.Med.48:409-21, Zitvogel and Kroemer. (2012) OncoImmunol.1:1223-25).It has been confirmed that as immunomodulator PD-L1 antibody as monotherapy or and its It is effective when the antibody combination of its immunosuppression molecule.

VEGFR is receptor tyrosine kinase, and with PDGF and fibroblast growth factor (FGF) belong to it is identical by Body family.KDR (also referred to as VEGFR2) is the receptor in conjunction with VEGF isotype A, C, D and E.It is in endothelial cell differentiation and VEGF It works in mitogenesis, angiogenesis and permeability enhancement effect.KDR is 200kDa glycoprotein, by extracellular domain In 7 Ig sample rings, transmembrane domain and be made of two intracellular tyrosine kinase structural domains that kinases insert separates.The Two and the 3rd Ig sample ring is the high-affinity part binding structural domain of VEGF, and first and the 4th Ig sample ring adjust ligand knot respectively Conjunction and Receptor dimerization.Compared with the Kd of the 25pM of VEGFR1, the Kd of VEGF combination KDR is 75-250pM.KDR is mainly in blood vessel It is expressed on the cell surface of endothelial cell.KDR exists in hematopoietic cell, vascular smooth muscle cells (VSMC) and some pernicious thin The cell surface of born of the same parents.

Angiogenesis is the highly complex process for growing new blood vessel, is related to capillary endothelial cell from pre-existing Vascular proliferation and migration and tissue infiltration, cell assembling is at tubular structure, the tubular assembly and closed circuit vascular system that are newly formed Connection, and the capillary newly formed are mature.

Angiogenesis is critically important in normal physiological processes, including embryonic development, ovarian follicular growth and wound healing.Excessive blood Pipe generation also results in tumor disease and non-neoplastic disease (such as age-related macular degeneration (AMD), diabetic keratopathy view Nethike embrane disease and neovascular glaucoma) in neovascularization.It has been confirmed that using Lucentis (ranibizumab)The anti-angiogenic therapy of target vascular therapy endothelial growth factors (VEGF) is effective to delay AMD progress.

Angiogenesis plays an important role in the growth and transfer of primary tumor, this is because growth and transfer depend on The formation of new blood vessel.When not having neovascularization caused by angiogenesis, there is necrosis, apoptosis or cannot grow into bright in tumour Aobvious size.Tumor Angiongesis is related to several processes, including from previous existing blood vessel activated endothelial cell, be proliferated, move Shifting and tissue infiltration.These processes generate angiogenesis growth factor such as VEGF by tumour cell and its surrounding substrate and draw Hair.

KDR has become the important target of anti-cancer therapies.Because of the VEGF of many tumors secrete rises, and KDR molecule Quantity keeps relative constant, so targeting KDR increases a possibility that inhibiting VEGF signal transduction, to inhibit tumour growth.

Summary of the invention

Provide the bispecific binding protein in conjunction with human PD-L 1 and people KDR.In certain embodiments, double special Property binding protein is in conjunction with PD-L1 and blocks the interaction of PD-L1 and PD-1.By the phase interaction for blocking PD-L1 and PD-1 With such bispecific binding protein can be used for reducing or inhibiting immunosupress.By the interaction of blocking VEGF and KDR, Such bispecific binding protein can be used for reducing or inhibiting angiogenesis.Bispecific binding protein is because in a kind of medicament It is combined with the ability for inhibiting both immunosupress and angiogenesis and particularly useful.

In one embodiment, the bispecific antibody in conjunction with human PD-L 1 and people KDR is provided.Bispecific is anti- Body includes the first antigen binding site in conjunction with human PD-L 1 and the second antigen binding site in conjunction with people KDR.It additionally provides The nucleic acid molecules of encoding bispecific antibody and expression vector comprising the nucleic acid, can be thin in protokaryon or eucaryon host The nucleic acid is expressed in born of the same parents, so as to cause the generation of the bispecific antibody.It additionally provides comprising double special for recombinating generation The host cell of the expression vector of heterogenetic antibody.

There is provided herein a kind of bispecific antibodies, and it includes the scFv for combining PD-L1, and the scFv is with combination KDR's Antibody connection.In one embodiment, the carboxyl terminal of the heavy chain constant domain of the IgG of PD-L1 scFv and combination KDR Connection.In another embodiment, the carboxyl terminal of the light chain constant domain of the IgG of PD-L1 scFv and combination KDR connects It connects.In another embodiment, PD-L1 scFv is connect with the amino terminal of the heavy-chain variable domains for the IgG for combining KDR. In another embodiment, PD-L1 scFv is connect with the amino terminal of the light variable domains for the IgG for combining KDR.

There is provided herein a kind of bispecific antibodies, and it includes the scFv for combining KDR, and the scFv is with combination PD-L1's Antibody connection.In one embodiment, the carboxyl terminal of the heavy chain constant domain of the IgG of KDR scFv and combination PD-L1 Connection.In another embodiment, the carboxyl terminal of the light chain constant domain of the IgG of KDR scFv and combination PD-L1 connects It connects.In another embodiment, KDR scFv is connect with the amino terminal of the heavy-chain variable domains for the IgG for combining PD-L1. In another embodiment, PD-L1 scFv is connect with the amino terminal of the light variable domains for the IgG for combining PD-L1.

In one embodiment, in conjunction with the amino acid sequence of the scFv of PD-L1 are as follows:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPAS VSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDE ADYYCSSYTSSSTRVFGTGTKVTVLGQP(SEQ ID NO:1).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the scFv of KDR are as follows:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMGWVRQAPGKGLEWVSSIYPSGGATNYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARGNYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVSP GQTASITCSGEKLGDEYASWYQQKPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAW DSSTLLFGGGTKLTVLGQP(SEQ ID NO:2).CDR is underlined.

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMGWVRQAPGKGLEWVSSIYPQGGATSYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARGNYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPGTLSLS PGEGATLSCRASQSVSSNYFGWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQ QFDSLPLTFGGGTKVEIK(SEQ ID NO:3).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the heavy-chain variable domains of the IgG of PD-L1 are as follows:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYRMFWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSS(SEQ ID NO:4).CDR adds lower stroke Line.

In one embodiment, in conjunction with the amino acid sequence of the light variable domains of the IgG of PD-L1 are as follows:

QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSK SGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL(SEQ ID NO:5).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the heavy-chain variable domains of the IgG of KDR are as follows:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMGWVRQAPGKGLEWVSSIYPSGGATNYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARGNYFDYWGQGTLVTVSS(SEQ ID NO:6).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the light variable domains of the IgG of KDR are as follows:

QSVLTQPPSVSVSPGQTASITCSGEKLGDEYASWYQQKPGQSPVLVIYQDNKRPSGIPERFSGSNSGN TATLTISGTQAMDEADYYCQAWDSSTLLFGGGTKLTVL(SEQ ID NO:7).CDR is underlined.

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMGWVRQAPGKGLEWVSSIYPQGGATSYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARGNYFDYWGQGTLVTVSS(SEQ ID NO:8).CDR is underlined.

DIQMTQSPGTLSLSPGEGATLSCRASQSVSSNYFGWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDSAVYYCQQFDSLPLTFGGGTKVEIK(SEQ ID NO:9).CDR is underlined.

In one embodiment, the bispecific antibody includes the heavy-chain variable domains in conjunction with human PD-L 1, and There are three complementary determining region (CDR) for tool, and wherein the amino acid sequence of CDR1 is GFTFSAYRMF (SEQ ID NO:10), CDR2's Amino acid sequence is SIYPSGGITFYADSVKG (SEQ ID NO:11), and the amino acid sequence of CDR3 is IKLGTVTTVDY (SEQ ID NO:12)。

In one embodiment, the bispecific antibody includes the light variable domains in conjunction with human PD-L 1, and There are three CDR for tool, and wherein the amino acid sequence of CDR1 is TGTSSDVGAYNYVS (SEQ ID NO:13), the amino acid sequence of CDR2 It is classified as DVSNRPS (SEQ ID NO:14), and the amino acid sequence of CDR3 is SSYTSSSTRV (SEQ ID NO:15).

In one embodiment, the bispecific antibody includes the heavy-chain variable domains in conjunction with people KDR and has Three CDR, wherein the amino acid sequence of CDR1 is GFTFSWYVMG (SEQ ID NO:16), and the amino acid sequence of CDR2 is SIYPSGGATNYADSVKG (SEQ ID NO:17), and the amino acid sequence of CDR3 is GNYFDY (SEQ ID NO:18).

In one embodiment, the bispecific antibody includes the light variable domains in conjunction with people KDR and has Three CDR, wherein the amino acid sequence of CDR1 is SGEKLGDEYAS (SEQ ID NO:19), and the amino acid sequence of CDR2 is QDNKRPS (SEQ ID NO:20), and the amino acid sequence of CDR3 is QAWDSSTLL (SEQ ID NO:21).

In one embodiment, the bispecific antibody includes the heavy-chain variable domains in conjunction with people KDR and has Three CDR, wherein the amino acid sequence of CDR1 is GFTFSWYVMG (SEQ ID NO:22), and the amino acid sequence of CDR2 is SIYPQGGATSYADSVK (SEQ ID NO:23), and the amino acid sequence of CDR3 is GNYFDY (SEQ ID NO:24).

In one embodiment, the bispecific antibody includes the light variable domains and tool in conjunction with people KDR There are three CDR, and wherein the amino acid sequence of CDR1 is RASQSVSSNYFG (SEQ ID NO:25), and the amino acid sequence of CDR2 is GASSRAT (SEQ ID NO:26), and the amino acid sequence of CDR3 is QQFDSLPLT (SEQ ID NO:27).

In one embodiment, in conjunction with the amino acid sequence of the IgG heavy chain of PD-L1 are as follows:

(SEQ ID NO:28).CDR is underlined.In some instances, LL (runic) can sport other residues, example Such as AA.

In one embodiment, in conjunction with the amino acid sequence of the IgG light chain of PD-L1 are as follows:

QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSK SGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFY PGAVTVAWKADSSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC(S) (SEQ ID NO:29).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the IgG heavy chain of KDR are as follows:

(SEQ ID NO:30).CDR is underlined.In some instances, LL (runic) can sport other residues, such as AA.

In one embodiment, in conjunction with the amino acid sequence of the IgG light chain of KDR are as follows:

QSVLTQPPSVSVSPGQTASITCSGEKLGDEYASWYQQKPGQSPVLVIYQDNKRPSGIPERFSGSNSGN TATLTISGTQAMDEADYYCQAWDSSTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC(S)(SEQ ID NO:31).CDR is underlined.

(SEQ ID NO:32).CDR is underlined.In some instances, LL (runic) can sport other residues, such as AA.

DIQMTQSPGTLSLSPGEGATLSCRASQSVSSNYFGWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDSAVYYCQQFDSLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:33).CDR is underlined.

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYLMKWVRQAPGKCLEWVSYIGSSGGFTAYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAMYYCAREDDFGAMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDIQMTQSPSSLSASVGDRVTITCRASQTVSKYFNWFQQKPGEAPKLLIYATSTLQSGVPSRFSGSGYGTEFT LTISSLQPEDFATYYCQQSYTTPWTFGCGTKVEIK(SEQ ID NO:57).CDR is underlined.Also have and to underline It is two cysteines from G mutation, i.e., " C ".

In one embodiment, the bispecific antibody include in conjunction with human PD-L 1 heavy-chain variable domains and Tool there are three complementary determining region (CDR), wherein the amino acid sequence of CDR1, CDR2 and CDR3 be respectively as follows: GFTFSWYLMK, YIGSSGGFTAYADSVKG and EDDFGAMDV (SEQ ID NO:58-60).

In one embodiment, the bispecific antibody include in conjunction with human PD-L 1 light variable domains and Tool there are three complementary determining region (CDR), wherein the amino acid sequence of CDR1, CDR2 and CDR3 be respectively as follows: RASQTVSKYFNW, ATSTLQS and QQSYTTPWT (SEQ ID NO:61-63).

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYLMKWVRQAPGKGLEWVSYIGSSGGFTAYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAMYYCAREDDFGAMDVWGQGTTVTVSS(SEQ ID NO:64).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the heavy chain of the IgG of PD-L1 are as follows:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYLMKWVRQAPGKGLEWVSYIGSSGGFTAYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAMYYCAREDDFGAMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:66).CDR is underlined.It also added with underscore is two the third ammonia from two leucine LL mutation Acid, i.e. " AA ".

DIQMTQSPSSLSASVGDRVTITCRASQTVSKYFNWFQQKPGEAPKLLIYATSTLQSGVPSRFSGSGYG TEFTLTISSLQPEDFATYYCQQSYTTPWTFGQGTKVEIK(SEQ ID NO:65).CDR is underlined.

In one embodiment, in conjunction with the amino acid sequence of the light chain of the IgG of PD-L1 are as follows:

DIQMTQSPSSLSASVGDRVTITCRASQTVSKYFNWFQQKPGEAPKLLIYATSTLQSGVPSRFSGSGYG TEFTLTISSLQPEDFATYYCQQSYTTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:67).CDR is underlined.

The present invention also provides the conjugates of bispecific binding protein, such as, but not limited to imaging agent, therapeutic agent or thin Cellular toxicity agent.

The present invention also provides the combinations comprising bispecific binding protein and at least one pharmaceutically acceptable carrier Object.

In one embodiment, egg can be merged in conjunction with people KDR in conjunction with people PDL1 and also by providing It is white.Fusion protein may include the part in conjunction with human PD-L 1 and the part in conjunction with people KDR.In one embodiment, it merges Part of the albumen in conjunction with human PD-L 1 is antibody or its PD-L1 binding fragment.In one embodiment, fusion protein and people The part that KDR is combined is antibody or its KDR binding fragment.In one embodiment, portion of the fusion protein in conjunction with human PD-L 1 Dividing is antibody or its PD-L1 binding fragment, and part of the fusion protein in conjunction with people KDR is antibody or its KDR binding fragment.

A kind of method for inhibiting people PD1 and human PD-L 1 interaction in subject is provided, this method includes applying effectively The bispecific antibody disclosed herein or its segment of amount.Additionally provide a kind of immunosuppressive side that inhibition human PD-L 1 mediates Method comprising apply a effective amount of bispecific antibody disclosed herein or its segment or fusion protein disclosed herein.

Additionally provide it is a kind of stimulation to expression human PD-L 1 cell or tissue immune response method comprising to by Examination person applies a effective amount of bispecific antibody disclosed herein or its segment or fusion protein disclosed herein.In certain realities It applies in scheme, the cell or tissue for expressing human PD-L 1 is neoplastic cell or infected cell.

Provide a kind of method of neutralization people KDR or mouse KDR activation comprising make cell and a effective amount of pair of the invention Specific antibody or the contact of its segment.

Additionally provide a kind of method for inhibiting angiogenesis comprising apply a effective amount of double spies of the invention to subject Heterogenetic antibody or its segment.

Additionally provide a kind of method for reducing tumour growth comprising apply a effective amount of double spies of the invention to subject Heterogenetic antibody or its segment.

Disclosed herein is a kind of methods of tumor disease for treating subject comprising applies to subject a effective amount of Bispecific antibody as disclosed herein or its segment, wherein the tumorous type disease is selected from lung cancer, colorectal cancer nephrocyte Cancer, spongioblastoma, oophoroma, bladder cancer, gastric cancer, Huppert's disease, non-small cell lung cancer and cancer of pancreas.

There is provided herein the non-limiting embodiments of the invention below with number:

1. a kind of bispecific binding protein, it includes the firstth areas in conjunction with human PD-L 1 and second in conjunction with people KDR Area.

2. being bispecific antibody according to bispecific binding protein described in embodiment 1.

3. being fusion protein according to bispecific binding protein described in embodiment 1.

4. a kind of bispecific antibody, it includes IgG, IgA, IgE or IgD and scFv.

5. it includes the IgG for combining PD-L1 and combining KDR's according to bispecific antibody described in embodiment 4 scFv。

6. it includes the IgG for combining KDR and combining PD-L1's according to bispecific antibody described in embodiment 4 scFv。

7. the bispecific antibody according to embodiment 5 or 6, it includes combine human PD-L 1 and have that there are three mutually The heavy-chain variable domains for determining area (CDR) are mended, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 10-12 or respectively SEQ ID NO:58-60.

8. the bispecific antibody according to embodiment 5 or 6, it includes combine human PD-L 1 and have that there are three CDR Light variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 be respectively SEQ ID NO:13-15 or be respectively SEQ ID NO:61-63。

9. the bispecific antibody according to embodiment 5 or 6, it includes combine people KDR and have that there are three CDR's Heavy-chain variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:16-18.

10. the bispecific antibody according to embodiment 5 or 6, it includes combine people KDR and have that there are three CDR Light variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:19-21.

11. the bispecific antibody according to embodiment 5 or 6, it includes combine people KDR and have that there are three CDR Heavy-chain variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:22-24.

12. the bispecific antibody according to embodiment 5 or 6, it includes combine people KDR and have that there are three CDR Light variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:25-27.

13. the bispecific antibody according to embodiment 5 or 6, it includes heavy chain and light chain, wherein the heavy chain and Light chain includes heavy chain/light chain pair corresponding sequence selected from the group being made up of: SEQ ID NO:34 and 31, SEQ ID NO: 30 and 35, SEQ ID NO:36 and 31, SEQ ID NO:30 and 37, SEQ ID NO:38 and 33, SEQ ID NO:32 and 39, SEQ ID NO:40 and 33, SEQ ID NO:32 and 41, SEQ ID NO:42 and 29, SEQ ID NO:28 and 43, SEQ ID NO:44 and 29, SEQ ID NO:28 and 45, SEQ ID NO:46 and 29, SEQ ID NO:28 and 47, SEQ ID NO:48 and 29, SEQ ID NO:28 and 49, SEQ ID NO:51 and 50, SEQ ID NO:52 and 50, SEQ ID NO:53 and 50, SEQ ID NO:54 and 29, SEQ ID NO:55 and 29 and SEQ ID NO:56 and 29.

14. it is a kind of treat need to reduce immunosupress or reduce angiogenesis patient method comprising to need this The patient that sample reduces immunosupress or angiogenesis apply bispecific binding protein described in any one of embodiment 1-3 or Bispecific antibody described in any one of embodiment 4-13.

15. a kind of method for the treatment of cancer comprising applied described in any one of embodiment 1-3 to patient in need Bispecific binding protein or any one of embodiment 4-13 described in bispecific antibody.

16. according to method described in embodiment 15, wherein the cancer be selected from lung cancer, colorectal cancer clear-cell carcinoma, at Spongiocytoma, oophoroma, bladder cancer, gastric cancer, Huppert's disease, non-small cell lung cancer and cancer of pancreas.

17. a kind of isolated nucleic acid molecules encode bispecific combination egg described in any one of embodiment 1-3 It is white, bispecific antibody described in any one of embodiment 4-13 or its polypeptide chain.

18. a kind of carrier, it includes nucleic acid molecules described in embodiment 17.

19. a kind of host cell of culture, it includes carriers described in embodiment 18.

20. a kind of method for generating polypeptide, the method includes cultivating under conditions of allowing the nucleic acid molecules to express Host cell described in embodiment 19.

21. any in bispecific binding protein described in a kind of any one of embodiment 1-3 or embodiment 4-13 The conjugate of bispecific antibody described in, wherein the bispecific binding protein or the bispecific antibody be selected from The medicament of imaging agent, therapeutic agent and cytotoxic agent is conjugated.

22. a kind of pharmaceutical composition, it includes

Bispecific binding protein described in any one of embodiment 1-3, described in any one of embodiment 4-13 Conjugate and pharmaceutically acceptable carrier described in bispecific antibody or embodiment 21.

It is explained in the following description the details of one or more embodiments of the invention.From description and claims It sees, other features, objects, and advantages of the present invention will be evident.

Detailed description of the invention

Figure 1A, 1B, 1C and 1D show the bispecific antibody that can be constructed comprising the combined area PD-L1 and the combined area KDR Four kinds of possible modes.In each case, the bispecific antibody includes IgG, one of combined area that IgG includes It is covalently attached with the scFv comprising other combined areas.IgG uses conventional both arms antibody plot and display；ScFv is elongated oval. Figure 1A depicts the scFv connecting with the carboxyl terminal of IgG heavy chain constant domain.Figure 1B is depicted and IgG chain constant structure The scFv of the carboxyl terminal connection in domain.Fig. 1 C depicts the scFv connecting with the amino terminal of IgG light variable domains.Fig. 1 D Depict the scFv connecting with the amino terminal of IgG heavy-chain variable domains.

Fig. 2 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:34 and 31) of bispecific antibody, wherein claiming For the carboxyl terminal of the heavy chain constant domain of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1A1 of D7A8 Connection.

Fig. 3 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:30 and 35) of bispecific antibody, wherein claiming For the carboxyl terminal of the light chain constant domain of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1A1 of D7A8 Connection.

Fig. 4 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:36 and 31) of bispecific antibody, wherein claiming For the amino terminal of the heavy-chain variable domains of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1A1 of D7A8 Connection.

Fig. 5 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:30 and 37) of bispecific antibody, wherein claiming For the amino terminal of the light variable domains of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1A1 of D7A8 Connection.

Fig. 6 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:38 and 33) of bispecific antibody, wherein claiming For the carboxyl end of the heavy chain constant domain of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1C4A7 of D7A8 End connection.

Fig. 7 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:32 and 39) of bispecific antibody, wherein claiming For the carboxyl end of the light chain constant domain of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1C4A7 of D7A8 End connection.

Fig. 8 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:40 and 33) of bispecific antibody, wherein claiming For the amino end of the heavy-chain variable domains of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1C4A7 of D7A8 End connection.

Fig. 9 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:32 and 41) of bispecific antibody, wherein claiming For the amino end of the light variable domains of the KDR specific IgG antibodies of the PD-L1 specificity scFv and referred to as B1C4A7 of D7A8 End connection.

Figure 10 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:42 and 29) of bispecific antibody, wherein claiming For the carboxyl terminal of the heavy chain constant domain of the PD-L1 specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1A1 Connection.

Figure 11 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:28 and 43) of bispecific antibody, wherein claiming Carboxyl terminal for the light chain constant domain of the KDR specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1A1 connects It connects.

Figure 12 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:44 and 29) of bispecific antibody, wherein claiming Amino terminal for the heavy-chain variable domains of the KDR specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1A1 connects It connects.

Figure 13 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:28 and 45) of bispecific antibody, wherein claiming Amino terminal for the light variable domains of the KDR specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1A1 connects It connects.

Figure 14 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:46 and 29) of bispecific antibody, wherein claiming For the carboxyl end of the heavy chain constant domain of the PD-L1 specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1C4A7 End connection.

Figure 15 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:28 and 47) of bispecific antibody, wherein claiming For the carboxyl terminal of the light chain constant domain of the KDR specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1C4A7 Connection.

Figure 16 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:48 and 29) of bispecific antibody, wherein claiming For the amino terminal of the heavy-chain variable domains of the KDR specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1C4A7 Connection.

Figure 17 shows the heavy chain and light-chain amino acid sequence (SEQ ID NO:28 and 49) of bispecific antibody, wherein claiming For the amino terminal of the light variable domains of the KDR specific IgG antibodies of the KDR specificity scFv and referred to as D7A8 of B1C4A7 Connection.

Figure 18 shows the structure of the bispecific antibody (BsAb) of VEGFR2 and PDL1.Anti-vegf R2 antibody B1A1 (with People, rat and monkey VEGFR2 combination) and B1C4A7 (in conjunction with people, mouse, rat and monkey VEGFR2) use as routine IgG1. Anti- PDL1 antibody D7A8 (in conjunction with mouse, rat and monkey PDL1) be reformatted as single chain variable fragment and invest B1A1 or The end c- of B1C4A7." LALA form ", which refers to, replaces two leucine residues (such as above SEQ by two alanine (AA) LL runic in ID NO:28,30 and 32) make the antibody of its CH2 region mutation.

Figure 19 analyzes the SDS-PAGE of bispecific antibody after showing Protein A purification.

Figure 20 shows the size exclusion chromatography figure of the bispecific antibody in UPLC system through Protein A purification (under 280nm UV trace).

Figure 21 shows the DSC scanning result of bispecific antibody in PBS buffer solution.

Figure 22 is shown in mice serum in the Western blotting result of the bispecific antibody of 37 processing 5 days.

Figure 23 shows the bispecific antibody of the EC50 for measuring people and mouse VEGFR2 and people and mouse PDL1 The dose response ELISA result of A7-A8.

Figure 24 shows the agent for measuring bispecific antibody A7-A8 to the IC50 of VEGF-VEGFR2 and PDL1-PD1 Quantitative response blocks ELISA result.

Figure 25 is shown and the combination of the people or mouse VEGFR2 and people or mouse PDL1 of cell expression.

Figure 26 show the bispecific antibody A7-A8 that is checked by surface plasma body resonant vibration (Biacore) with by The interaction of body hVEGFR2 and hPDL1.

Figure 27, which is shown, is intersecting the scheme for combining the compound in ELISA on the fixed surface hVEGFR2 and in solution.

Figure 28 is shown combines ELISA to check by intersecting, and bispecific antibody A7-A8 can be tied simultaneously with VEGFR2 and PDL1 It closes.

Figure 29 shows bispecific antibody A7-A8 to VEGFR2 and KDR-PAE (hVEGFR2) and EOMA (mVEGFR2) In downstream molecules inhibited by the VEGF phosphorylation stimulated.

Figure 30 shows the secretion of cell factor IL2 and INF γ in the presence of bispecific antibody A7-A8.

Figure 31 shows the bispecific antibody of the EC50 for measuring people and mouse VEGFR2 and people and mouse PDL1 The dose response ELISA result of A1-A8.

Figure 32 shows the agent for measuring bispecific antibody A1-A8 to the IC50 of VEGF-VEGFR2 and PDL1-PD1 Quantitative response blocks ELISA result.

Figure 33 is shown and the combination of the people or mouse VEGFR2 and people or mouse PDL1 of cell expression.

Figure 34 show the bispecific antibody A1-A8 that is checked by surface plasma body resonant vibration (Biacore) with by The interaction of body hVEGFR2 and hPDL1.

Figure 35 is shown combines ELISA to check by intersecting, and bispecific antibody A1-A8 can be tied simultaneously with VEGFR2 and PDL1 It closes.

Figure 37 shows the secretion of cell factor IL2 and INF γ in the presence of bispecific antibody A1-A8 and A7-A8.

Figure 38 shows the CT26 result of study of BsAb (A7-A8).

Figure 39 A and 39B show the MC38 result of study of BsAb (A7-A8).

Figure 40 shows the SDS-PAGE of BsAb (B1A1-A11) and BsAb (B1C4A7-A11) variant as a result, wherein observing To degradation band.

Figure 41 shows the SDS-PAGE of BsAb (A11-B1A1) and BsAb (D7A8-B1A1) variant as a result, wherein in side Degradation band is not observed after to change.

Figure 42 is shown and the combination of PDL1 and VEGFR2.

Figure 43 display blocks the interaction of ligand and receptor.

Figure 44 shows that BsAb (A11-B1A1), BsAb (A11-B1A1) _ 30 and BsAb (A11-B1A1) _ 30cc are common Sequence of light chain (SEQ ID NO:50) and three sequence of heavy chain (SEQ ID NO:51-53).

Figure 45 shows BsAb (D7A8-B1A1), BsAb (D7A8-B1A1) _ 30 and BsAb (D7A8-B1A1) _ 30cc Common light chain sequence (SEQ ID NO:29) and three sequence of heavy chain (SEQ ID NO:54-56).

Specific embodiment

The interaction of PD-1 and PD-L1 on immunocyte inhibit the proliferation of immunocyte and cell factor to generate.PD- L1 is also derivable and up-regulation in various tissues (including cancer).PD-1 and PD-L1 works in immunosupress together. There is provided herein in conjunction with human PD-L 1 and blocking its novel bispecific binding protein with the interaction of people PD-1, such as The antigen-binding fragment of bispecific antibody or such bispecific antibody.

Bispecific antibody is also in conjunction with people KDR and blocks the interaction of itself and people VEGF.In some embodiments, The bispecific antibody block ligand in conjunction with KDR (for example, one of VEGF-A, VEGF-C, VEGF-D or VEGF-E or A variety of combination).In some embodiments, in the bispecific antibody and the activation of KDR.The bispecific antibody can For treating tumor disease, including such as entity and non-physical knurl and hyperproliferative disorder.It thus provides neutralizing KDR The method of activation inhibits tumour growth, the method including inhibiting tumor-associated vessels to generate, and treatment angiogenesis related diseases The method of disease.

Additionally provide the kit containing bispecific antibody or antibody fragment in conjunction with PDL1 and KDR.

The bispecific antibody is not limited by the KDR any specific mechanism inhibited.A kind of bispecific antibody is abided by The mechanism followed is not necessarily identical as the mechanism that another bispecific antibody is followed.Some possible mechanism include preventing VEGF Ligand is in conjunction with KDR extracellular binding domains and prevents Receptor dimerization or oligomerization.However, can not rule out other mechanism.

The bispecific antibody inhibits KDR activation.The measurement that KDR inhibits is the tyrosine kinase activity drop of receptor It is low.Known method measurement, such as the autophosphorylation level of measurement receptor can be used in tyrosine kinase inhibition.It can also pass through Inhibition or adjusting to the phosphorylation event of natural or synthetic KDR substrate and the other components of KDR signal transduction pathway are observed pair The inhibition of KDR.For example, ELISA measurement in or can be used on Western blotting has specificity to resist phosphotyrosine Body detects phosphorylation.Panek etc., J.Pharmacol.Exp.Thera., 283:1433-44 (1997) and Batley etc., Life Sci., 62:143-50 (1998) describe some measuring methods to tyrosine kinase activity.

Also it can use in vivoassay method.For example, can be inhibited by mitogenesis measuring method existing and being not present Receptor tyrosine kinase inhibition is observed in the case where agent using the cell line through receptor ligand stimulation.For example, being stimulated through VEGF HUVEC cell (ATCC) can be used for measuring KDR inhibition.Another method is related to using such as injection intracorporal human tumour of mouse Cell tests the inhibition to the growth of tumour cell of VEGF expression.See, e.g. U.S. Patent No. 6,365,157 (Rockwell etc.).

Bispecific binding protein (such as bispecific antibody) disclosed herein can pass through methods known in the art Preparation.Such method may involve the use of the nucleic acid of coding binding protein (such as bispecific antibody) or part thereof.It can be used for making The carrier of standby binding protein disclosed herein (such as bispecific antibody) includes being suitable for expressing protein in HEK293 cell Transient expression vector, such as pBh1 (Dyax) or pcDNA^TM3.4Carrier (Thermo Fisher Scientific).Suitable for expressing the stable expressed vector of protein, such as CHO-S (Thermo in mammalian cells Fisher Scientific) in pCHO.1 or CHO-K (Lonza) in GS carrier.Those skilled in the art can refer to following Publication obtains the guidance for preparing binding protein disclosed herein (such as bispecific antibody) or part thereof: Lu D and Zhu Z. (2014)；Construction and production of an IgG-like tetravalent bispecific antibody,IgG-single-chain Fv fusion.Human Monoclonal antibodies,methods and protocols；Humanna press；ISSN 1064-3745；Marvin J.S.,Zhu Z.(2006)Bispecific antibodies for dual-modality cancer therapy:killing two signaling cascades with one stone.Curr.Opin.Drug Discov.Devel.9,184-193；Lu, D., Zhang, H., Koo, H. etc. (2005)A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity.J.Biol.Chem.280,19665–19672；Demarest S.J (2011)Emerging antibody combinations in oncology.mAbs 3,338-351；Spiess C., Zhai Q.,Carter P.(2015)Alternative molecular formats and therapeutic applications for bispecific antibodies.Molecular Immunology 67,95-106； Croasdale R. etc. (2012) Development of tetravalent IgG1 dual targeting IGF-1R- EGFR antibodies with potent tumor inhibition.Archives of Biochem.and Biophys.526,206-218。

According to the amino acid sequence of Kabat and Chothia mark system banner heavy chain and light chain CDR set forth herein.Root The first two heavy chain CDR is identified according to Kabat and Chothia common system, provides different but overlapping position for CDR.To perhaps The comparison of multiple chain and light chain shows the significant similitude between many CDR sequences.It is therefore contemplated that many CDR can be with It is mixed and matched between sequence.

Bispecific binding protein or bispecific antibody as described herein can have one or more amino acid substitutions, lack It loses, be inserted into and/or add.In certain embodiments, the bispecific antibody or albumen can comprising heavy chain disclosed above One of one of structure changes domain and above-mentioned light variable domains.In certain embodiments, the bispecific antibody Or its binding fragment includes one or more variable domains disclosed above, amino acid sequence and variable knot disclosed above Structure domain sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% are identical. In order to weaken effector function, the bright of the position of 234 or 235 or both (in the CH2 structural domains) corresponding to IgG1 can be made Histidine mutations are alanine (such as above-mentioned LALA form or LALA mutation).Furthermore it is possible to be mutated in two structural domains, Such as one or more disulfide bond are introduced between two variable domains, so as to improve thermal stability.For example, corresponding to SEQ ID The residue of the position 44 and 248 of NO:57 can sport cysteine to introduce disulfide bond.

It is unless otherwise indicated or clear from the context, otherwise term " antibody " as used herein may include whole antibody and Its any antigen-binding fragment (i.e. antigen-binding portion thereof) or its is single-stranded.In one embodiment, " antibody " refers to comprising logical Cross the glycoprotein or its antigen-binding fragment of disulfide bond at least two weight (H) chains interconnected and two light (L) chain.Every (abbreviated herein as V by heavy chain variable region for heavy chain_H) and heavy chain constant region composition.In certain naturally occurring IgG, IgD and IgA In antibody, heavy chain constant region is made of three structural domain, that is, CH1, CH2 and CH3.It is every light in certain naturally occurring antibody (abbreviated herein as V by light chain variable region for chain_L) and constant region of light chain composition.Constant region of light chain is by a structural domain, that is, C_LComposition. V_HAnd V_LArea can be further subdivided into the hypervariable region of referred to as complementary determining region (CDR), and it is higher to be scattered with conservative, referred to as skeleton area (FR) region.Each V_HAnd V_LIt is made of three CDR and four skeleton areas (FR), it is in the following order from aminoterminal to c-terminus Arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.Heavy chain and light chain variable region contain and the combination of antigen interactions Structural domain.Antibody constant region can include siberian crabapple with the combination of mediated immunity globulin and host tissue or the factor, the factor The first component (Clq) of the various cells (such as effector cell) of system and classical complement system.

As used herein, " antibody " uses in the sense that most wide, and specifically includes monoclonal antibody (including overall length Monoclonal antibody), polyclonal antibody, multi-specific antibody (such as bispecific antibody) and antibody fragment, as long as it shows Required bioactivity.Example includes human antibody, humanized antibody and chimeric antibody.As used herein, " antibody fragment " can A part comprising complete antibody, generally include complete antibody antigen binding domain and/or variable region and/or keep FcR combine The antibody Fc district of ability.The example of antibody fragment includes linear antibodies；Single-chain antibody molecules；It is mostly special with being formed by antibody fragment Heterogenetic antibody.Preferably, antibody fragment remains the entire constant region of IgG heavy chain and including IgG light chain.

As used herein, " antigen-binding portion thereof " or " antigen-binding fragment " of term antibody refers to that the holding of antibody is special One or more segments of the opposite sex in conjunction with the ability of antigen (such as human PD-L 1 or KDR).Term antibody " antigen-binding portion thereof/ The example for the binding fragment that segment " is covered includes (i) Fab segment-by V_L、V_H、C_LWith the monovalent fragment of CH1 structural domain composition； (ii) 2 segment of F (ab')-is included in the bivalent fragment for two Fab segments that hinge area is connected by disulfide bond；(iii) by V_HWith The Fd segment of CH1 structural domain composition；(iv) by the V of antibody single armed_LAnd V_HThe Fv segment of structural domain composition, and (v) by V_HStructural domain The dAb segment (Ward etc. (1989) Nature341:544-546) of composition.Independent complementary determining region (CDR) is connect by synthesis The combination of two or more independent CDR of head connection, if it is possible in conjunction with antigen, then may include the antigen binding knot of antibody Structure domain.

" people " antibody (HuMAb) refers to that the antibody with variable region, middle skeleton area and CDR region are derived from people's racial immunity Globin sequence.In addition, if antibody contains constant region, then constant region also originates from human germline immunoglobulin's sequence.The present invention Human antibody may include the amino acid residue of not human germline immunoglobulin's sequential coding (for example, by external random or fixed Point mutagenesis introduces mutation, or passes through internal somatic mutation).However, as used herein, term " human antibody " is not intended to wrap It includes the wherein CDR sequence from another mammalian species (such as mouse) germline and has been transplanted to resisting in human skeleton's sequence Body.The use synonymous with " full people " antibody of term " people " antibody.

" humanization " antibody refer to some, most of outside wherein non-human antibody (such as mouse antibodies) CDR structural domain or All amino acid are originated from the antibody of the corresponding amino acid replacement of human immunoglobulin(HIg).In an implementation of antibody humanization's form In scheme, some, most of or all amino acid outside CDR structural domain by the amino acid replacement from human immunoglobulin(HIg), And some, most of or all amino acid in one or more CDR regions are constant.Amino acid it is a small amount of addition, missing, insertion, Replace or modification is allowed, as long as they do not eliminate the ability of antibody combination specific antigen." humanization " antibody is kept and original Antigentic specificity as antibody class.

" chimeric antibody " refers to the antibody that wherein variable region is originated from a species and constant region is originated from another species, such as its The middle variable region antibody of constant region from human antibody from mouse antibodies." heterozygosis " antibody refers to different types of heavy chain And light chain, such as the antibody of mouse (parent) heavy chain and humanization light chain, or vice versa.

" bispecific " refers to protein (such as antibody) in conjunction with PD-L1 and KDR." bispecific antibody " is that have two The artificial hybrid antibody of a different heavy chains/light chain pair generates two antigen binding sites to not synantigen with specificity. Bispecific antibody can be generated by a variety of methods, the connection of fusion or Fab' segment including protein.See, e.g. Songsivilai and Lachmann (1990) Clin.Exp.Immunol.79:315-321；Kostelny etc. (1992) J.Immunol.148,1547-1553。

Binding specificity can be conjugated by using other methods known in the art in bispecific molecule described herein It is prepared by component part.For example, each binding specificity part of bispecific molecule can individually generate, then sew each other It closes.A variety of coupling agents or crosslinking agent can be used for covalently being conjugated.Example includes albumin A, carbodiimide, N- succinimido-S- Acetyl group-thiacetate (SATA), 5,5 '-two thiobis (2- nitrobenzoic acid) (DTNB), two maleimide of adjacent phenylene Amine (oPDM), N- succinimido -3- (2)-pyridyl group two are thio) propionic ester (SPDP) and sulfosuccinimide base 4- (N- maleimidomehyl) hexamethylene -1- carboxylate (sulfo group-SMCC) is (see, for example, Karpovsky etc. (1984) J.Exp.Med.160:1686；Liu, MA etc. (1985) Proc.Natl.Acad.Sci. (USA) 82:8648).Other method packets Include Paulus (1985) Behring Ins.Mitt.No.78,118-132；Brennan etc. (1985) Science 229:81- 83) and (1987) J.Immunol.139:2367-2375 such as Glennie) described in those methods.Agent, which is preferably conjugated, is SATA and sulfo group-SMCC can both be obtained from Pierce Chemical Co. (Rockford, IL).Antibody can also pass through The sulfydryl of two heavy chain C-terminal hinge areas is bonded and is conjugated.In an especially preferred embodiment, by hinge before conjugation Area is modified to containing odd number sulfhydryl residue, preferably one.

Alternatively, two basic change part can encode in same vehicle, and expresses and assemble in identical host cell.This The bispecific molecule of text description can be the single chain molecule comprising two single-chain antibodies, or answering at least two antibody chains Object is closed, or combinations thereof.The method for preparing bispecific molecule can be at such as U.S. Patent No. 5,260,203；United States Patent (USP) No. 5,455,030；U.S. Patent No. 4,881,175；U.S. Patent No. 5,132,405；U.S. Patent No. 5,091,513 Number；U.S. Patent No. 5,476,786；U.S. Patent No. 5,013,653；U.S. Patent No. 5,258,498；It is special with the U.S. It is found in benefit the 5,482,858th.Art recognized methods can be used in the combination of bispecific molecule and its particular target Confirmation, such as enzyme linked immunosorbent assay (ELISA) known in the art or as described herein (ELISA), radiommunoassay (RIA), facs analysis, bioassay (such as growth inhibition) or Western blotting measurement.

" inhibit receptor " means to weaken and/or inactivate the ability of receptor transduction signal, for example, by weaken and/or inactivate by The inherent kinase activity of body.

" identity " refers to the quantity or percentage for the same position that two amino acid or nucleic acid sequence share, that takes into account The quantity in the vacancy for needing to introduce for two sequences of optimal comparison and the length in each vacancy.

Term " peptide ", " polypeptide " and " protein " is interchangeable for describing the row of amino acid residue in polymer herein Column.In addition to rare amino acid and synthesizing amino acid-like substance, peptide, polypeptide or protein can be by the naturally occurring of 20 kinds of standards Amino acid composition.They can be any amino acid chain, regardless of length or posttranslational modification how (for example, glycosylation or Phosphorylation).

In some cases, can by select to keep following aspect influence on the substitution without significant difference come into Row amino acid substitution: (a) replace the structure of peptide backbone in region, (b) charge or hydrophobicity of the molecule in target position；Or (c) side chain Volume.For example, naturally occurring residue can be grouped based on side chain properties；(1) hydrophobic amino acid (nor-leucine, first sulphur ammonia Acid, alanine, valine, leucine and isoleucine)；(2) Neutral hydrophilic amino acids (cysteine, serine and Soviet Union's ammonia Acid)；(3) acidic amino acid (aspartic acid and glutamic acid)；(4) (asparagine, histidine, relies glutamine basic amino acid Propylhomoserin and arginine)；(5) amino acid (glycine and proline) of chain orientation is influenced；(6) aromatic amino acid (tryptophan, Tyrosine and phenylalanine).The substitution carried out in these groups can be considered as conservative replaces.Substituted example includes but is not limited to Valine substituted for alanine, lysine for arginine, glutamin for asparagine, glutamate for aspartate, silk Propylhomoserin substitution cysteine, asparagine for glutamine, aspartate for glutamate, proline take glycine, smart ammonia Acid replaces histidine, leucine for isoleucine, and isoleucine replaces leucine, arginine for lysine, and leucine takes For methionine, leucine for phenylalanine, glycine for proline, threonin for serine, serine, which replaces, revives Propylhomoserin, tyrosine for tryptophan, phenylalanine for tyrosine and/or leucine for valine.

Method and computer program for measuring sequence similarity is publicly available, including but not limited to GCG program Packet (Devereux etc., Nucleic Acids Research 12:387,1984), BLASTP, BLASTN, FASTA (Altschul etc., J.Mol.Biol.215:403 (1990) and ALIGN program (2.0 editions).Well known Smith Waterman is calculated Method can also be used for measurement similitude.Blast program is from NCBI and other sources publicly available (BLAST Manual, Altschul Deng NCBI NLM NIH, Bethesda, Md.20894；On http://www.ncbi.nlm.nih.gov/blast/ BLAST2.0).In relatively sequence, these methods consider various substitutions, deletions, and other modifications.Conservative replaces are usually wrapped Include the substitution with the following group: glycine, alanine；Valine, isoleucine, leucine；Aspartic acid, glutamic acid, asparagine, Glutamine；Serine, threonine；Lysine, arginine；With phenylalanine, tyrosine.

Bispecific antibody provided herein further include by direct mutagenesis, affinity maturation method, phage display or Chain improves those of binding characteristic bispecific antibody.It can be by being mutated CDR and screening with required feature Affinity and specificity are modified or improved to antigen binding site.It is mutated CDR in many ways.A kind of method be make it is single residual Base or residue combinations randomization, so that finding institute in specific position in other aspects in the group of identical antigen binding site There are 20 kinds of amino acid.Alternatively, by fallibility PCR method on a series of CDR residues induced mutation (see, for example, Hawkins Deng J.Mol.Biol., 226:889-896 (1992)).For example, being carried containing heavy chain and the phage display of light-chain variable region gene Body can be proliferated in the mutant strain of Escherichia coli (E.coli) (see, for example, Low etc., J.Mol.Biol., 250:359- 368(1996)).These method of mutagenesis illustrate many methods well known by persons skilled in the art.

In order to minimize immunogenicity, the bispecific antibody of people's constant domain sequence is preferably comprised.Bispecific Antibody can be, and may include or can combine any immunoglobulin class, for example, IgG, IgM, IgA, IgD or IgE and its The member of subclass.Antibody isotype be can choose with improved effect subfunction (for example, complement-dependent cytotoxicity (CDC) and anti- Body dependent cellular cytotoxicity (ADCC)).

Certain embodiments of bispecific binding protein are related to using PD-L1 and KDR binding antibody segment.Fv is to have contained Bulk wight chain and light variable domains, the minimal segment including all six hypervariable loops (CDR).Lack constant domain, varistructure Domain noncovalent associations.Can be used allows V_HAnd V_LStructural domain, which associates, to be formed the connector of antigen binding site and connects heavy chain and light chain It is connected into single polypeptide chain (" scFv " or " scFv ").In one embodiment, the connector is (Gly-Gly-Gly-Gly- Ser)₃.Since scFv segment lacks the constant domain of whole antibody, so more much smaller than whole antibody.ScFv segment is also without just The interaction of normal heavy-chain constant domains and other biomolecule, this may be unwanted in certain embodiments.

Containing V_H、V_LWith optional C_L、C_H1 or the antibody fragments of other constant domains can also be used for bispecific binding protein In.Fab is known as by the antibody monovalent fragment that papain digestion generates and lacks heavy chain hinge region.By pepsin digestion It generates, referred to as F (ab')₂Segment remain heavy chain hinge and be divalent.Such segment can also recombinate generation.Perhaps Mostly other useful antigen binding antibody fragments are known in the art, including but not limited to double-chain antibody, three chain antibodies, unijunction Structure domain antibodies and other unit prices and multivalent forms.

In one embodiment, it is measured by surface plasma body resonant vibration, bispecific binding protein is for example double special Property antibody the combined area PD-L1 association rate constants (Kon) be at least about 10²M^-1s^-1；At least about 10³M^-1s^-1；At least about 10⁴M^-1s^-1；At least about 10⁵M^-1s^-1；Or at least about 10⁶M^-1s^-1.In one embodiment, pass through surface plasma body resonant vibration Measurement, the association rate constants (Kon) of the combined area PD-L1 are between 10²M^-1s^-1With 10³M^-1s^-1Between；Between 10³M^-1s^-1With 10⁴M^-1s^-1Between；Between 10⁴M^-1s^-1With 10⁵M^-1s^-1Between；Or between 10⁵M^-1s^-1With 10⁶M^-1s^-1Between.

In one embodiment, it is measured by surface plasma body resonant vibration, bispecific binding protein is for example double special Property antibody the combined area KDR association rate constants (Kon) be at least about 10²M^-1s^-1；At least about 10³M^-1s^-1；At least about 10⁴M^-1s^-1；At least about 10⁵M^-1s^-1；Or at least about 10⁶M^-1s^-1.In one embodiment, it is measured by surface plasma body resonant vibration, The association rate constants (Kon) of the combined area KDR are between 10²M^-1s^-1With 10³M^-1s^-1Between；Between 10³M^-1s^-1With 10⁴M^-1s^-1It Between；Between 10⁴M^-1s^-1With 10⁵M^-1s^-1Between；Or between 10⁵M^-1s^-1With 10⁶M^-1s^-1Between.

In one embodiment, it is measured by surface plasma body resonant vibration, bispecific binding protein is for example double special Property antibody the combined area PD-L1 dissociation rate constant (Koff) be at most about 10^-3s^-1；At most about 10^-4s^-1；At most about 10^-5s^-1；Or at most about 10^-6s^-1.In one embodiment, it is measured by surface plasma body resonant vibration, the dissociation of the combined area PD-L1 Rate constant (Koff) is 10^-3s^-1To 10^-4s^-1；10^-4s^-1To 10^-5s^-1；Or 10^-5s^-1To 10^-6s^-1。

In one embodiment, it is measured by surface plasma body resonant vibration, bispecific binding protein is for example double special Property antibody the combined area KDR dissociation rate constant (Koff) be at most about 10^-3s^-1；At most about 10^-4s^-1；At most about 10^-5s^-1； Or at most about 10^-6s^-1.In one embodiment, it is measured by surface plasma body resonant vibration, the dissociation rate of the combined area KDR Constant (Koff) is 10^-3s^-1To 10^-4s^-1；10^-4s^-1To 10^-5s^-1；Or 10^-5s^-1To 10^-6s^-1。

In one embodiment, the dissociation of the combined area PD-L1 of bispecific binding protein such as bispecific antibody Constant (K_D) it is at most about 10^-7M；At most about 10^-8M；At most about 10^-9M；At most about 10^-10M；At most about 10^-11M；At most about 10^-12M；Or at most 10^-13M.In one embodiment, the PD-L1 of bispecific binding protein such as bispecific antibody is combined Dissociation constant (the K in area and its target_D) it is 10^-7M to 10^-8M；10^-8M to 10^-9M；10^-9M to 10^-10M；10^-10M to 10^-11M；10^-11M to 10^-12M；Or 10^-12M to 10^-13M。

In one embodiment, the dissociation of the combined area KDR of bispecific binding protein such as bispecific antibody is normal Number (K_D) it is at most about 10^-7M；At most about 10^-8M；At most about 10^-9M；At most about 10^-10M；At most about 10^-11M；At most about 10^- ¹²M；Or at most 10^-13M.In one embodiment, the combined area KDR of bispecific binding protein such as bispecific antibody with Dissociation constant (the K of its target_D) it is 10^-7M to 10^-8M；10^-8M to 10^-9M；10^-9M to 10^-10M；10^-10M to 10^-11M；10^-11M is extremely 10^-12M；Or 10^-12M to 10^-13M。

Bispecific binding protein described herein can be the also conjugation comprising imaging agent, therapeutic agent or cytotoxic agent Object.In one embodiment, imaging agent is radioactive label, enzyme, fluorescent marker, luminescent marking, bioluminescence marker, magnetism Label or biotin.In another embodiment, the radioactive label are as follows:³H、¹⁴C、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I、¹⁷⁷Lu or¹⁵³Sm.In further embodiment, the therapeutic agent or cytotoxic agent are antimetabolite, alkylating agent, antibiosis Element, growth factor, cell factor (such as immune stimulating cytokines), anti-angiogenic agent, antimitotic agent, anthracycline, Toxin or apoptosis agent.As discussed below, immune stimulating cytokines are especially important.

It provides and combines human PD-L 1 to inhibit immunosupress and inhibit the molecule of angiogenesis herein in connection with people KDR.? In one embodiment, the PD-L1 binding structural domain and secondary antibody knot of such molecular combinations first antibody combination human PD-L 1 Close the KDR binding structural domain of people KDR.

The PD-L1 bound fraction of the molecule can be the antigen-binding domains of antibody, such as heavy chain and light chain variable Structural domain pair.There is provided herein suitable heavy chain of antibody and light variable domains and include their antibody.Bispecific knot The PD-L1 bound fraction of hop protein can be any medicament that in conjunction with PD-L1 and blocking immunity inhibits.These include anti-PD-L1 Antibody and segment are not limited to those disclosed herein antibody, and (human PD-L 1 is naturally matched for the peptide from people PD1 and protein Body).As disclosed herein, the combined area PD-L1 is connect with the region of people KDR is combined.

Therefore, in certain embodiments, hybrid molecule is provided, it includes in conjunction with human PD-L 1 and block and people PD1 In conjunction with region, and in conjunction with people KDR and block region in conjunction with people KDR.The combined area PD-L1 and KDR can be by connecing Head is connected as a polypeptide.It thus provides the human PD-L 1 combined area being connect with the combined area people KDR.

Another aspect provides encoding such antibodies or the nucleic acid molecules of its chain.Nucleic acid can reside in complete thin In born of the same parents, cell lysate or in partial purification or substantially pure form.When by standard technique, including alkali/SDS processing, CsCl striping, column chromatography method, agarose gel electrophoresis and other technologies well known in the art, from other cellular components or its In its pollutant (such as other cellular nucleic acids or protein) when purifying, nucleic acid is by " separation " or " causing to be substantially pure ". Referring to F.Ausubel etc. edits (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience,New York.Nucleic acid of the invention can be such as DNA or RNA, and And it can contain or can be free of intron sequences.In a preferred embodiment, the nucleic acid is cDNA molecule.

Standard molecular biological technique can be used and obtain nucleic acid of the invention.For hybridoma (for example, being exempted from by carrier Epidemic disease globulin gene transgenic mice preparation hybridoma) expression antibody, standard PCR amplification or cDNA clone can be passed through Technology obtains the cDNA of the light chain and heavy chain that encode the antibody generated by hybridoma.For by immunoglobulin gene libraries (example Such as, using display technique of bacteriophage) obtain antibody, the nucleic acid of encoding antibody can be recycled from the library.The nucleic acid and Carrier containing the nucleic acid can be used for expressing above-mentioned antibody or its chain.

As used herein, term " carrier " means that the nucleic acid molecules for another nucleic acid being attached thereto can be transported.It is a kind of The carrier of type is " plasmid ", refers to the circular double stranded DNA ring that wherein can connect additional DNA section.Another type of load Body is viral vectors, wherein additional DNA section may be coupled in viral genome.Certain carriers can be introduced into wherein The host cell bacteria carrier and mammal episomal vector of bacterial origin of replication (for example, with) in independently replicate.Its Its carrier (for example, non-add type mammalian vector) can be integrated into host cell gene group after being introduced into host cell, To be replicated together with host genome.Moreover, certain carriers can instruct the expression for the gene being operatively connected therewith.This Class carrier is referred to herein as " recombinant expression carrier " (or being referred to as " expression vector ").In general, in recombinant DNA technology The expression vector for having practicability is usually in plasmid form.In the present specification, " plasmid " and " carrier " is used interchangeably, because of matter Grain is most common carrier format.However, further including the expression vector for playing identical function of other forms, such as viral vectors (for example, replication defect type retrovirus, adenovirus and adeno-associated virus).

As used herein, term " recombinant host cell " (or referred to as " host cell ") mean comprising non-naturally-occurring in The cell of nucleic acid in cell, and can be the cell for introducing recombinant expression carrier thereto.It should be understood that such term Not only mean particular subject cell, but also means the offspring of such cell.Because since mutation or environment are influenced in the next generation In be likely to occur certain modifications, so in fact such offspring may be different from parental cell, but be included in used herein In the range of term " host cell ".

It should be understood that bispecific binding protein usually will be in addition when being used for prophylactic or therapeutic purposes in mammals Composition forms application comprising at least one pharmaceutically acceptable carrier.Suitable pharmaceutically acceptable carrier includes, Such as one of water, salt water, phosphate buffered saline (PBS), dextrose, glycerol, sucrose, polysorbate, ethyl alcohol etc. or it is a variety of and A combination thereof.Pharmaceutically acceptable carrier also may include a small amount of auxiliary substance, such as wetting agent or emulsifier, preservative or buffering Agent enhances shelf-life or the validity of bispecific binding protein.

In method described herein, the bispecific binding protein of therapeutically effective amount such as bispecific antibody is applied To mammal in need.As used herein, term administering " means by the way that any of sought result may be implemented Method is by bispecific binding protein such as bispecific antibody delivery to mammal.Application can be, for example, it is intravenous or Intramuscular application.It, can also be with although bispecific binding protein such as bispecific antibody is particularly useful to human administration It is administered to other mammals.As used herein, the term " mammal " be intended to include but be not limited to people, laboratory animal, Domestic pets and domestic animal." therapeutically effective amount " means bispecific binding protein such as bispecific antibody, when being administered to lactation When animal, the amount of curative effect needed for effectively generating.

Bispecific binding protein such as bispecific antibody can be used for inhibiting tumour and other tumor diseases, Yi Jizhi Treat other pathological conditions relevant to immunosupress.Treatable tumour includes primary tumor, metastatic tumo(u)r and refractory Property tumour.Refractory neoplasm includes for being carried out with individual chemotherapeutant, individual antibody, individual radiation or combinations thereof The reactionless or resistant tumour for the treatment of.Refractory neoplasm, which also covers, to be seemed to be suppressed and with such pharmaceutical treatment, but It is up to after stopping the treatment 5 years, the tumour recurred sometimes up to 10 years or for more time.Bispecific binding protein is for example double Specific antibody can effectively treat vascularized tumors and non-vascularization or the substantially not yet tumour of vascularization.

The example of treatable solid tumor includes breast cancer, lung cancer, colorectal cancer, cancer of pancreas, glioma and leaching Bar tumor.Some examples of such tumour include epidermoid, squamous tumor, such as head and neck neoplasm, colorectal carcinoma, forefront adenoncus Tumor, tumor of breast, lung tumors (including cellule and non-fire power), pancreatic neoplasm, thyroid tumors, ovarian neoplasm And liver neoplasm.Other examples include Kaposi sarcoma (Kaposi's sarcoma), CNS neoplasm, neuroblastoma, Capillary hemangioblastomas, meningioma and metastatic encephaloma, melanoma, human primary gastrointestinal cancers and kidney and sarcoma, rhabdomyosarcoma, at Spongiocytoma (preferably glioblastoma multiforme) and leiomyosarcoma.Bispecific binding protein such as bispecific is anti- Body includes to the example of its effective vascularized skin cancers can be by inhibiting malignant keratinocytes such as people's malignant keratinocytes Squamous cell carcinoma, basal-cell carcinoma and the cutaneum carcinoma for growing to treat.

The example of non-solid tumors includes to the unresponsive leukaemia of cell factor, Huppert's disease and lymthoma.It is white Some examples of blood disease include acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic Leukaemia (ALL), chronic lymphocytic leukemia (CLL), fragility of erythrocytes leukaemia or monocytic leukemia.Lymthoma Some examples include Huo Qijin (Hodgkin's) and non-Hodgkin's (non-Hodgkin's) lymthoma.

Bispecific binding protein such as bispecific antibody is also used for treatment virus infection.PD-1 expression in T cell It is associated with the virus load in HIV and HCV infection patient, and PD-1 expression has been accredited as virus-specific CD8⁺T is thin The mark that born of the same parents exhaust.For example, PD-1⁺CD8⁺T cell shows that the relevant T cell of impaired effector function and PD-1 exhausts that this can By blocking PD-1/PD-L1 interaction to restore.This leads to virus-specific CD8⁺The immunity that T cell mediates is restored, Show that interrupting PD-1 signal transduction using antagonistic antibodies has restored T cell effector function.It is blocked based on PD-1/PD-L1 Immunotherapy does not only result in the T cell breaking tolerance of tumour antigen, and additionally provides and reactivate virus-specific effect T Cell and the strategy for eradicating the pathogen in chronic viral infection.Therefore, antibody of the invention and hybrid protein can be used for treating Chronic viral infection, including but not limited to HCV and HIV and Lymphocyte function-associated antigen-1 (LCMV).

Bispecific binding protein such as bispecific antibody can advantageously be administered to together with second medicament in need Patient.For example, in some embodiments, by bispecific binding protein such as bispecific antibody together with antitumor agent It is administered to subject.In some embodiments, bispecific binding protein such as bispecific antibody and angiogenesis are pressed down Preparation is administered to subject together.In some embodiments, by bispecific binding protein such as bispecific antibody and anti- Scorching agent or immunosuppressor are applied together.

Antitumor agent includes cytotoxic chemotherapy agent, targeting small molecule and biomolecule, and radiation.Chemotherapy The non-limiting example of agent includes cis-platinum (cisplatin), Dacarbazine (dacarbazine) (DTIC), actinomycin D (dactinomycin), Irinotecan (irinotecan), mechlorethamine (mustargen), streptozotocin (streptozocin), cyclophosphamide, Carmustine (carmustine) (BCNU), lomustine (lomustine) (CCNU), Doxorubicin (doxorubicin) (adriamycin (adriamycin)), daunorubicin (daunorubicin), procarbazine (procarbazine), mitomycin (mitomycin), cytarabine (cytarabine), Etoposide (etoposide), Methotrexate (MTX) (methotrexate), 5 FU 5 fluorouracil, vincaleukoblastinum (vinblastine), vincristine (vincristine), Bleomycin (bleomycin), taxol (paclitaxel) (Taxol, taxol), docetaxel (docetaxel) (Tai Suo Supreme Being, taxotere), Aldesleukin (aldesleukin), asparaginase, busulfan (busulfan), carboplatin (carboplatin), Cladribine (cladribine), Dacarbazine, floxuridine (floxuridine), fludarabine (fludarabine), hydroxycarbamide, ifosfamide, interferon-' alpha ', Leuprorelin (leuprolide), megestrol acetate (megestrol), melphalan (melphalan), purinethol, plicamycin (plicamycin), mitotane (mitotane), Pegaspargase (pegaspargase), Pentostatin (pentostatin), pipobroman (pipobroman), plicamycin (plicamycin), streptozotocin, tamoxifen (tamoxifen), Teniposide (teniposide), Testolactone (testolactone), thioguanine, phosphinothioylidynetrisaziridine (thiotepa), uracil mastard, vinorelbine (vinorelbine), benzene Butyric acid mustargen (chlorambucil), Taxol and combinations thereof.

Targeting small molecule and biomolecule include but is not limited to the inhibitor of signal transduction pathway component, such as tyrosine-kinase Enzyme adjustment agent and receptor tyrosine kinase inhibitors, and the medicament in conjunction with tumour specific antigen.Involved by tumour occurs Growth factor receptors non-limiting example be platelet derived growth factor (PDGFR), insulin-like growth factor (IGFR), The receptor and Epidermal Growth Factor Receptor Family of nerve growth factor (NGFR) and fibroblast growth factor (FGFR) by Body, including EGFR (erbB1), HER2 (erbB2), erbB3 and erbB4.

EGFR antagonist include with EGFR or EGFR ligand binding, and inhibit the antibody of ligand binding and/or receptor activation. For example, the medicament can block receptor dimer and the heterodimer of other EGFR family members to be formed.The ligand of EGFR includes Such as the EGF (HB-EGF) and β regulatory protein (betaregullulin) of the bis- regulatory proteins of EGF, TGF- α, heparin-binding.EGFR Antagonist can be with combined outside EGFR extracellular portion, this can inhibit or not inhibit the combination of ligand or internal combustion tyrosine Kinase domain.EGFR antagonist further includes for example inhibiting EGFR by inhibiting the function of the component of EGFR signal transduction pathway The medicament of dependent signals transduction.Example in conjunction with the EGFR antagonist of EGFR includes but is not limited to biomolecule for example to EGFR There are specific antibody (and its functional equivalent) and small molecule for example to directly act on the synthesis of the cytoplasmic domain of EGFR Kinase inhibitor.

Small molecule and biostatic agent include EGF-R ELISA (EGFR) inhibitor, including Gefitinib (gefitinib), Erlotinib (erlotinib) and Cetuximab (cetuximab), HER2 inhibitor (such as toltrazuril Monoclonal antibody (trastuzumab), trastuzumab-Maitansine (trastuzumabemtansine) (trastuzumab-DM1；T-DM1) and Handkerchief trastuzumab (pertuzumab)), anti-VEGF antibody and segment (such as bevacizumab (bevacizumab)), inhibit CD20 Antibody (such as Rituximab (rituximab), ibritumomab tiuxetan (ibritumomab)), anti-vegf R antibody (such as thunder Not Lu Dankang (ramucirumab) (IMC-1121B), IMC-1C11 and CDP791), anti-PDGFR antibody and Imatinib (imatinib).Small molecule kinase inhibitors can to specific tyrosine kinase have specificity or two or more The inhibitor of kinases.For example, compound N-(3,4- bis- chloro- 2- fluorophenyl) -7- ({ [(3aR, 6aS) -2- methyl octahydro ring penta [c] pyrroles -5- base] methyl } oxygroup) -6- (methoxyl group) quinazoline -4- amine (also referred to as XL647, EXEL-7647 and KD-019) It is several receptor tyrosine kinases (RTK), including the external of EGFR, EphB4, KDR (VEGFR), Flt4 (VEGFR3) and ErbB2 Inhibitor, and cause tumour to the inhibitor of SRC kinases involved in the unresponsive approach of certain TKI.Of the invention In one embodiment, the treatment to subject in need includes the rho- kinase inhibitor and application KD-019 of application Formulas I.

Dasatinib (Dasatinib) (BMS-354825；Bristol-Myers Squibb, New York) it is another Orally bioavailable ATP site competition Src inhibitor.Dasatinib also targets Bcr-Abl, and (FDA approval is for chronic Myelomatosis (CML) or Philadelphia Chromosome Positive (Ph+) acute lymphatic leukemia (ALL) patient) and c- Kit, PDGFR, c-FMS, EphA2 and SFK.The oral tyrosine kinase inhibitor of other two kinds of Src and Bcr-Abl is that rich relax is replaced Buddhist nun (bosutinib) (SKI-606) and saracatinib (saracatinib) (AZD0530).

In one embodiment, bispecific binding protein such as bispecific antibody is combined with antivirotic for controlling Treat chronic viral infection.For example, following medicament can be used for HCV.HCV protease inhibitor includes but is not limited to wave west Pu Wei (boceprevir), telavi (telaprevir) (VX-950), ITMN-191, SCH-900518, TMC-435, BI- 201335, MK-7009, VX-500, VX-813, BMS790052, BMS650032 and VBY376.HCV non-structural protein 4B (NS4B) inhibitor includes but is not limited to clemizole (clemizole) and other NS4B-RNA binding inhibitors, including but not It is limited to benzimidazole RBI (B-RBI) and indazole RBI (I-RBI).HCV Non structural protein 5 A (NS5A) inhibitor includes but unlimited In BMS-790052, A-689, A-831, EDP239, GS5885 and PP1461.HCV polymerase (NS5B) inhibitor includes but not It is limited to nucleoside analog (such as cut down Lip river his shore (valopicitabine), R1479, R1626, R7128), nucleotide analog (such as IDX184, PSI-7851, PSI-7977 and non-nucleoside like object (such as Filibuvir (filibuvir), HCV-796, VCH-759、VCH-916、ANA598、VCH-222(VX-222)、BI-207127、MK-3281、ABT-072、ABT-333、 GS9190,BMS791325).In addition it is possible to use Ribavirin (ribavirin) or ribavirin analogs such as Ta Liweilin (Taribavirin) (Wei rummy pyridine (viramidine)；ICN3142), mizoribine (Mizoribine), U.S. mooring basin cloth (Merimepodib) (VX-497), mycophenolate (Mycophenolatemofetil) and mycophenolic acid (Mycophenolate).

When bispecific antibody of the invention is applied together with second medicament, the first and second medicaments can successively or together When apply.Every kind of medicament can be applied with single dose or multi-dose, and the dosage can be used by any time top application, including but not It is limited to twice daily, once a day, once a week, once every two weeks and monthly.

The invention also includes the applications of the auxiliary of bispecific antibody.Auxiliary application means in addition to having applied to treat disease Or outside the first medicament of disease symptoms, second medicament also is applied to patient.In some embodiments, auxiliary application includes to trouble Person applies second medicament, wherein the disease or disease symptoms are not treated or do not treated sufficiently in the application of the first medicament.Other In embodiment, auxiliary application includes that second medicament is administered to the patient that disease is effectively treated by applying the first medicament.

In certain embodiments, the bispecific binding protein such as bispecific for applying a dosage to subject is anti- Body, once a day, it is every other day primary, per once a few days, once every three days, once a week, twice a week, three-times-weekly or often Biweekly.In other embodiments, the bispecific binding protein example of two, three or four dosage is applied to subject Such as bispecific antibody, once a day, per once a few days, once every three days, once a week or once every two weeks.In some implementations In scheme, the bispecific binding protein of a dosage is applied such as bispecific antibody 2 days, 3 days, 5 days, 7 days, 14 days or 21 It.In certain embodiments, the bispecific binding protein of a dosage is applied such as bispecific antibody 1 month, 1.5 The moon, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or longer time.

Method of administration includes but is not limited to parenteral, intradermal, vitreum is interior, intramuscular, intraperitoneal, intravenous, subcutaneous, nose Interior, Epidural cavity, intravaginal, transdermal, transmucosal, rectum, passes through sucking or part at oral, sublingual, intranasal, intracerebral, especially Ear, nose, eye or dermal administration.Administration mode is decided in its sole discretion by practitioner.In most cases, application will lead to bispecific Binding protein such as bispecific antibody is discharged into blood flow.Treatment for eye disease, it is preferably double special in intravitreal administration Property binding protein such as bispecific antibody.

In specific embodiments, it may be necessary to local application bispecific binding protein such as bispecific antibody.This Can be accomplished by the following way, such as, but not limited to by local infusion, part applies, by injection, by means of conduit or By means of implantation material, the implantation material is porous, non-porous or gel-like material, including film, such as silicone rubber membrane or fiber.Herein In the case of class, the application property of can choose ground targeted local tissue, bispecific binding protein such as bispecific antibody is substantially It will not be discharged into blood flow.

Pulmonary administration can also be used, such as prepared by using inhalator or atomizer, and with Alevaire, or by It is perfused in fluorocarbon or synthetic lung surfactant preparation.In certain embodiments, for example sweet with conventional adhesive and medium Bispecific binding protein such as bispecific antibody is configured to suppository by oily three esters.

In another embodiment, bispecific binding protein such as bispecific antibody especially exists in vesica Delivering is (referring to Langer, 1990, Science 249:1527-1533 in liposome；Treat etc., in Liposomes in the In Therapy of Infectious Disease and Bacterial infection, Lopez-Berestein and Fidler (editor), Liss, New York, the 353-365 pages (1989)；Lopez Berestein, ibid, 317-327 Page；In general it is same as above).

In another embodiment, bispecific binding protein such as bispecific antibody delivers in controlled release system (see, e.g. Goodson, in Medical Applications of Controlled Release, ibid, volume 2, The 115-138 pages (1984)).It can be used in Langer, discussed in the summary of 1990, Science 249:1527-1533 The example of controlled release system.In one embodiment, pump can be used (referring to Langer, ibid；Sefton,1987,CRC Crit.Ref.Biomed.Eng.14:201；Buchwald etc., 1980, Surgery 88:507；Saudek etc., 1989, N.Engl.J.Med.321:574).In another embodiment, polymer material can be used (referring to Medical Applications of Controlled Release, Langer and Wise (editor), CRC Pres., Boca Raton, Florida(1974)；Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (editor), Wiley, New York (1984)；Ranger and Peppas, 1983, J.Ma cromol.Sci.Rev.Macromol.Chem.23:61；Also refer to Levy etc., 1985, Science 228:190；During Deng 1989, Ann.Neurol.25:351；Howard etc., 1989, J.Neurosurg.71:105).

The purpose that above-mentioned administration time table is merely to illustrate that is provided, is not construed as limiting.The ordinary skill of this field Personnel will readily appreciate which dosage and administration time table are suitable.

It should be understood that with it is contemplated that those skilled in the art can be changed principle disclosed herein, and it is intended to It is such modification including within the scope of this disclosure.

In entire application, various publications are referred to.These publications is hereby incorporated by reference in its entirety, with more The status of disclosure fields is comprehensively described.Following examples further illustrate the disclosure, but should not be construed as to appoint Where formula limits the scope of the present disclosure.

U.S. Provisional Patent Application Serial No. 61/710,420；62/061,097；61/927,907 and international monopoly Shen It please number PCT/US2013/063754；PCT/US2015/011657；This is integrally incorporated by reference with PCT/US2015/054569 Text.

Embodiment

Expression and physical characterization of the embodiment 1 for the bispecific antibody of VEGFR2 and PDL1

This embodiment describes the transient expressions for the bispecific antibody that VEGFR2 and PDL1 is directed in HEK293, and pass through Protein A purification；Concentration class analysis (passing through SEC-UPLC), serum stability experiment and heat stabilization test (are surveyed by using DSC Measure Tm).

It introduces

By VEGFR2 directly fixed from DyaxFab310 phagemid library elutriation, separation antibody λ R3B1 (is directed to VEGFR2).By matching the heavy chain in light chain library and λ R3B1, light chain shuffled library is constructed.It is sieved to people and mouse VEGFR2 After choosing, the B1C4 that can be combined with both people and mouse VEGFR2 is isolated.By soft mutation method to its light chain CDR3 and heavy chain CDR1 and CDR2 are further modified, wherein only 10% residue mutations are another 19 kinds of amino acid (not including cysteine). B1C4A7 is identified by screening soft mutated library under conditions of very strict, i.e., a kind of affinity is higher and more stable to be resisted Body.

Also anti-PDL1 antibody is identified by carrying out elutriation to the PDL1 from 310 phagemid library of Dyax Fab TccR3D7 and tccR3A11.It is isolated from the library tccR3D7_HCDR1 with higher affinity and compared with low oxidation sensitivity Antibody D7A8, wherein being located at three methionine amino acid random mutations in tccR3D7 heavy chain CDR1.

In order to construct BsAb, anti-PDL1 antibody D7A8 (or A11) is made to be reformatted as VH- (SG first₄)₅The direction-VL Single chain variable fragment (scFv)；Then it invests scFv (to be directed to by conventional antibody B1A1 or B1C4A7 that connector connects VEGFR2 the end c- (Fig. 1)).In order to weaken effector function, make (the IgG1-CH2 structure of the position B1A1 or B1C4A7 234 and 235 Domain) in leucine sport alanine.

The gene of bispecific antibody (A1-A8) and (A7-A8) are inserted into mammalian expression vector pBh1, and Transient expression in HEK293 cell line.Harvest supernatant simultaneously loads in albumin A column with purifying bispecific antibody.

The pure of bispecific antibody (A1-A8) and (A7-A8) is analyzed by using SDS-PAGE, SEC-UPLC and DSC Degree and stability.After Western blotting, the blood of both bispecific antibodies is checked by being incubated for BsAb in mice serum Clear stability.

Material and method

To be expressed and test monospecific and bispecific antibody

Expression and purifying

For expressing, purifying and the material of SDS-PAGE

For expressing, purifying and the material of SDS-PAGE is listed in the following table.

For expressing, purifying and the method for SDS-PAGE

Illustrated according to manufacturer, the mammal table of bispecific antibody will be carried by using Polyjet transfection reagent It transfects up to carrier into sequestered HEK293 cell.Harvest 6 days cultures and by the supernatant of filtering load to On the affinity column albumin A of AKTAxpress protein purification system connection.The antibodies buffer of purifying is changed to PBS and mistake Filter.Antibody concentration is obtained by measuring the absorbance of 280nm in Nanodrop photometer and calculating by using theoretical coefficient.

It is restoring under non reducing conditions, is preparing antibody samples by adding the carried dye with or without reducing agent.It will Sample cell heats 10 minutes at 100, and reduction and non-reducing sample are loaded to 4-12% together with molecular weight standardsOn NOVEX bis-tris gel.Gel electrophoresis about 50 minutes under 200v constant voltage.Then protein is used Dyestuff is gel-colored and decolourizes in QH2O, until observing protein band.(Figure 19) shows gel images

UPLC and DSC

Material for UPLC and DSC

Method for UPLC and DSC

Method for UPLC

Machine is programmed according to manufacturer's explanation.Sample is loaded in UPLC small samples bottle first, is loaded 10 μ g samples simultaneously inject in BEH200SEC column.With 0.4ml/ minutes velocity separation antibody in the PBS buffer solution of pH=7.4 10 minutes, and the antibody eluted from column is detected by TDA under 280nM wavelength.Each sample is injected at least twice. The area percentage at each peak is analyzed with the software Empower 3 that Waters is provided.

Method for DSC

The sample that concentration is 1mg/ml is loaded in 96 deep-well plates, then carries out dsc analysis using Nano DSC.It carries out Buffer (PBS) injection several times is to obtain enough sample blanks.Implement 15 minutes prescans before sample electrophoresis with true Protect accurate initial temperature.By from 25 to 100 monitorings, 60/ hour temperature ramp is realized.Before analysis from every kind of antibody In subtract the Thermogram of only buffer sample, and calculate Tm using NanoDSC software after deconvoluting.

Stability in mice serum

Material for mice serum Stability Determination

Method for mice serum Stability Determination

The bispecific antibody of each 200ng or Mono-specific antibodies are added in 90% mice serum, and incubated under 37 It educates 5 days.Antibody thermally treated in mice serum is loaded under the reducing conditions in 4-12%Bis-Tris protein gel And carry out electrophoresis.Illustrated on the Protein transfer to pvdf membrane that will be separated according to manufacturer.After the closing of 3%PBS lotion, by film HRP- anti-human igg (H+L) antibody with 1/2000 is incubated with.After being washed three times with PBST, react film with ECL first.? In different exposure time point shooting picture to obtain high quality graphic in ImageQuant LAS4000.

As a result

Expression and purifying

The mammalian expression vector for carrying bispecific antibody is transfected to sequestered HEK293 cell.Pass through albumin A Purifying bispecific antibody, and buffer is changed to PBS.Being measured and being passed through at 280nm by Nanodrop photometer makes With theoretical coefficient calculating antibody concentration.As a result it is shown in following table.

The result shows that bispecific antibody A1-A8 and A7-A8 can in HEK-293 cell transient expression.In albumin A The BsAb (A1-A8) and BsAb (A7- of 10mg are recovered over after purifying and buffer-exchanged in the 1L supernatant of 6th day harvest A8)。

SDS-PAGE analysis

The antibody of each 5 μ g is loaded in 4-12%NuPAGE gel under reduction or non reducing conditions.It is contaminated with protein Material dyes and uses H₂Gel images are shot after O decoloration.As a result it is shown in Figure 19.The result shows that being estimated by SDS-PAGE, two kinds The purity of BsAb is more than 95%.

SEC-UPLC analysis

SEC-UPLC analysis is carried out using following methods: column: ACQUITY UPLC BEH200SEC, and 1.7 μm, 4.6x150mm；UPLC system: the waters ACQUITY UPLC with TDA detector；Eluant, eluent: PBS, pH=7.4；Flow velocity: 0.4mL/ minutes；Injection: 10 μ g；Wavelength: 280nm；Every curve lists the percentage of main peak.Two are carried out to each sample Secondary injection.Size exclusion chromatography figure (the ultraviolet light rail under 280nm of bispecific antibody in UPLC system through Protein A purification Mark) result be shown in Figure 20.Percentage of the peak area relative to retention time is summarized in table.It also lists in table and surveys twice The average peak area of amount.

It was found that being detected BsAb (A1-A8) and BsAb (A7-A8) lower than 2% He respectively by SEC-HPLC analysis 1% concentration class.It has also been found that the concentration class of BsAb (A7-A8) increases, but still is lower than 3% if concentration is higher.

Pass through dsc measurement Tm

The DSC scanning result in PBS buffer solution to bispecific antibody is shown in Figure 21 and following table.

Table: the melting temperature of the bispecific antibody from DSC scanning

The result shows that being about about 60 by the Tm1 of BsAb (A7-A8)-LALA of DSC scanning survey, it is lower than parental antibody The minimum Tm1 of B1C4A7 and D7A8-LALA (the two is about 69).The minimum Tm1 of BsAb (A7-A8) is generated by scFvD7A8, ScFv antibody fragment is stablized not as good as IgG as report.The further engineering of scFv, such as in the heavy chain of D7A8 and light Longer connector is used between chain variable domains, or adds disulfide bond between two structural domains, its thermostabilization can be improved Property.

Serum stability

It is shown in Figure 22 in mice serum in the Western blotting result of the bispecific antibody of 37 processing 5 days.Such as Shown in figure, under the reducing conditions after handling BsAb (A1-A8)-LALA and BsAb (A7-A8) under 37-LALA5 days, do not observe To short heavy chain and light chain bands.Therefore, the results showed that both BsAb (A1-A8)-LALA and BsAb (A7-A8)-LALA are 37 Under be stable for up in mice serum 5 days.

The vitro characterization of 2 bispecific antibody of embodiment (A7-A8)

It (is directed to This embodiment describes the vitro characterization of bispecific antibody A7-A8 and with its parental antibody B1C4A7 VEGFR2) compare with the effect of dsD7A8-LALA (being directed to PDL1).

It introduces

Previous embodiment according to the report, by HEK293 transient expression obtain and pass through the pure double special of Protein A purification Property antibody A 7-A8, pass through SEC-UPLC analysis detection to lower than 1% higher molecular weight assemble.BsAb (A7-A8) is in 37 mouse It is also stable for up in serum 5 days.

Individually together with its parental antibody B1C4A7 (being directed to VEGFR2) and dsD7A8-LALA (being directed to PDL1), it has studied It can be with the VEGFR2 and PDL1 of people and the bispecific antibody A7-A8 of mouse species cross reaction and the expression of soluble and cell The binding ability of (people and mouse species) inhibits the combination of ligand (VEGF and PDL1) and each autoreceptor (VEGFR2 and PD1), resistance Disconnected VEGFR2/ downstream molecules phosphorylation and stimulating cytokine IL2 and INF γ secretion.

Material and method

Antibody to be tested

Dose response combination ELISA

Material for dose response combination ELISA

Method for dose response combination ELISA

Antigen VEGFR2-Fc (people and mouse) and PDL1-Fc (people and mouse) Immulon 2HB is coated by 1 μ g/ml to put down On plate, under 4 overnight.With 3%PBSM blocking of plates.The bispecific of serial dilution and Mono-specific antibodies are added to closing Plate in, and be incubated at room temperature about 1 hour.After washing flat board, the anti-human igg Fab specificity (HRP of addition HRP conjugation Anti- hFab) antibody (1/5000 dilution in 3%PBSM).Then washing flat board again adds TMB reaction buffer to develop the color. With 1N H₂SO₄Terminate reaction after, with microplate reader at OD450 read plate.

Dose response competitive ELISA

Material for dose response competitive ELISA

Method for dose response competitive ELISA

According to the manufacturer's instructions, with biotin labeling people and mouse PDL1-Fc.

Ligand VEGF (people and mouse) and PDL1 (people and mouse) are coated on Immulon2HB plate by 1 μ g/ml, 4 It is lower to stay overnight.With 3%PBSM blocking of plates.By the antibody of serial dilution and people or mouse VEGFR2 (final concentration of 0.5 μ g/ml) or It mixes, and is incubated at room temperature 1 hour in the closed 96 hole round bottom plate of PBSM with the people of biotin labeling and mouse PDL1.It will Antagonist-receptor mixture is transferred to respectively on the closed plate for being coated with ligand, and is incubated for again at room temperature 1 hour.Washing is flat After plate, add respectively HRP- anti-human igg Fc specific antibody (for VEGF-VEGFR2) or Streptavidin-HRP (for PD1-PDL1) (1/5000 dilution in 3%PBSM).Then washing flat board again adds TMB reaction buffer to develop the color.With 1NH₂SO₄Terminate reaction after, with microplate reader at OD450 read plate.

Dynamic analysis is carried out by Biacore

Biacore material

Title	Company	Model/catalog number
			Biacore	GE	T200
BiaEvaluation software V1	GE	T200
			Recombinant human VEGF R2	R&Dsystems	357-KD-050
S series CM5 chip	GE	BR1005-30
			HBSEP	GE	BR1006-69

Biacore method

Surface prepares

It is combined in Biacore T200 instrument (GE Healthcare) using surface plasma body resonant vibration (SPR) evaluation Affinity.Make CM5 chip balance in the electrophoretic buffer HBSEP (electrophoretic buffer) of 10 μ l/ml.It is infused with 1:1 NHS/EDC Two flow chambers 5 minutes for penetrating liquid activation CM5 chip.It is diluted in the 10mM sodium acetate buffer of pH5 with 5 μ g/ml HVEGFR2 fixes the second flow chamber, to reach the immobilized protein of 40-100RU.Inject 5 minutes ethanol amines then to seal Close surface.

Sample electrophoresis

With l/ minutes progress electrophoresis of 30 μ in HBSEP electrophoretic buffer.The serial dilution in electrophoretic buffer by sample, Concentration range is 1.5-100nM.By in sample injection to two flow chambers, the association time is 3 minutes and Dissociation time is 10 points Clock.It is regenerated every time in conjunction with the 20mMHCL injected 30 seconds after circulation.The sensing figure of each concentration is obtained, and uses Bia- Evaluation software subtracts blank flow chamber, and derived curve is fitted to 1:1 Langmuir binding model.

Intersect and combines ELISA

For intersecting the material for combining ELISA

For intersecting the method for combining ELISA

According to the manufacturer's instructions, people PDL1- is marked with the biotin labeling reagent box from Thermo-Fisher Fc。

Human VEGFR-3 2 is coated on Immulon 2HB plate by 1 μ g/ml, under 4 overnight.With 3%PBSM blocking of plates. The hPDL1 of 1nM bispecific antibody or Mono-specific antibodies and biotin labeling is mixed in closed round 96 orifice plate of PBSM It closes, and is incubated at room temperature 1 hour.Mixture is transferred on the closed plate for being coated with hVEGFR2 of PBSM, and at room temperature It is incubated for again 1 hour.After washing flat board, addition HRP- Streptavidin (1/5000 dilution in 3%PBSM).Washing is flat again Plate, and TMB reaction buffer is added to develop the color.Use 1NH₂SO₄Terminate reaction after, with microplate reader at OD450 read plate.

Cell combination measurement

Material for cell combination

Method for cell combination

PAE-KDR cell (containing glutamine, be supplemented in the DMEM of 10% heat inactivation FBS), EOMA cell are (containing paddy Glutamine is supplemented in the DMEM of 10% heat inactivation FBS), MDA-MB-231 cell (contain glutamine, be supplemented with 10% heat In the RPMI1640 for inactivating FBS) and B16-F10 cell (containing glutamine, be supplemented with the RPMI1640 of 10% heat inactivation FBS In) growth is until 90% converges.Cell is harvested, is washed first, then presses 5 × 10⁵A cell/ml is resuspended in ice-cold In FACS buffer solution (PBS, 1%BSA).100 μ l cell suspending liquids are added in each hole of 96 hole round bottom microtiter plates And it is centrifuged (1200rpm, 5 minutes).The bispecific antibody of serial dilution and Mono-specific antibodies are added in cell, and with Cell is incubated for 1 hour under 4.By in the 200 ice-cold FACS buffer solutions of μ l 1500rpm be centrifuged 5 minutes and wash cell 3 times. Cell is resuspended in the PE- anti-human antibody of 100 μ l (1/100), and is incubated in the dark under 4 at least 30 minutes.It will Cell washs 3 times, is resuspended in 200 μ lPBS and reads fluorescent value.

Phosphorylation assay

Material for phosphorylation assay

Solution for phosphorylation assay

1. lysis buffer: 50mM Tris (pH 7.5), 150mM NaCl, 1%Triton x100,1mM EDTA, egg White Protease Inhibitor Cocktail (1:500): phosphatase inhibitor cocktail 2 (1:200) and phosphatase inhibitor cocktail 3 (1: 200)

2. strip buffer: 100mM 2 mercapto ethanol, 2% lauryl sodium sulfate and 62.5mM Tris-HCl pH 6.7

3. load buffer: 100% glycerol of 4ml, the 1M Tris/HCl of 2.4ml pH6.8,0.8g SDS, 4mg bromine phenol Blue, 0.5ml2- mercaptoethanol and 3.1ml ddH₂O。

Method for phosphorylation assay

By the PAE/KDR (0.4x10 in 1ml 10%FBS/DMEM⁶It is a) cell or 1.5ml 10%FBS DMEM culture EOMA (1x10 in base⁶It is a) cell is added in 12 orifice plates, in 37,5%CO₂It is incubated for 4 hours until cell attachment.Removal training It supports base and adds serum-free DMEM so that cell overnight starvation.Second day, the antibody of serial dilution is added in cell, first In 37,5%CO₂Lower to be incubated for 20 minutes, VEGF is then added into cell, and (ultimate density is divided for PAE/KDR and EOMA It Wei not 30ng/ml and 50ng/ml；By cell mixture in 37,5%CO₂Under be incubated for again 10 minutes.It is washed with serum free medium Cell is primary.Then, 100 μ l lysis buffers are added and cell is moved to 1.5ml Eppendorf tube from culture dish It is incubated in (Eppendorf tubes) and on ice about 2 hours.By treated sample (lazed sample) 10 under 4, 000rpm is centrifuged 10 minutes and collects supernatant.Protein concentration is measured by Bio-Rad Protein Assay Kit.Then, 30 μ g proteins are mixed and boiled 5 minutes with 8 μ l load buffers.Mixture is loaded on 4-12%NUPAGE gel, Gel electrophoresis is with protein isolate matter.Protein and marker are transferred on pvdf membrane overnight together.Second day, with 3%PBS cream Then anti-phosphoric acid-VEGFR2 (1/1000) or anti-phosphoric acid-MAPK are added on film by liquid close membrane.Film was incubated under 4 Night.After film is washed twice with 0.1%T-PBS, add the HRP of the anti-rabbit antibody conjugate of 1:1500, and at room temperature with film one It rises and is incubated for 1 hour.Film is washed three times with PBST, add ECL later and is taken pictures with ImageQuant LAS4000 (for phosphorus For acid-protein).

The secondary antibodies previously added are stripped by the way that film to be incubated under 50 to 45 minutes together with strip buffer.By film It is washed twice with PBST, is then incubated together with rabbit-anti KDR antibody or anti-MAPK antibody (1:400 dilutes in 10ml 3%PBSM) It educates.Film and rabbit antibody are incubated overnight under 4.Film is washed again, and HRP- anti-rabbit antibody (1:1500 dilution) is added to On film.After being incubated at room temperature 1 hour, three times by film washing, ECL is added later and is shot in ImageQuant LA4000 Luminous photo (for gross protein).

Cytokine secretion measurement

Material for phosphorylation assay

Method for phosphorylation assay

Using Histopaque-1077 according to manufacturer specification from LeukoPak (containing high concentration haemocyte, including Monocyte, lymphocyte, blood platelet, blood plasma and red blood cell enrichment of leukocytes remove method) in separate PBMC.PBMC is by every Hole 2x10⁴A cell is containing IMDM (being supplemented with 2mM glutamine, 25mM HEPES, 3.024g/L sodium bicarbonate) and 10% It is cultivated in 96 orifice plates of FBS, and with 0.01 μ g/mL in the presence of the bispecific antibody of serial dilution or its parental antibody SEB is activated 5 to 7 days.At the 6th day or the 7th day, collects supernatant and be used for user's IFN-γ and human IL-2 Quantikine ELISA kit illustrates measurement IL-2 and IFN γ according to manufacturer.

As a result

In conjunction with the dose response of people and mouse VEGFR2 and PDL1

By BsAb (A7-A8)-LALA of serial dilution, individually (it is directed to its parent's Mono-specific antibodies B1C4A7 VEGFR2) and dsD7A8-LALA (be directed to PDL1) be respectively added to be coated with together hVEGFR2 or mVEGFR2 or hPDL1 and In 96 orifice plates of mPDL1 and it is incubated at room temperature 1 hour.Later by the anti-human igg Fab specific antibody of plate and HRP conjugation (goat) is incubated with.Washing flat board adds peroxidase substrate and reads A450nm.Data point is replication twice Average value ± standard deviation (S.D.).By using from GraphPad Prism be known as " log (agonist) vs. reaction -- The program of variable slope (four parameters) " calculates the EC50 for not synantigen, and is listed in the top of each figure.As a result it is shown in Figure 23 In.

The result shows that BsAb (A7-A8)-LALA can be with recombinant human VEGF R2, mouse VEGFR2, people PDL1 and mouse PDL1 is combined strongly.It is 0.128nM that EC50 is directed to hVEGFR2 respectively, is 0.598Nm for mVEGFR2, is for hPDL1 0.079nM and for mPDL1 be 0.137nM；B1C4A7 is respectively 0.107nM for hVEGFR2 EC50 related to mVEGFR2's And 0.466nM；DsD7A8 is directed to hPDL1 and mPDL1, respectively 0.121nM and 0.172nM.

Dose response for measuring IC50 measures

In order to blocking VEGF-VEGFR2 interaction, by BsAb (A7-A8)-LALA of serial dilution, individually with its parent Mono-specific antibodies B1C4A7 (be directed to VEGFR2) and dsD7A8-LALA (for PDL1) one reinstate fixed amount any people or Mouse VEGFR2-Fc (being finally 0.5 μ g/ml) is incubated for 1 hour in solution at room temperature, is transferred to mixture is coated with later It is incubated in 96 orifice plates of people VEGF or mouse VEGF and again 1 hour.It is flat by being incubated for HRP- anti-human igg Fc specific antibody Plate quantifies the amount of the h/mVEGFR2 in conjunction with fixed h/mVEGF.

It is individually single with its parent by BsAb (A7-A8)-LALA of serial dilution in order to block PD1-PDL1 to interact Specific antibody B1C4A7 (be directed to VEGFR2) and dsD7A8-LALA (for PDL1) one reinstate fixed amount through biotin labeling People or mouse PDL1-Fc (finally be 0.5 μ g/ml) be incubated for 1 hour in solution at room temperature, mixture is transferred to later It applies in 96 orifice plates of someone PDL1 or mouse PDL1 and is incubated for again 1 hour.By with HRP- Streptavidin be incubated for plate come Quantify the amount of the h/mPDL in conjunction with fixed h/mPD1.

As a result it is shown in Figure 24.Data point is the average value ± standard deviation (S.D.) of replication twice.By using The program for being known as " log (inhibitor) vs. reaction -- variable slope (four parameters) " from GraphPad Prism is calculated for not With the IC50 of interaction, and it is listed in the top of each figure.

The result shows that BsAb (A7-A8) can also block strongly ligand VEGF or PDL1 and its respective receptor VEGFR2 and PD1 is combined.The IC50 of hVEGF-hVEGFR2 interaction, is 1.29nM for BsAb (A7-A8)-LALA, and for B1C4A7 For 1.72nM.The IC50 of mVEGF-mVEGFR2 interaction is 0.801nM for BsAb (A7-A8)-LALA, and for B1C4A7 is 0.973nM.The IC50 of hPDL1-hPD1 interaction is 2.51nM for BsAb (A7-A8)-LALA, and for DsD7A8-LALA is 3.04nM.The IC50 of mPDL1-mPD1 interaction is 5.59nM for BsAb (A7-A8)-LALA, and It is 2.80nM for dsD7A8-LALA.

And the combination of the VEGFR2 and PDL1 of cell expression

First by the BsAb of serial dilution (A7-A8)-LALA, individually with its parental antibody B1C4A7 (be directed to VEGFR2) and DsD7A8 (being directed to PDL1) is added to together in following cell line: KDR-PAE (hVEGFR2), EOMA (mVEGFR2), MDA-MB- 231 (hPDL1) and B16-F10 (mPDL1) are then incubated for 1 hour under 4.Later, by the anti-human igg Fc of cell and PE label Specific antibody is incubated with.Washing cell simultaneously reads fluorescent value.The fluorescence intensity of antibody on cell surface antigen is calculated, and is drawn Make the chart relative to antibody concentration.As a result it is shown in Figure 25.By using from GraphPad Prism be known as " log (swash Dynamic agent) vs. reaction -- variable slope (four parameters) " program calculate the EC50 for not synantigen, and be listed in the top of each figure.

The result shows that human VEGFR-3 2, mouse VEGFR2, people PDL1 that BsAb (A7-A8)-LALA can also be expressed with cell It is combined with mouse PDL1.BsAb (A7-A8)-LALA is respectively for the EC50 value of KDR/PAE, MDA-MB-231 and B16-F10 0.533nM, 0.223nM and 0.122nM；B1C4A7 is 0.522nM for the related EC50 of KDR/PAE, and dsD7A8 is for MDA- The value of MB-231 and B16-F10 is respectively 0.159nM and 0.042nM.BsAb (A7-A8)-LALA expresses EOMA The measurement of the EC50 of mVEGFR2 by and the PDL1 that expresses of EOMA combination interference (Figure 25).

The EC50 and IC50 of bispecific antibody A7-A8 and its parental antibody B1C4A7 and dsD7A8-LALA are listed in the table below In.Very close EC50 and IC50 between BsAb (A7-A8)-LALA parental antibody B1A1 or dsD7A8-LALA associated therewith Show that BsAb (A7-A8)-LALA is effective as B1A1 or dsD7A8-LALA.

Table: the EC50 (for combination) and IC50 of BsAb (A7-A8) (for blocking)

*: measurement by and the combination of PDL1 expressed of EOMA interfere

Dynamic analysis

HVEGFR-Fc or hPDL1-Fc is fixed to S series CM5 sensor at pH5 using standard amine-coupling chemistry method On chip.Use 1X HBSEP as electrophoretic buffer, is injected antibody within l/ minutes with 1.5 to 100nM range concentration by 30 μ Onto immobilization surface.Time of contact (association stage) is 3 minutes.Dissociation time is 6-10 minutes.After each combination circulation, It is injected 20mM HCL 30 seconds, is regenerated with 30ul/ minutes flow velocitys.The sensing figure of each concentration is obtained, and is used Derived curve is fitted to 1:1 Langmuir binding model by Biaevaluation software.As a result it is shown in Figure 26.Association rate (ka), dissociation rate (kd) and the KD calculated are listed in the top of each figure.

This dynamic analysis carried out by Biacore shows that BsAb (A1-A8)-LALA and VEGFR2 or PDL1 is fast Speed is associated and is slowly dissociated with VEGFR2 or PDL1.Machine measurement is had exceeded with the slowly dissociation rate of VEGFR2 and PDL1 Range.BsAb (A7-A8) is respectively about 47pM and 1.2pM for the KD value of hVEGFR2 and hPDL1, with the 52pM of B1C4A7 and The 7.7pM of dsD7A8-LALA is suitable.

ELISA is combined with two intersecting for target

Intersect using scheme 1 shown in Figure 27 and combines ELISA.As shown, can be in immobilization hVEGFR2 table Compound 2 and 3 is found on face, but only can detecte compound 3 with Streptavidin-HRP.It can find in the solution Compound 1 will be washed off during processing.

By BsAb (A7-A8)-LALA of serial dilution, individually (it is directed to its parent's Mono-specific antibodies B1C4A7 VEGFR2) and dsD7A8-LALA (being directed to PDL1) people PDL1-Fc through biotin labeling for reinstating fixed amount (is finally 0.5 μ g/ml) it is incubated for 1 hour in solution at room temperature, mixture is transferred to later in 96 orifice plates for being coated with hVEGFR2 and again It is incubated for hour.In conjunction with BsAb/ biotin-PDL1 compound can be detected by HRP- Streptavidin (such as institute in scheme 1 Show).As a result it is shown in Figure 28.Data point is the average value ± standard deviation (S.D.) of six measurements.

As shown, combining ELISA to check that bispecific antibody A7-A8 can be with VEGFR2 and PDL1 simultaneously by intersecting In conjunction with.This result shows that the conformation of bispecific antibody rear constant in conjunction with the first target；Therefore, what the first target combined is double Specific antibody can combine the second target.

Phosphorylation assay

Serum starved cells and different amounts of antibody are incubated at room temperature 30 minutes, the hVEGF of 30ng/ml is then used The mVEGF (for EOMA) of (for KDR-PAE) or 50ng/ml are stimulated 15 minutes again.Lytic cell simultaneously measures egg White matter concentration.30 μ g proteins are decomposed with SDS PAGE, and are carried out using the antiphosphotyrosine antibody for receptor and MAPK Immunoblotting assay.Use the chemiluminescence detection signal of enhancing.As a result it is shown in Figure 29.

It is checked by testing the ability of its blocking human VEGFR-3 2 or mouse VEGFR2 and downstream molecules MAPK phosphorylation The effect of BsAb (A7-A8)-LALA.Figure 29 is shown, in the presence of BsAb (the A7-A8)-LALA and B1C4A7 of 100 or 10nM Under, the phosphoprotein band of human VEGFR-3 2, mVEGFR2 and MAPK wants much weaker.The band handled with BsAb (A7-A8) and B1C4A7 Density variation very little can not be distinguished with eyes.

Cytokine secretion measurement

By BsAb (A7-A8)-LALA of serial dilution, individually with its parental antibody B1C4A7 (be directed to VEGFR2) and DsD7A8 (being directed to PDL1) is added to together in the PBMC (from donor BG) with 0.01 μ g/mLSEB activation.6th day, in collection Clear liquid illustrates to measure IL-2 and IFN γ according to manufacturer by using Duoset kit.As a result it is shown in Figure 30.Data point It is the average value ± standard deviation (S.D.) measured three times and is calculate by the following formula:

(reading that sample readout-does not stimulate)/(reading that the reading-of SEB stimulation does not stimulate) * 100

The result shows that in the presence of BsAb (A7-A8)-LALA and dsD7A8, cell factor in PBMC when being stimulated through SEB The secretion of IL2 and INF γ is much higher than anti-vegf R2 antibody B1C4A7 (Figure 30).It is handled with BsAb (A7-A8)-LALA and dsD7A8 There is no difference.

In short, the above results prove BsAb (A7-A8)-LALA keep its parental antibody B1C4A7 (for VEGFR2) and The effect of dsD7A8-LALA (being directed to PDL1).

The vitro characterization of 3 bispecific antibody of embodiment (A1-A8)

It (is directed to This embodiment describes the vitro characterization of bispecific antibody A1-A8 and with its parental antibody B1A1 VEGFR2) compare with the effect of dsD7A8-LALA (being directed to PDL1).

It is just as in the previous examples reported, by HEK293 transient expression obtain and pass through Protein A purification Pure bispecific antibody A1-A8 is assembled by SEC-UPLC analysis detection to the higher molecular weight lower than 1%.It is incubated under 37 After 5 days, BsAb (A1-A8) is also highly stable in mice serum.

Individually together with its parental antibody B1A1 (being directed to VEGFR2) and dsD7A8-LALA (being directed to PDL1), having studied can With the VEGFR2 and PDL1 of bispecific antibody A1-A8 and the expression of soluble and cell with people and mouse species cross reaction In conjunction with, inhibit ligand (VEGF and PDL1), with receptor (VEGFR2 and PD1) combine, blocking VEGF R2/ downstream molecules phosphorylation and The ability of stimulating cytokine IL2 and INF γ secretion.

Material and method

Outside using following antibody, including bispecific antibody A1-A8, material and method and above-described embodiment herein Those are identical described in 2.

As a result

In conjunction with the dose response of people and mouse VEGFR2 and PDL1

The dose response to people and mouse VEGFR2 and PDL1 is measured in the same manner described above.As a result it is shown in Figure 31.

The result shows that BsAb (A1-A8)-LALA can be combined strongly with recombinant human VEGF R2, people PDL1 and mouse PDL1. It is 0.128nM that EC50 is directed to hVEGFR2 respectively, is 0.088nM for hPDL1 and is 0.160nM for mPDL1；B1A1 for The related EC50 of hVEGFR2 is 0.127nM, and dsD7A8 is respectively 0.121nM and 0.172nM for hPDL1 and mPDL1.

Dose response for measuring IC50 measures

It is measured in the above described manner.As a result it is shown in Figure 32.Data point is the average value ± standard of replication twice Deviation (S.D.).It is known as " log (inhibitor) vs. reaction -- variable slope (four ginsengs by using from GraphPad Prism Number) " program calculate the IC50 for being directed to different interactions, and be listed in the top of each figure.

The result shows that BsAb (A1-A8) can block strongly ligand VEGF or PDL1 and its respective receptor VEGFR2 and PD1 is combined.The IC50 of hVEGF-hVEGFR2 interaction, is 1.19nM for BsAb (A1-A8)-LALA, and is for B1A1 1.60nM.The IC50 of hPDL1-hPD1 interaction, is 2.51nM for BsAb (A1-A8)-LALA, and for dsD7A8- LALA is 3.04nM.The IC50 of mPDL1-mPD1 interaction is 3.20nM for BsAb (A1-A8)-LALA, and for DsD7A8-LALA is 2.80nM.

And the combination of the VEGFR2 and PDL1 of cell expression

It is combined measurement in the above described manner.As a result it is shown in Figure 33.

The result shows that human VEGFR-3 2, people PDL1 and mouse PDL1 that BsAb (A1-A8)-LALA can also be expressed with cell are tied It closes.BsAb (A1-A8)-LALA for the EC50 value of KDR/PAE, MDA-MB-231 and B16-F10 be respectively 0.779nM, 0.402nM and 0.113nM；B1A1 is 0.177nM for the related EC50 of KDR/PAE, dsD7A8 for MDA-MB-231 and B16-F10 is respectively 0.159nM and 0.042nM.(Figure 33).

The EC50 and IC50 of bispecific antibody A1-A8 and its parental antibody B1A1 and dsD7A8-LALA are listed in the following table. Very close EC50 and IC50 show between BsAb (A1-A8)-LALA parental antibody B1A1 or dsD7A8-LALA associated therewith BsAb (A1-A8)-LALA is effective as B1A1 or dsD7A8-LALA.

Table: the EC50 (for combination) and IC50 of BsAb (A1-A8) (for blocking)

*: EOMA expresses both mVEGFR2 and mPDL1

Dynamic analysis

Kinetic determination is carried out in the above described manner.As a result it is shown in Figure 34.Association rate (ka), dissociation rate (kd) and meter The KD of calculation is listed in the top of each figure.

Dynamic analysis (Figure 34) display carried out by Biacore, BsAb (A1-A8)-LALA and VEGFR2 or PDL1 It quickly associates and is rested on VEGFR2 or PDL1 for a long time.Slowly dissociation rate exceeds in conjunction with VEGFR2 and PDL1 Machine measurement range.BsAb (A1-A8) is respectively about 200pM and 3.2pM for the KD value of hVEGFR2 and hPDL1, with B1A1 164pM and dsD7A8-LALA 7.7pM it is suitable.

ELISA is combined with two intersecting for target

It is combined measurement in the above described manner.As a result it is shown in Figure 35.Data point is the average value ± standard of six measurements Deviation (S.D.).

As shown, combining ELISA to check by intersecting, bispecific antibody A18 can be tied simultaneously with VEGFR2 and PDL1 It closes (Figure 36).This result shows that the conformation of bispecific antibody rear constant in conjunction with the first target；Therefore, the first target combines Bispecific antibody can combine the second target.

Phosphorylation assay

It is measured in the above described manner.As a result it is shown in Figure 36.

BsAb (A1-A8)-is checked by testing the ability of its blocking human VEGFR-3 2 and downstream molecules MAPK phosphorylation The effect of LALA.Figure 36 shows exist in BsAb (A1-A8)-LALA and BsAb (the A7-A8)-LALA of 1.5 μ g/ml (7.5nM) Under, the phosphoprotein band of both VEGFR2 and MAPK are more much weaker than the band that no antibody is handled.In the experiment, parental antibody Concentration is 1.5 μ g/ml (10nM)；Therefore, Mono-specific antibodies more more than bispecific antibody are used in this experiment.

Cytokine secretion measurement

Cytokine secretion measurement is carried out in the above described manner.By BsAb (the A1-A8)-LALA and BsAb (A7- of serial dilution A8) it is added in the PBMC (from donor BG) with 0.01 μ g/mLSEB activation.6th day, supernatant is collected, by using Duoset kit illustrates measurement IL-2 and IFN γ according to manufacturer.Data point is the average value ± mark of replication twice Quasi- deviation (S.D.).As a result it is shown in Figure 37.

The result shows that in the presence of BsAb (A1-A8)-LALA and BsAb (A7-A8)-LALA, PBMC when being stimulated through SEB The secretion of middle cell factor IL2 and INF γ is higher than control antibodies B1A1 and B1C4A7 (Figure 37).

In short, the above results prove BsAb (A1-A8)-LALA keep its parental antibody B1A1 (for VEGFR2) and The effect of dsD7A8-LALA (being directed to PDL1).

4 antitumor action I of embodiment

In this embodiment, effect of the above-mentioned BsAb (A1-A8) to CT26 mouse colon cancer cell is checked.

More specifically, CT26 of the 0.1mL in serum-free RPMI-1640 culture medium is subcutaneously injected in the bottom right veutro in mouse Mouse colon cancer cell (0.3x10⁶A cell/mouse), it is used for tumor development.When tumour mean size reaches about 75-125mm³ When, mouse is randomly assigned as 5 experimental groups according to tumor size.Every group is made of 13 mouse.As shown in the table, antibody is used Handle mouse.Each dosage is adjusted to 200 μ l with PBS, and twice a week, is lasted up to by intraperitoneal injection to mouse Three weeks.Weight and gross tumor volume are measured twice a week.As a result it is shown in following table.

The result shows that B1C4A7, D7A8, a combination thereof and BsAb (A7-A8) reduce gross tumor volume.It was found that in this CT26 In model, the combination of two kinds of antibody (B1C4A7 and D7A8) and BsAb (A7-A8) in terms of reducing gross tumor volume individually than two kinds Antibody it is more effective.Because all antibody are human antibody, handle more than 2 weeks, develop immunogenicity.Therefore, it reports The result after handling up to 3 weeks is accused.As a result it is also shown that not finding weight significant decrease in reason group in where in office, (data are not It shows).

5 antitumor action II of embodiment

In this embodiment, effect of the above-mentioned BsAb (A1-A8) to MC38 mouse colon cancer cell is checked.

Every mouse inoculates MC38 mouse colon cancer cell (1 × 10 in bottom right veutro⁶A cell/mouse), it is used for Tumor development.When tumour mean size reaches about 90mm³When, mouse is randomly assigned as 9 experimental groups according to tumor size.Often Group is made of 13 mouse.As shown in Figure 39 A and 39B, antibody treated mice is used.Each dosage is adjusted to 200 μ l with PBS, And twice a week, three weeks are lasted up to animal by intraperitoneal injection.Weight and gross tumor volume are measured twice a week.As a result It is shown in Figure 39 A and 39B.

As shown, the antibody B1C4A7 for VEGFR2 only shows middle equivalent force.In contrast, anti-PDL1 antibody The combination of D7A8, BsAb (A7-A8) and B1C4A7 and D7A8 completely inhibit tumour growth, and do not observe between three groups Significant difference.Since all antibody are human antibody, so processing developed immunogenicity more than 2 weeks.After reporting treatment Up to 3 weeks results.In addition, not observing significant weight loss in three weeks for all mouse (data are not shown). All antibody (Figure 39 A) and LALA form (Figure 39 B) in the form of conventional IgG1 construct, wherein 234 He of leucine in CH2 235 sport alanine to weaken effector function.Significant difference is not observed between both forms.

The vitro characterization of the other bispecific antibodies of embodiment 6

In this embodiment, generate and have checked other example (i.e. heavy chains-of bispecific antibody as depicted in fig. 1A C-terminal fusion).VEGFR2 is directed to for this purpose, generating using anti-PDL1 antibody A 11 or D7A8 and anti-vegf R2 antibody B1A1 or B1C4A7 With the different orientation of the bispecific antibody of PDL1.These bispecific antibodies, related component and binding specificity are listed in the table below In.In bispecific antibody, following two connector is used:

15GS:GGGGSGGGGSGGGGS (SEQ ID NO:68)

30GS:GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:69).

In addition, mutation is introduced into scFv to add disulfide bond between two variable domains.Also into CH2 structural domain Mutation is introduced to weaken effector function (IgG in the form of LALA), wherein (such as SEQ ID NO:28,30 and of the LL residue in CH2 Runic in 32) become AA.

Analyze the purity and stability of these bispecific antibodies in the above described manner by SDS-PAGE and SEC-UPLC.Knot Fruit is shown in Figure 40 and 41.

As shown in figure 40, some degradation bands of BsAb (B1A1-A11) and BsAb (B1C4A7-A11) variant are observed.

In contrast, orientation is not seen after changing in BsAb (A11-B1A1) and BsAb (D7A8-B1A1) variant (Figure 41) Observe degradation band.

SEC-UPLC the result shows that, when being added to disulfide bond, bispecific antibody is more stable.For example, for BsAb (A11-B1A1) for _ 30cc, monomer accounts for 95.5% or more, and for A11IgG, BsAb (A11-B1A1) and BsAb (A11- B1A1 for) _ 30, monomer accounts for 97.8%, 94.3% and 93.8% or more respectively.Similarly, in BsAb (D7A8-B1A1) variant In, for BsAb (D7A8-B1A1) _ 30cc, monomer accounts for 97.3% or more, and for D7A8IgG, BsAb (D7A8-B1A1) and BsAb (D7A8-B1A1) _ 30, monomer account for respectively more than 99%, 86.6% and 90.2%.

The combination of these bispecific antibodies and PDL1 and VEGR2 are analyzed in the same manner described above.As a result it is shown in Figure 42. It was found that these are effective as parental antibody for the bispecific antibody of PDL1 and VEGR2.

The interaction for blocking ligand and receptor is also had checked in the above described manner.As a result it is shown in Figure 43.It was found that these are double Specific antibody is effective as parental antibody.

Biacore analysis is carried out in the above described manner.As a result it is shown in following table.

Claims

1. a kind of bispecific binding protein, it includes the firstth areas in conjunction with human PD-L 1 and the secondth area in conjunction with people KDR.

2. bispecific binding protein as described in claim 1 is bispecific antibody.

3. bispecific binding protein as described in claim 1 is fusion protein.

4. a kind of bispecific antibody, it includes IgG, IgA, IgE or IgD and scFv.

5. bispecific antibody as claimed in claim 4, it includes the scFv of the IgG for combining PD-L1 and combination KDR.

6. bispecific antibody as claimed in claim 4, it includes the scFv of the IgG for combining KDR and combination PD-L1.

7. it includes combine human PD-L 1 and have to determine there are three complementary such as bispecific antibody described in claim 5 or 6 The heavy-chain variable domains in area (CDR), wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:10-12 Or respectively SEQ ID NO:58-60.

8. it includes combine human PD-L 1 and have the light chain there are three CDR such as bispecific antibody described in claim 5 or 6 Variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:13-15 or respectively SEQ ID NO:61-63。

9. such as bispecific antibody described in claim 5 or 6, it includes combine people KDR and have that there are three the heavy chains of CDR can Structure changes domain, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:16-18.

10. it includes combine people KDR and have the light chain there are three CDR such as bispecific antibody described in claim 5 or 6 Variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:19-21.

11. it includes combine people KDR and have the heavy chain there are three CDR such as bispecific antibody described in claim 5 or 6 Variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:22-24.

12. it includes combine people KDR and have the light chain there are three CDR such as bispecific antibody described in claim 5 or 6 Variable domains, wherein the amino acid sequence of CDR1, CDR2 and CDR3 are respectively SEQ ID NO:25-27.

13. such as bispecific antibody described in claim 5 or 6, it includes heavy chain and light chain, wherein the heavy chain and described light Chain includes heavy chain/light chain pair corresponding sequence selected from the group being made up of: SEQ ID NO:34 and 31, SEQ ID NO:30 With 35, SEQ ID NO:36 and 31, SEQ ID NO:30 and 37, SEQ ID NO:38 and 33, SEQ ID NO:32 and 39, SEQ ID NO:40 and 33, SEQ ID NO:32 and 41, SEQ ID NO:42 and 29, SEQ ID NO:28 and 43, SEQ ID NO:44 With 29, SEQ ID NO:28 and 45, SEQ ID NO:46 and 29, SEQ ID NO:28 and 47, SEQ ID NO:48 and 29, SEQ ID NO:28 and 49, SEQ ID NO:51 and 50, SEQ ID NO:52 and 50, SEQ ID NO:53 and 50, SEQ ID NO:54 With 29, SEQ ID NO:55 and 29 and SEQ ID NO:56 and 29.

14. a kind of treat needs to reduce immunosupress or the method for the patient that reduces angiogenesis, the method includes to needs The patient for reducing immunosupress or angiogenesis in this way applies bispecific binding protein of any of claims 1-3 Or bispecific antibody described in any one of claim 4-13.

15. a kind of method for the treatment of cancer comprising of any of claims 1-3 double to patient in need application Bispecific antibody described in any one of binding proteins specific or claim 4-13.

16. method as claimed in claim 15, wherein the cancer is thin selected from lung cancer, colorectal cancer clear-cell carcinoma, collagen Born of the same parents' tumor, oophoroma, bladder cancer, gastric cancer, Huppert's disease, non-small cell lung cancer and cancer of pancreas.

17. a kind of isolated nucleic acid molecules, coding bispecific binding protein of any of claims 1-3, power Benefit requires bispecific antibody described in any one of 4-13 or its polypeptide chain.

18. a kind of carrier, it includes the nucleic acid molecules described in claim 17.

19. a kind of host cell of culture, it includes the carriers described in claim 18.

20. a kind of method for generating polypeptide, the method includes cultivating right under conditions of allowing the nucleic acid molecules to express It is required that host cell described in 19.

21. a kind of any one of bispecific binding protein of any of claims 1-3 or claim 4-13 institute The conjugate for the bispecific antibody stated, wherein the bispecific binding protein or the bispecific antibody with selected from imaging The medicament of agent, therapeutic agent and cytotoxic agent is conjugated.

22. a kind of pharmaceutical composition, it includes

It is double special described in any one of bispecific binding protein of any of claims 1-3, claim 4-13 Conjugate described in heterogenetic antibody or claim 21, and

Pharmaceutically acceptable carrier.