Trispecific binding proteins, methods and uses thereof

Abstract

The present disclosure provides trispecific and / or trivalent binding proteins that comprise four polypeptide chains forming three antigen-binding sites, wherein the three antigen-binding sites specifically bind one or more target proteins, wherein a first pair of the polypeptide chains forming the binding protein have dual variable domains with a cross orientation, and wherein a second pair of the polypeptide chains have a single variable domain forming a single antigen-binding site. In some embodiments, the binding protein comprises a binding site that binds a CD28 polypeptide, a binding site that binds a CD3 polypeptide, and a binding site that binds a third polypeptide, such as a tumor target protein. The present disclosure also relates to methods for preparing trispecific and / or trivalent binding proteins and uses of such binding proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 831,572, filed April 9, 2019, and European Application No. EP19306311.2, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0003] Submission of Sequence Listings as ASCII Text Files

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 183952032040SEQLIST.TXT, Record Date: April 6, 2020, Size: 526KB). Technical Field

[0005] The present disclosure relates to trispecific and / or trivalent binding proteins comprising four polypeptide chains forming three antigen-binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possesses dual variable domains in a cross-shaped orientation. The present disclosure also relates to methods for preparing trispecific and / or trivalent binding proteins and uses of such binding proteins. Background Art

[0006] Biotherapeutics based on monoclonal antibodies have become the important approach for the development of new drugs.Monoclonal antibody technology provides specific targeting, accurate signal transduction delivery and / or useful load for specific cell populations, and provides lasting biological action by its Fc function.The effort aspect antibody engineering has allowed to develop bispecific antibodies that the specificity of two monoclonal antibodies is combined, for various biological applications, thereby expanded the scope of antibody drug development.Newly discovered neutralizing antibodies with improved breadth and effectiveness can provide more selections for the biotherapeutics that are developed for the treatment of complicated diseases (such as cancer, arthritis and / or inflammatory disorders).

[0007] Immuno-oncology is a promising, emerging therapeutic approach for cancer management. The immune system is the first line of defense against cancer development and progression. There is now ample evidence that T cells can control tumor growth and prolong survival in cancer patients both in the early and late stages of the disease. However, tumor-specific T cells can be limited in many ways, hindering their ability to control the disease.

[0008] As part of human adaptive immunity, T cell immunity plays a key role in controlling viral infection and cancer, potentially eliminating infected and malignant cells, leading to clearance of viral infection or cure of cancer. In chronic infectious diseases such as herpes virus infection (HSV, CMV, EBV, etc.), HIV and HBV, the virus establishes its persistence in the human body through a variety of mechanisms including immunosuppression, T cell depletion and establishment of latency. However, viral infection usually induces viral antigen-specific immunity including antigen-specific CD8 T cells, which can easily recognize infected cells for control or killing through cytokine release or cytotoxic T cell (CTL)-mediated killing processes.

[0009] Therefore, activation and / or expansion of viral antigen-specific T cells in vivo and / or ex vivo may provide a therapeutic strategy against chronic viral infections.

[0010] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0011] In order to meet these and other needs, the present invention provides a trispecific binding protein (e.g., antibody) that forms three antigen binding sites. These binding proteins can specifically bind to one, two or three antigen targets or target proteins, such as CD28, CD3 and tumor target proteins. Some tumors express specific antigens. For example, HER2 amplification and overexpression can be found in the molecular subtypes of breast cancer, and are also found in gastric cancer, ovarian cancer, lung cancer and prostate cancer. The optimal activation of T cells requires two factors: 1. antigen recognition and 2. co-stimulation. In the case of using trispecific HER2 / CD28xCD3 trispecific binding proteins as described herein, signal 1 is provided by the agonist anti-CD3 binding site, and signal 2 is provided by the agonist anti-CD28 binding site. The trispecific binding protein recruits T cells to tumors via the binding site of HER2, CD38 or another tumor target protein, and activates the engaged T cells via anti-CD3 and CD28. The resulting activation induces the killing potential of the immune cells to nearby tumor cells. Furthermore, anti-CD3 binding sites are described that possess high affinity binding to human CD3 polypeptide and eliminate potential manufacturing disadvantages (e.g., deamidation sites).

[0012] This article further provides an anti-CD38 / CD28xCD3 trispecific antibody, which has been developed and evaluated for its potential in activating T cells and subsequently proliferating and / or amplifying antigen-specific T cells. These trispecific Abs can effectively amplify CD4 and CD8 effector and memory populations in vitro, and the population includes antigen-specific CD8 T central memory cells and effector memory cells. Specifically, the in vitro amplification of CMV, EBV, HIV-1, influenza-specific CD8 central memory cells and effector memory cells has been demonstrated. The anti-CD38 / CD28xCD3 trispecific antibodies described herein exhibit novel properties by engaging CD3 / CD28 / CD38, thereby providing a signaling pathway that stimulates and amplifies T cells, which can provide an effective strategy for treating chronic infectious diseases (such as HSV, CMV, EBV, HIV-1 and HBV infection).

[0013] To meet these and other needs, provided herein are binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins having at least one antigen binding site that binds to a CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells, subsequently activate T cells, and promote the killing of adjacent cancer cells by the activated T cells through the granzyme / perforin mechanism, thereby providing a novel approach comparable to anti-CD38 antibodies such as (daratumumab) has a different mode of action for anti-tumor activity. Furthermore, the ability to bind both human and cynomolgus monkey CD38 polypeptides allows for easy testing of the binding protein in preclinical toxicology studies, for example to assess its safety profile for future clinical use.

[0014] In some embodiments, provided herein are binding proteins comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0015] V L2 -L1-V L1 -L2-C L [I]

[0016] And the second polypeptide chain comprises a structure represented by the following formula:

[0017] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0018] And the third polypeptide chain comprises a structure represented by the following formula:

[0019] VH3 -C H1 -Hinge-C H2 -C H3 [III]

[0020] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0021] V L3 -C L [IV]

[0022] in:

[0023] V L1 is the first immunoglobulin light chain variable domain;

[0024] V L2 is the second immunoglobulin light chain variable domain;

[0025] V L3 is the third immunoglobulin light chain variable domain;

[0026] V H1 is the first immunoglobulin heavy chain variable domain;

[0027] V H2 is a second immunoglobulin heavy chain variable domain;

[0028] V H3 is the third immunoglobulin heavy chain variable domain;

[0029] C L is the immunoglobulin light chain constant domain;

[0030] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0031] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0032] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0033] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0034] L1, L2, L3, and L4 are amino acid linkers;

[0035] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0036] Where VH1 and V L1 Forming a first antigen binding site;

[0037] Where V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2 the domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), in which X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65); and

[0038] Where V H3 and V L3 Formation of the third antigen binding site.

[0039] In some embodiments, the first binding site binds to a CD28 polypeptide. H1 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO: 49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO: 50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO: 51); and the V L1 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO: 52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO: 53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO: 54). In some embodiments, the V H1The domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91), and / or the V L1 The domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92).

[0040] In some embodiments, the V L2The CDR-L1 sequence of the domain comprises an amino acid sequence selected from the group consisting of: QSLVHQNAQTY (SEQ ID NO: 59), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61), and QSLVHENLRTY (SEQ ID NO: 62). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the V H2 The domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93), and / or the V L2The domain includes an amino acid sequence selected from the following: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO:96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the V H2 The domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302), and / or the V L2The domain includes an amino acid sequence selected from the following: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO:96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO: 97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 97.In some embodiments, the binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.

[0041] In some embodiments, the third antigen binding site binds to a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, the tumor target proteins of the present disclosure include, but are not limited to, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2,Receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin White), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1 R, IL2Rβ, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0042] In some embodiments, the third antigen binding site binds to human CD38 polypeptide. H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and the V L3 The V domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the V H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and the V L3 The V domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, the V H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO: 25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO: 26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO: 27); and the V L3 The V domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO: 28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO: 30). In some embodiments, the V H3The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO: 31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO: 32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO: 33); and the V L3 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO: 34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO: 36).

[0043] In some embodiments, the V H3 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and the V L3 The V domain comprises a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42). H3 The V domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO: 45); and the V L3 The V domain comprises a CDR-L1 sequence comprising an amino acid sequence of QGIRND (SEQ ID NO: 46), a CDR-L2 sequence comprising an amino acid sequence of AAS (SEQ ID NO: 47), and a CDR-L3 sequence comprising an amino acid sequence of LQDYIYYPT (SEQ ID NO: 48). In some embodiments, the V H3The domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79), and / or the V L3 The V domain comprises the amino acid sequence of DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80). In some embodiments, the V H3 The domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81), and / or the V L3 The domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82). In some embodiments, the V H3 The domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83), and / or the V L3 The V domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84). H3The domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87), and / or the V L3 The V domain comprises the amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88). In some embodiments, the V H3 The domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89), and / or the V L3 The V domain comprises the amino acid sequence of AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90). In some embodiments, the V H3 The domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85), and / or the V L3 The domain comprises the amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO: 86).

[0044] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:160 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:161 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:162 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:163 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:163. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:168 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:169 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:170 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:171 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:171. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:179.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:188.

[0045] In some embodiments, the third antigen binding site binds to a human HER2 polypeptide. H3 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4) or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7) or SRWGGSGFYAMDY (SEQ ID NO: 8); and the V L3The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and the V L3 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and the V L3 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and the V L3The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V H3 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and the V L3 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V H3 The domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and the V L3 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the V H3 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and the V L3 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 The domain contains the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:75) or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:76), and / or the V L3 The domain contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, the V H3The domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or the V L3 The V domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the V H3 The domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), and / or the V L3 The V domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the V H3 The domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75), and / or the V L3 The V domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the V H3The domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), and / or the V L3 The V domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the V H3 The domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and / or the V L3 The V domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the V H3 The domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or the V L3 The domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78).

[0046] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:104, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:105, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:106, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:107, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:107. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:115.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:116 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:119. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:123. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:127.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293.In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.

[0047] In some embodiments that may be combined with any other embodiment described herein, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, and L4 are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO: 68), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, at least one of L1, L2, L3, or L4 comprises the sequence DKTHT (SEQ ID NO: 66). In some embodiments, L1, L2, L3, and L4 comprise the sequence DKTHT (SEQ ID NO: 66).

[0048] In some embodiments that may be combined with any other embodiments described herein, the hinge-C H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein the hinge-C H2 -C H3 The hinge-C domains of the second and third polypeptide chains each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A. H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein the hinge-C H2 -C H3The domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C H2 -C H3 The domain is human IgG4 hinge-C H2 -C H3 domain, and wherein the hinge-C H2 -C H3 The hinge-C domains each comprise an amino acid substitution at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-C domains of the second and third polypeptide chains are H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, and wherein the hinge-C H2 -C H3 The hinge-C domains of the second and third polypeptide chains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. H2 -C H3 The domain is human IgG1 hinge-C H2 -C H3 domain, and wherein the hinge-C H2 -C H3 The hinge-C domains each comprise an amino acid substitution at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-C domain of the second polypeptide chain H2 -C H3 The structural domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-C H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-C H2-C H3 The structural domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C H2 -C H3 The domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[0049] In some embodiments, provided herein are isolated nucleic acid molecules comprising a nucleotide sequence encoding the binding protein of any of the above embodiments. In some embodiments, provided herein are expression vectors comprising the nucleic acid molecules of any of the above embodiments. In some embodiments, provided herein are isolated host cells comprising the nucleic acid molecules of any of the above embodiments or the expression vectors of any of the above embodiments. In some embodiments, the host cells are mammalian cells or insect cells.

[0050] In some embodiments, provided herein is a pharmaceutical composition comprising the binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0051] In some embodiments, provided herein are methods for preventing and / or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of at least one binding protein or pharmaceutical composition of any one of the above embodiments. In some embodiments, provided herein are binding proteins or pharmaceutical compositions according to any one of the above embodiments for use in a method for preventing and / or treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition. In some embodiments, provided herein are binding proteins or pharmaceutical compositions according to any one of the above embodiments for use in the manufacture of a medicament for preventing and / or treating cancer in a patient.

[0052] In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent.In some embodiments, the patient is a human.

[0053] In some embodiments, the third antigen binding site binds to a human CD38 polypeptide, and wherein the cancer cells from the individual or patient express CD38. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma. In some embodiments, prior to administration of the binding protein, the patient has been treated with daratumumab without a washout period.

[0054] In some embodiments, the third antigen binding site binds to a human HER2 polypeptide, and wherein the cancer cells from the individual or patient express HER2. In some embodiments, the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

[0055] In some embodiments, provided herein are methods for expanding virus-specific memory T cells, comprising contacting the virus-specific memory T cells with a binding protein, wherein the binding protein comprises four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0056] V L2 -L1-V L1 -L2-C L [I]

[0057] And the second polypeptide chain comprises a structure represented by the following formula:

[0058] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0059] And the third polypeptide chain comprises a structure represented by the following formula:

[0060] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0061] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0062] V L3 -C L [IV]

[0063] in:

[0064] V L1 is the first immunoglobulin light chain variable domain;

[0065] V L2 is the second immunoglobulin light chain variable domain;

[0066] V L3 is the third immunoglobulin light chain variable domain;

[0067] V H1 is the first immunoglobulin heavy chain variable domain;

[0068] V H2 is a second immunoglobulin heavy chain variable domain;

[0069] V H3 is the third immunoglobulin heavy chain variable domain;

[0070] C L is the immunoglobulin light chain constant domain;

[0071] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0072] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0073] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0074] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0075] L1, L2, L3, and L4 are amino acid linkers;

[0076] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0077] Where V H1 and V L1 Forms the first antigen binding site that binds to CD28 polypeptide, wherein V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the VL2 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V H3 and V L3 A third antigen binding site is formed that binds to the CD38 polypeptide.

[0078] In some embodiments, provided herein are binding proteins comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0079] V L2 -L1-V L1 -L2-C L [I]

[0080] And the second polypeptide chain comprises a structure represented by the following formula:

[0081] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0082] And the third polypeptide chain comprises a structure represented by the following formula:

[0083] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0084] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0085] V L3 -C L [IV]

[0086] in:

[0087] V L1 is the first immunoglobulin light chain variable domain;

[0088] V L2 is the second immunoglobulin light chain variable domain;

[0089] V L3 is the third immunoglobulin light chain variable domain;

[0090] V H1 is the first immunoglobulin heavy chain variable domain;

[0091] V H2 is a second immunoglobulin heavy chain variable domain;

[0092] V H3 is the third immunoglobulin heavy chain variable domain;

[0093] C L is the immunoglobulin light chain constant domain;

[0094] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0095] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0096] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0097] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0098] L1, L2, L3, and L4 are amino acid linkers;

[0099] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0100] Where V H1 and V L1 Forms the first antigen binding site that binds to CD28 polypeptide, wherein V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V H3 and V L3 A third antigen binding site is formed that binds to the CD38 polypeptide for use in expanding virus-specific memory T cells.

[0101] In some embodiments, the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cells with the binding protein causes activation and / or proliferation of the virus-specific memory T cells.

[0102] In some embodiments, provided herein are methods of expanding T cells, the methods comprising contacting the T cells in vitro or ex vivo with a binding protein, wherein the binding protein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0103] V L2 -L1-V L1 -L2-C L [I]

[0104] And the second polypeptide chain comprises a structure represented by the following formula:

[0105] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0106] And the third polypeptide chain comprises a structure represented by the following formula:

[0107] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0108] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0109] V L3 -C L [IV]

[0110] in:

[0111] VL1 is the first immunoglobulin light chain variable domain;

[0112] V L2 is the second immunoglobulin light chain variable domain;

[0113] V L3 is the third immunoglobulin light chain variable domain;

[0114] V H1 is the first immunoglobulin heavy chain variable domain;

[0115] V H2 is a second immunoglobulin heavy chain variable domain;

[0116] V H3 is the third immunoglobulin heavy chain variable domain;

[0117] C L is the immunoglobulin light chain constant domain;

[0118] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0119] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0120] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0121] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0122] L1, L2, L3, and L4 are amino acid linkers;

[0123] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0124] Where V H1 and V L1 Forms the first antigen binding site that binds to CD28 polypeptide, wherein V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V H3 and V L3 A third antigen binding site is formed that binds to the CD38 polypeptide.

[0125] In some embodiments, provided herein are binding proteins comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0126] V L2 -L1-V L1 -L2-C L [I]

[0127] And the second polypeptide chain comprises a structure represented by the following formula:

[0128] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0129] And the third polypeptide chain comprises a structure represented by the following formula:

[0130] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0131] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0132] V L3 -C L [IV]

[0133] in:

[0134] V L1is the first immunoglobulin light chain variable domain;

[0135] V L2 is the second immunoglobulin light chain variable domain;

[0136] V L3 is the third immunoglobulin light chain variable domain;

[0137] V H1 is the first immunoglobulin heavy chain variable domain;

[0138] V H2 is a second immunoglobulin heavy chain variable domain;

[0139] V H3 is the third immunoglobulin heavy chain variable domain;

[0140] C L is the immunoglobulin light chain constant domain;

[0141] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0142] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0143] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0144] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0145] L1, L2, L3, and L4 are amino acid linkers;

[0146] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0147] Where V H1 and V L1 Forms the first antigen binding site that binds to CD28 polypeptide, wherein V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V H3 and V L3 A third antigen binding site is formed that binds a CD38 polypeptide for use in a method for expanding T cells.

[0148] In some embodiments, the T cell is a memory T cell or an effector T cell. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or comprises a polynucleotide encoding CAR on its cell surface.

[0149] In some embodiments, provided herein are methods for treating chronic viral infections, comprising administering to an individual or patient in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0150] V L2 -L1-V L1 -L2-C L [I]

[0151] And the second polypeptide chain comprises a structure represented by the following formula:

[0152] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0153] And the third polypeptide chain comprises a structure represented by the following formula:

[0154] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0155] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0156] V L3 -C L [IV]

[0157] in:

[0158] V L1 is the first immunoglobulin light chain variable domain;

[0159] V L2 is the second immunoglobulin light chain variable domain;

[0160] V L3 is the third immunoglobulin light chain variable domain;

[0161] V H1 is the first immunoglobulin heavy chain variable domain;

[0162] V H2 is a second immunoglobulin heavy chain variable domain;

[0163] V H3 is the third immunoglobulin heavy chain variable domain;

[0164] C L is the immunoglobulin light chain constant domain;

[0165] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0166] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0167] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0168] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0169] L1, L2, L3, and L4 are amino acid linkers;

[0170] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0171] Where V H1 and V L1 Forms the first antigen binding site that binds to CD28 polypeptide, wherein V H2 and V L2Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2 The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V H3 and V L3 A third antigen binding site is formed that binds to the CD38 polypeptide.

[0172] In some embodiments, provided herein are binding proteins comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0173] V L2 -L1-V L1 -L2-C L [I]

[0174] And the second polypeptide chain comprises a structure represented by the following formula:

[0175] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0176] And the third polypeptide chain comprises a structure represented by the following formula:

[0177] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0178] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0179] V L3 -C L [IV]

[0180] in:

[0181] V L1 is the first immunoglobulin light chain variable domain;

[0182] V L2 is the second immunoglobulin light chain variable domain;

[0183] V L3 is the third immunoglobulin light chain variable domain;

[0184] V H1 is the first immunoglobulin heavy chain variable domain;

[0185] V H2 is a second immunoglobulin heavy chain variable domain;

[0186] V H3 is the third immunoglobulin heavy chain variable domain;

[0187] C L is the immunoglobulin light chain constant domain;

[0188] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0189] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0190] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0191] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0192] L1, L2, L3, and L4 are amino acid linkers;

[0193] wherein the polypeptide of Formula I and the polypeptide of Formula II form a crossover light chain-heavy chain pair; and

[0194] Where V H1 and V L1 Forms the first antigen binding site that binds to CD28 polypeptide, wherein V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, wherein the V H2The structural domain comprises: a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2 The domain comprises: a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64); and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65), wherein V H3 and V L3 A third antigen binding site is formed that binds to a CD38 polypeptide. The binding protein is used in a method for treating chronic viral infection, wherein the method comprises administering an effective amount of the binding protein to an individual or patient in need thereof.

[0195] In some embodiments, the individual or patient is a human. In some embodiments, the binding protein is administered to the individual or patient in the form of a pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier. In some embodiments, administration of the binding protein results in activation and / or proliferation of virus-specific memory T cells in the individual or patient.

[0196] In some embodiments that may be combined with any other embodiment described herein, the memory T cells are CD8+ or CD4+ memory T cells. In some embodiments, the memory T cells are central memory T cells (T CM ) or effector memory T cells (T EM ).

[0197] In some embodiments that may be combined with any other embodiment described herein, the virus is human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus (HBV), human papillomavirus (HPV), Epstein-barr virus (EBV), human foamy virus (HFV), herpes simplex virus 1 (HSV-1), or herpes simplex virus 2 (HSV-2).

[0198] In some embodiments that may be combined with any other embodiments described herein, the CD28 polypeptide is a human CD28 polypeptide, wherein the CD3 polypeptide is a human CD3 polypeptide, and wherein the CD38 polypeptide is a human CD38 polypeptide.

[0199] In some embodiments, provided herein are vector systems comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of the binding protein of any of the embodiments above. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein.

[0200] In some embodiments, provided herein are kits comprising one, two, three, or four polypeptide chains of a binding protein according to any of the above embodiments. In some embodiments, the kit further comprises instructions for using the polypeptide chains or binding proteins according to any of the methods or uses described herein, such as those described above.

[0201] In some embodiments, provided herein are kits comprising one, two, three, or four polynucleotides according to any of the above embodiments. In some embodiments, provided herein are kits of polynucleotides comprising one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 200; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 200; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 208; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 212; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: A fourth polynucleotide of the polynucleotide sequence of NO:216;(h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 220; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 224; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 228; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 236; (ii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 240; (iii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 244; (iv) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 248. a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 247 and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 248;(p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 252; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 256; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 260; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 268; (ii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 272; or (iii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 276.

[0202] It should be understood that one, some or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become clear to those skilled in the art. These and other embodiments of the present invention are further described by the subsequent detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0203] Figure 1A provides a schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and HER2. The first pair of polypeptides possesses dual variable domains (VH1-VH2 and VL2-VL1) in a cross-orientation that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides possesses single variable domains (VH3 and VL3) that form a single antigen-binding site that recognizes HER2. The trispecific binding protein shown in Figure 1A utilizes a constant region with a "knob-into-hole" mutation, where the knob is located on the second pair of polypeptides with a single variable domain.

[0204] Figure 1B provides the fold change (relative to the parent) of the binding affinity of the anti-CD28 / CD3 / HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3 and anti-CD28 binding domains. The mutation of 3233QQ to QEQ (from top to bottom) refers to the mutation in residues 32-35 of the VL domain introduced into the anti-CD3 binding site (indicated by *); the remaining mutations are introduced into the VH or VL domain of the trastuzumab anti-HER2 binding site (indicated by #; according to Kabat numbering). For mutations in the anti-HER2 binding site, mutation 30Q is introduced into the VL domain, and the remaining mutations are introduced into the VH domain. Binding affinity was measured by ELISA, and the values ​​provided are relative to the parent trispecific antibodies.

[0205] Figure 1C provides binding curves for the binding of the indicated trispecific antibodies to human HER2, human CD28, and CD3, as determined by ELISA.

[0206] Figure ID provides a proposed mechanism of action for HER2 / CD28xCD3 trispecific antibody-mediated T cell activation and killing of HER2+ cancer cells.

[0207] Figure 2A provides a schematic diagram of a trispecific binding protein comprising four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and CD38. The first pair of polypeptides possesses dual variable domains (VH1-VH2 and VL2-VL1) in a cross-orientation that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides possesses single variable domains (VH3 and VL3) that form a single antigen-binding site that recognizes CD38. The trispecific binding protein shown in Figure 2A utilizes an IgG4 constant region with a "knob-in-hole" mutation, with the knob located on the second pair of polypeptides having a single variable domain.

[0208] Figures 2B to 2E show the binding affinities of the CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains to the target antigens human CD38 (Figure 2B), cynomolgus CD38 (Figure 2C), human CD3 (Figure 2D), and human CD28 (Figure 2E), as measured by ELISA.

[0209] Figure 3 shows an SPR competition assay for binding of daratumumab and anti-CD38 monospecific antibodies with the indicated anti-CD38 binding domains to CD38. If the antibody recognizes an epitope on CD38 that is different from the epitope recognized by daratumumab, injection of the antibody results in an increase in the SPR signal. If the antibody recognizes an epitope that overlaps with daratumumab, injection of the antibody does not increase the SPR signal.

[0210] Figures 4A to 4B show the in vitro cell killing activity of the CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with indicated anti-CD38 binding domains against human multiple myeloma NCI-H929 cells (CD38+ / CD28+). The assay was performed in the presence of 5 nM isotype control antibody (Figure 4A) or daratumumab (Figure 4B). In the presence of daratumumab, the trispecific antibody continued to exhibit cell killing activity.

[0211] Figures 5A to 5B show the in vitro cell killing activity of the CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated anti-CD38 binding domains against human lymphoma OCI-Ly19 cells (CD38+ / CD28-). The assay was performed in the presence of 5 nM isotype control antibody (Figure 5A) or daratumumab (Figure 5B). Daratumumab caused a reduction in the cell killing activity of the anti-CD38 / CD28xCD3 trispecific antibody.

[0212] Figures 6A to 6J show the characterization of in vitro T cell subset expansion in response to CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with indicated alternative anti-CD38 binding domains in PBMCs collected from donor D infected by CMV. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup xΔCD3mid IgG4 FALA). T cell colonies were measured at indicated time points (D3 refers to the 3rd day; D7 refers to the 7th day). The indicated trispecific antibodies were tested at indicated 0.2nM and 1nM concentrations. Flow cytometry was used to quantify CMV-specific CD8+ T cells (Figures 6A to 6B), CMV-specific T cm CD8+ cells (Figure 6C to Figure 6D) and CMV-specific T em CD8+ cells (Fig. 6E to Fig. 6F). In addition, CMV-specific T cells were quantified at the indicated time points. cm (Figure 6G to Figure 6H) and T em (Fig. 6I to Fig. 6J) Percentage of CD8+ cells. All trispecific antibodies tested promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics.

[0213] Figure 7 A to Figure 7 J show the characterization of the in vitro T cell subset expansion in response to the CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4FALA trispecific antibody with indicated anti-CD38 binding domains in PBMCs collected from donor E infected by CMV. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup xΔCD3mid IgG4 FALA). The antibodies shown from top to bottom in the legend are shown in the figure from left to right. T cell colonies were measured at the indicated time points (D3 refers to the 3rd day; D7 refers to the 7th day). The indicated trispecific antibodies were tested at indicated 0.2nM, 1nM and 2nM concentrations. Flow cytometry was used to quantify CMV-specific CD8+ T cells (Figure 7 A to Figure 7 B), CMV-specific T cm CD8+ cells (Figure 7C to Figure 7D) and CMV-specific T em CD8+ cells (Fig. 7E to Fig. 7F). In addition, CMV-specific T cells were quantified at the indicated time points. cm (Figure 7G to Figure 7H) and T em(FIG. 7I to FIG. 7J) Percentage of CD8+ cells. All trispecific antibodies tested promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-response manner with varying potency and kinetics.

[0214] Figure 8 A to Figure 8 J show the characterization of the in vitro T cell subset expansion in response to the CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with indicated alternative anti-CD38 binding domains in PBMCs collected from donor C infected by EBV. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (ΔCD38VH1 / ΔCD28sup xΔCD3mid IgG4 FALA). T cell colonies were measured at indicated time points (D3 refers to the 3rd day; D7 refers to the 7th day). The indicated trispecific antibody was tested at indicated 0.2nM and 1nM concentrations. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figure 8 A to Figure 8 B), CMV-specific T cm CD8+ cells (Figure 8C to Figure 8D) and CMV-specific T em CD8+ cells (Fig. 8E to Fig. 8F). In addition, EBV-specific T cells were quantified at the indicated time points. cm (Figure 8G to Figure 8H) and T em (FIG. 8I to FIG. 8J) Percentage of CD8+ cells. All trispecific antibodies tested promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-response manner with varying potency and kinetics.

[0215] Figure 9 A to Figure 12 show the characterization of the in vitro T cell subset expansion in response to the CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with indicated alternative anti-CD38 binding domains in PBMCs collected from donor D infected by EBV. Using the trispecific antibody lacking the CD38VH1 anti-CD38 binding domain as a negative control (ΔCD38VH1 / ΔCD28sup xΔCD3mid IgG4 FALA). T cell colonies were measured at the indicated time points (D3 refers to the 3rd day; D7 refers to the 7th day). The indicated trispecific antibody was tested at indicated 0.2nM and 1nM concentrations. Flow cytometry was used to quantify EBV-specific CD8+T cells (Figure 9 A to Figure 9 B), EBV-specific T cm CD8+ cells (Figure 9C to Figure 9D) and EBV-specific T em CD8+ cells (Fig. 9E to Fig. 9F). In addition, EBV-specific T cells were quantified at the indicated time points. cm(Figures 9G to 10) and T em (Figures 11 to 12) Percentage of CD8+ cells. All trispecific antibodies tested promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with varying potency and kinetics.

[0216] Figures 13A to 13D show the changes in tumor volume (Figure 13A) and body weight (Figure 13B) over time (days) in NSG mice bearing ZR-75-1 tumors transplanted with in vitro expanded human CD3+ T cells. Groups of 10 mice were treated with vehicle or Her2 / CD28 x CD3 trispecific antibody at the indicated doses. Arrows indicate the days of administration. Tumor volume is depicted as mean ± SEM, mm 3 Body weight changes are depicted as % change, mean ± SEM. The X-axis shows the number of days after implantation of ZR-75-1 cells. Tumor volume (mm) of individual mice in each treatment group over time 3 ) are shown in Figure 13C. Tumor weight (mg) for each treatment group is shown in Figure 13D. ** = p < 0.001; *** = p < 0.0003 (two-way ANOVA, control vs. 100 and 10 ug / kg).

[0217] Figures 14A to 14C show the effect of Her2 / CD28 x CD3 trispecific antibody treatment on T cells from whole blood. Figure 14A shows the analysis of hCD45+, CD8+, CD4+ and mCD45+ cells by flow cytometry. Figure 14B shows the effect of control or Her2 / CD28 x CD3 trispecific antibody treatment (at the indicated dose) on hCD45+, CD8+, CD4+ and mCD45+ cell counts. Figure 14C shows the effect of control or Her2 / CD28 x CD3 trispecific antibody treatment (at the indicated dose) on human cell ratios (CD4+ / CD45+ and CD8+ / CD45+). For each x-axis parameter shown in Figures 14B and 14C, the conditions are (from left to right): control, 100ug / kg trispecific antibody, 10ug / kg trispecific antibody, 1ug / kg trispecific antibody and 0.1ug / kg trispecific antibody. The percentages shown in FIG. 14B and FIG. 14C are based on the control sample relative to 100 ug / kg.

[0218] Figure 15 A to Figure 15 C show the effect of Her2 / CD28 x CD3 trispecific antibody treatment on tumor infiltrating lymphocytes (TIL) as examined by immunohistochemistry (IHC). Arrows indicate the tumor infiltrating T cells identified in ZR-75-1 breast tumors. The upper figure is a 1X amplification; the lower figure is a 20X amplification. In two groups of images, staining of human CD45, human CD4 and human CD8 is shown from left to right. Tumors from mice treated with vehicle control (Figure 15 A), 100ug / kg trispecific antibody (Figure 15 B) or 0.1ug / kg trispecific antibody (Figure 15 C) are shown.

[0219] Figures 16A to 16C show quantification of the effect of Her2 / CD28 x CD3 trispecific antibody treatment on TILs as measured by IHC. Each point represents a tumor from a single mouse; rectangles represent group means; and error bars indicate standard deviations. * = p < 0.05, compared to the vehicle control group (ANOVA). The number of CD45+ (Figure 16A), CD4+ (Figure 16B) or CD8+ (Figure 16C) cells is shown. In Figure 16C, due to the presence of excessive nonspecific signals in the CD8 IHC slides, the $ area quantification method was used instead of the cell counting algorithm for CD8+ cells.

[0220] Figures 17A to 17F show in vitro cell lysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by a Her2 / CD28 x CD3 trispecific antibody ("ctl") having a wild-type trastuzumab antigen-binding domain and an anti-CD3 antigen-binding domain without 32 / 35QQ mutations in the VL domain, compared to Her2 / CD28 x CD3 trispecific antibodies with mutations in the VL domains of the anti-HER2 and anti-CD3 arms (numbered as shown in Table 1). Cell killing activity is depicted against cell lines with different HER2 expression: high HER2-expressing HCC1954 (Figure 17A), medium HER2-expressing BT20 (Figure 17C), and low HER2-expressing MDA-MD-231 (Figure 17E). Shown are graphs depicting cell killing against target cells HCC1954 (FIG. 17B), BT20 (FIG. 17D), and MDA-MD-231 (FIG. 17F) as a function of antibody concentration, comparing binding protein #2 to ctl, or comparing binding proteins #1 and #5 to ctl.

[0221] Figures 18A and 18B summarize the mean EC50 (pM) of in vitro cell killing of experimental or control Her2 / CD28 x CD3 trispecific antibodies against the indicated breast cancer (Figure 18A) or gastric cancer cell lines (Figure 18B). The amino acid sequences of the indicated trispecific antibodies are provided in Table 1. DETAILED DESCRIPTION

[0222] The present disclosure provides trispecific and / or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possesses dual variable domains in a cross-shaped orientation.

[0223] General Definition

[0224] Unless otherwise indicated, as used in accordance with this disclosure, the following terms shall be understood to have the following meanings: Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0225] It is to be understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0226] As used herein, term " polynucleotide " refers to a single-stranded or double-stranded nucleic acid polymer having a length of at least 10 nucleotides. In certain embodiments, the nucleotides constituting the polynucleotides can be modified forms of ribonucleotides or deoxyribonucleotides or nucleotides of any type. Such modifications include base modifications (such as bromouridine), ribose modifications (such as cytarabine and 2', 3'-dideoxyribose) and internucleotide connection modifications (such as phosphorothioate, phosphorodithioate, selenophosphate (phosphoroselenoate), diselenphosphate, thioanilide phosphate (phosphoroanilothioate), selenophosphate (phoshoraniladate) and phosphoramidate (phosphoroamidate)). Term " polynucleotide " specifically includes the DNA of single-stranded and double-stranded forms.

[0227] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that: (1) is not associated with all or a portion of a polynucleotide with which it is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0228] An "isolated polypeptide" is a polypeptide that: (1) is free of at least some other polypeptides with which it is normally found, (2) is substantially free of other polypeptides from the same source (e.g., from the same species), (3) is expressed by cells from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other substances with which the isolated polypeptide is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with portions of polypeptides with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or non-covalent interactions) with polypeptides that are not associated with the isolated polypeptide in nature, or (7) does not occur in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA or other RNA, be of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other applications).

[0229] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light" chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity to a target antigen. The amino terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids, which are typically responsible for antigen recognition. The carboxyl terminal portion of each chain typically defines the constant domains responsible for effector function. Therefore, in naturally occurring antibodies, a full-length heavy chain immunoglobulin polypeptide includes one variable domain (V H ) and three constant domains (C H1 、C H2 and C H3 ), wherein the V H The domain is located at the amino terminus of the polypeptide and the C H3 The variable domain is located at the carboxyl terminus, and the full-length light chain immunoglobulin polypeptide includes a variable domain (V L ) and a constant domain (C L ), wherein the V L The domain is located at the amino terminus of the polypeptide and the C L The domain is located at the carboxyl terminus.

[0230] Human light chains are generally classified as kappa and lambda light chains, and human heavy chains are generally classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has multiple subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into multiple subclasses, including but not limited to IgA1 and IgA2. Within the full-length light and heavy chains, the variable domains and constant domains are generally joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, for example, Fundamental Immunology (Paul, W., ed., Raven Press, 2nd edition, 1989), which is incorporated by reference in its entirety for all purposes. The variable region of each light / heavy chain pair generally forms an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same overall structure of relatively conservative framework regions (FRs) engaged by three hypervariable regions (also referred to as complementary determining regions or CDRs). The CDRs from each pair of two chains are typically aligned by the framework regions, which can enable binding to specific epitopes. From amino-terminal to carboxyl-terminal, the light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0231] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined in different ways according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., 1989, Nature 342:877-83) found that, despite significant diversity at the amino acid sequence level, certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have similarities to the Kabat CDRs. Boundaries of CDR Overlap. Other boundaries that define CDRs that overlap with Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the systems herein but still overlap with Kabat CDRs, although such other CDR boundaries may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, but certain embodiments use CDRs defined according to Kabat or Chothia. The use of amino acid sequences to identify predicted CDRs is well known in the art, as described in Martin, AC "Protein sequence and structure analysis of antibody variable domains," In Antibody Engineering, Vol. 2. Kontermann R., Dübel S. eds. Springer-Verlag, Berlin, pp. 33-51 (2010).The amino acid sequence of the heavy chain and / or light chain variable domain can also be examined by other conventional methods (e.g., by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability) to identify the sequence of the CDR. The numbered sequences can be aligned by visual inspection or by using an alignment program (e.g., one of the CLUSTAL program suites) as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular modeling is conveniently used to correctly depict the framework and CDR regions and thereby correct the sequence-based alignment.

[0232] As used herein, the term "Fc" refers to a molecule comprising a sequence of non-antigen binding fragments derived from antibody digestion or produced by other means, the molecule being in monomeric or multimeric form, and the "Fc" may contain a hinge region. Although the original immunoglobulin source of natural Fc is preferably human origin and may be any immunoglobulin, IgG1 and IgG2 are preferred. Fc molecules are composed of monomeric polypeptides that can be linked into dimer or multimeric form by covalent (i.e., disulfide bond) and non-covalent association. The number of intermolecular disulfide bonds between the monomer subunits of natural Fc molecules ranges from 1 to 4, depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of Fc is a disulfide-bonded dimer derived from papain digestion of IgG. As used herein, the term "natural Fc" is universal in monomeric, dimer, and multimeric forms.

[0233] The F(ab) fragment usually contains a light chain and a heavy chain V H and C H1 domain, wherein the V H -C H1 The heavy chain portion is unable to form disulfide bonds with another heavy chain polypeptide. As used herein, a F(ab) fragment may also include a light chain comprising two variable domains separated by an amino acid linker, and a light chain comprising two variable domains separated by an amino acid linker and a C H1 A heavy chain domain.

[0234] The F(ab') fragment usually consists of a light chain and a portion of a heavy chain containing the more constant region (in C H1 with C H2 domains), allowing for the formation of an interchain disulfide bond between the two heavy chains to form a F(ab')2 molecule.

[0235] As used herein, the term "binding protein" refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen. Unless otherwise indicated, the trispecific binding proteins of the present disclosure typically comprise four polypeptide chains that form at least three antigen-binding sites, wherein the first polypeptide chain has a structure represented by the following formula:

[0236] V L2 -L1-V L1 -L2-C L [I]

[0237] And the second polypeptide chain has a structure represented by the following formula:

[0238] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0239] And the third polypeptide chain has a structure represented by the following formula:

[0240] V H3 -C H1 [III]

[0241] And the fourth polypeptide chain has a structure represented by the following formula:

[0242] V L3 -C L [IV]

[0243] in:

[0244] V L1 is the first immunoglobulin light chain variable domain;

[0245] V L2 is the second immunoglobulin light chain variable domain;

[0246] V L3 is the third immunoglobulin light chain variable domain;

[0247] V H1 is the first immunoglobulin heavy chain variable domain;

[0248] V H2 is a second immunoglobulin heavy chain variable domain;

[0249] V H3 is the third immunoglobulin heavy chain variable domain;

[0250] C L is the immunoglobulin light chain constant domain;

[0251] C H1 Immunoglobulin C H1 a heavy chain constant domain; and

[0252] The hinge is connected to the C H1 with C H2 immunoglobulin hinge region of the domain;

[0253] L1, L2, L3, and L4 are amino acid linkers;

[0254] And wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair.

[0255] A "recombinant" molecule is a molecule that has been prepared, expressed, produced, or isolated by recombinant means.

[0256] One embodiment of the present disclosure provides a binding protein with biological and immunological specificity for between one and three target antigens. Another embodiment of the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Another embodiment of the present disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain that forms such a binding protein. Yet another embodiment of the present disclosure provides a host cell that expresses such a binding protein (i.e., comprising a nucleic acid molecule or vector encoding a polypeptide chain that forms such a binding protein).

[0257] As used herein, the term "interchangeability" refers to the interchangeability of variable domains within a binding protein format and while maintaining folding and ultimately binding affinity. "Full interchangeability" refers to the ability to exchange V domains in a polypeptide chain of Formula I or a polypeptide chain of Formula II while maintaining full functionality of the binding protein (as evidenced by maintenance of binding affinity). H1 and V H2 The order of the two domains, and thus exchange V L1 and V L2 The ability to change the order of the domains (i.e., to reverse the order). H and V L It refers only to the position of the domain on a particular protein chain in its final form. For example, V H1 and V H2 Can be derived from V in the parent antibody L1 and V L2 domain and is placed into the V H1 and V H2 Position. Similarly, V L1 and V L2 Can be derived from V in the parent antibody H1 and VH2 domain and is placed in the V H1 and V H2 position. Therefore, V H and V L The designation refers to the current position and not the original position in the parent antibody. H and V L The domains are thus "exchangeable".

[0258] As used herein, the term "antigen" or "target antigen" or "antigen target" refers to a molecule or portion of a molecule that can be bound by a binding protein and that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[0259] The term "Her2" refers to human epidermal growth factor receptor 2, which is a member of the epidermal growth factor receptor family.

[0260] "CD3" is a cluster differentiation factor 3 polypeptide and is a T cell surface protein that is normally part of the T cell receptor (TCR) complex.

[0261] "CD28" is cluster of differentiation 28 polypeptide, and is a T cell surface protein that provides co-stimulatory signals for T cell activation and survival.

[0262] "CD38" is cluster of differentiation 38 polypeptide, and is a glycoprotein found on the surface of many immune cells.

[0263] The term "T cell engager" refers to binding proteins directed against the host's immune system (more specifically, against the cytotoxic activity of T cells) and against tumor target proteins.

[0264] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigenic target.

[0265] The term "monovalent binding protein" refers to a binding protein with one antigen binding site.

[0266] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigenic targets.

[0267] The term "bivalent binding protein" refers to a binding protein with two binding sites.

[0268] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigenic targets.

[0269] The term "trivalent binding protein" refers to a binding protein with three binding sites. In certain embodiments, a trivalent binding protein can bind to one antigenic target. In other embodiments, a trivalent binding protein can bind to two antigenic targets. In other embodiments, a trivalent binding protein can bind to three antigenic targets.

[0270] An "isolated" binding protein is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are substances that may interfere with the diagnostic or therapeutic use of the binding protein and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified to: (1) greater than 95% by weight of antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence as determined by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated binding protein includes the binding protein in situ within recombinant cells, as at least one component of the binding protein's natural environment will not be present.

[0271] As used herein, the terms "substantially pure" or "substantially purified" refer to a compound or species that is the predominant species present (i.e., more abundant on a molar basis than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition in which the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise greater than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In still other embodiments, the species is purified to substantial homogeneity (contaminant species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[0272] The term "epitope" includes any determinant, preferably a polypeptide determinant, that is capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, when the equilibrium dissociation constant is ≤10 -8 M, more preferably when the equilibrium dissociation constant is ≤10- 9 M, and most preferably when the dissociation constant is ≤ 10 -10 When M, the binding protein is said to specifically bind to the antigen.

[0273] The dissociation constant (K) of the binding protein D ) can be determined, for example, by surface plasmon resonance. Typically, surface plasmon resonance analysis measures the real-time binding interaction between a ligand (target antigen on a biosensor matrix) and an analyte (binding protein in solution) by surface plasmon resonance (SPR) using a BIAcore system (Pharmacia Biosensor; Piscataway, New Jersey (NJ)). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). As used herein, the term "K D ” refers to the dissociation constant of the interaction between a specific binding protein and the target antigen.

[0274] As used herein, the term "specifically binds" refers to a binding protein or antigen-binding fragment thereof with a specific binding affinity of at least about 1 x 10 - 6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 The ability to bind to an antigen containing the epitope with a Kd of M or greater, and / or the ability to bind to the epitope with an affinity that is at least two times greater than the affinity of the binding protein or antigen-binding fragment thereof for a nonspecific antigen.

[0275] In some embodiments, the antigen binding domains and / or binding proteins of the present disclosure "cross-react" with human and cynomolgus CD38 polypeptides (e.g., CD38 extracellular domain, human CD38 subtype A, human CD38 subtype E, and cynomolgus CD38). A binding protein that binds to antigen 1 (Ag1) "cross-reacts" with antigen 2 (Ag2) when the EC50 for the two antigens is in a similar range. In this application, a binding protein that binds to Ag1 cross-reacts with Ag2 when the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 20, and the affinities of the two antigens are measured using the same method.

[0276] As used herein, the term "linker" refers to one or more amino acid residues that are inserted between immunoglobulin domains to provide sufficient flexibility for the domains of the light chain and heavy chain to fold into a cross-dual variable region immunoglobulin. At the sequence level, the linker is inserted between the variable domains or between the variable domains and the constant domains. Because the approximate size of the immunoglobulin domains has been fully understood, the transition between the domains can be identified. The exact position of the domain transition can be determined by locating a peptide extension that does not form a secondary structure element (such as a beta sheet or an alpha helix), as confirmed by experimental data or as can be assumed by modeling or secondary structure prediction techniques. The linker described herein is referred to as L1, which is located between the light chain V L2 The C-terminus of the domain is closely related to the V L1 between the N-termini of the light chain V L1 The C-terminus of the domain L The heavy chain linker is called L3 and is located between the N-termini of the V H1 The C-terminus of the domain is closely related to the V H2 domain; and L4, which is located between the N-termini of the V H2 The C-terminus of the domain H1 between the N-termini of the domain.

[0277] As used herein, the term "vector" refers to any molecule (for example, nucleic acid, plasmid or virus) for transferring coding information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which another DNA segment can be inserted. Another type of vector is a viral vector, in which another DNA segment can be inserted into a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of the gene operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, the expression vectors used in recombinant DNA technology are typically in plasmid form. The terms "plasmid" and "vector" can be used interchangeably herein because plasmids are the most commonly used vector forms. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0278] As used herein, the phrase "recombinant host cell" (or "host cell") refers to a cell into which a recombinant expression vector has been introduced. Recombinant host cell or host cell is intended to refer not only to a specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may actually be different from the parental cell, but such cells are still included within the scope of the term "host cell" as used herein. Numerous host cell expression systems can be used to express the binding protein, including bacteria, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. In order to recombinantly express the binding protein, host cells are transformed or transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the binding protein polypeptide chain, such that the polypeptide chain is expressed in the host cell and preferably secreted into the culture medium in which the host cell is cultured, from which the binding protein can be recovered.

[0279] As used herein, the term "conversion" refers to the change of the genetic characteristics of a cell, and when a cell is modified and contains new DNA, the cell has been transformed. For example, if a cell is genetically modified from its native state, the cell is transformed. After conversion, the transforming DNA can be physically integrated into the chromosome of the cell and recombined with the DNA of the cell, or can be maintained as an additional element for a short period of time without being replicated, or can be replicated independently as a plasmid. When DNA is replicated along with cell division, it is believed that the cell has been stably transformed. As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA by a cell, and when exogenous DNA has been introduced into the cell membrane, the cell has been "transfected". A variety of transfection techniques are well known in the art. Such technology can be used to introduce one or more exogenous DNA molecules into suitable host cells.

[0280] As used herein and as applied to an object, the term "naturally occurring" refers to the fact that the object can be found in nature and has not been manipulated by humans. For example, a polynucleotide or polypeptide present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by humans is naturally occurring. Similarly, as used herein, "non-naturally occurring" refers to an object that is not found in nature or has been structurally modified or synthesized by humans.

[0281] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty conventional amino acids (e.g., d-amino acids); unnatural amino acids and analogs (such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unconventional amino acids) can also be suitable components of binding protein polypeptide chains. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, according to standard usage and convention, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction.

[0282] Naturally occurring residues can be divided into several classes based on common side-chain properties:

[0283] (1) Hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

[0284] (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

[0285] (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro;

[0286] (4) Aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;

[0287] (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0288] (6) Acidic: Asp, Glu;

[0289] (7) Basic: His, Lys, Arg;

[0290] (8) Residues that affect chain orientation: Gly, Pro;

[0291] (9) Aromatic: His, Trp, Tyr, Phe; and

[0292] (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0293] Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Non-conservative substitutions may involve exchanging a member of one of these classes for a member of another class.

[0294] The skilled person will be able to determine suitable variants of the binding protein polypeptide chain using well-known techniques. For example, one skilled in the art can identify suitable regions in the polypeptide chain that can be altered without destroying the activity by targeting regions that are thought to be unimportant for activity. Alternatively, one skilled in the art can identify residues and portions in the molecule that are conserved between similar polypeptides. Additionally, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0295] As used herein, the term "patient" includes both human and animal subjects.

[0296] As used herein, the term "treatment" or "treat" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those with the disorder and those susceptible to the disorder or those for which the disorder is to be prevented. In specific embodiments, the binding protein can be used to treat a person suffering from cancer or a person susceptible to cancer, or to improve cancer in a human subject. The binding protein can also be used to prevent cancer in a human patient. In specific embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, germinal center B cell lymphoma, or B cell acute lymphoblastic leukemia. In other embodiments, the binding protein can be used to treat a person suffering from an inflammatory disorder or a person susceptible to an inflammatory disorder, or to improve an inflammatory disorder in a human subject.

[0297] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0298] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

[0299] The terms "effective amount" and "therapeutically effective amount" when used with respect to a pharmaceutical composition comprising one or more binding proteins refer to an amount or dosage sufficient to produce the desired therapeutic result. More specifically, a therapeutically effective amount is an amount of binding protein sufficient to inhibit one or more clinically defined pathological processes associated with the condition being treated for a period of time. The effective amount can vary depending on the specific binding protein used and also depends on a variety of factors and circumstances related to the patient being treated and the severity of the disorder. For example, if the binding protein is to be administered in vivo, factors to be considered would include factors such as the patient's age, weight, and health status, as well as dose-response curves and toxicity data obtained in preclinical animal work. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is within the skill of those skilled in the art.

[0300] One embodiment of the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0301] Trispecific and / or trivalent binding proteins

[0302] Certain aspects of the present disclosure relate to trispecific and / or trivalent binding proteins comprising four polypeptide chains forming three antigen binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein possesses dual variable domains in a cross-orientation and wherein a second pair of polypeptides forming the binding protein possesses a single variable domain. Any CDR or variable domain of any antigen binding protein described herein can be used in the trispecific binding proteins of the present disclosure.

[0303] In some embodiments, each of the three antigen binding sites binds to a different target (e.g., a polypeptide antigen). In some embodiments, the trispecific binding protein comprises four polypeptide chains forming the three antigen binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula:

[0304] V L2 -L1-V L1 -L2-C L [I]

[0305] And the second polypeptide chain comprises a structure represented by the following formula:

[0306] V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II]

[0307] And the third polypeptide chain comprises a structure represented by the following formula:

[0308] V H3 -C H1 -Hinge-C H2 -C H3 [III]

[0309] And the fourth polypeptide chain comprises a structure represented by the following formula:

[0310] V L3 -C L [IV]

[0311] in:

[0312] V L1 is the first immunoglobulin light chain variable domain;

[0313] V L2 is the second immunoglobulin light chain variable domain;

[0314] V L3 is the third immunoglobulin light chain variable domain;

[0315] V H1 is the first immunoglobulin heavy chain variable domain;

[0316] V H2 is a second immunoglobulin heavy chain variable domain;

[0317] V H3 is the third immunoglobulin heavy chain variable domain;

[0318] C L is the immunoglobulin light chain constant domain;

[0319] C H1 Immunoglobulin C H1 heavy chain constant domain;

[0320] C H2 Immunoglobulin C H2 heavy chain constant domain;

[0321] C H3 Immunoglobulin C H3 heavy chain constant domain;

[0322] The hinge is connected to the C H1 with C H2 an immunoglobulin hinge region of a domain; and

[0323] L1, L2, L3, and L4 are amino acid linkers;

[0324] wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair.

[0325] It is contemplated that any antigen binding site described herein can be used in a trispecific binding protein of the present disclosure, for example, comprising four polypeptide chains having the above-described structure. For example, in some embodiments, a trispecific binding protein of the present disclosure comprises a V forming a first antigen binding site. H1 and V L1 Domain pair, V domain that forms a second antigen binding site that binds to CD3 polypeptide H2 and V L2 Domain pair and V forming the third antigen binding site H3 and V L3 Domain pairs. In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain that forms a first antigen binding site that binds to a CD28 polypeptide. H1 and V L1 Domain pair, V domain that forms a second antigen binding site that binds to CD3 polypeptide H2 and V L2 Domain pair and V forming the third antigen binding site H3 and V L3 In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain that forms a first antigen binding site. H1 and V L1 Domain pair, V domain that forms a second antigen binding site that binds to CD3 polypeptide H2 and V L2 Domain pair and V that forms a third antigen binding site that binds to tumor target proteins H3 and V L3 Domain pairs. In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain that forms a first antigen binding site that binds to a CD28 polypeptide. H1 and V L1 Domain pair, V domain that forms a second antigen binding site that binds to CD3 polypeptide H2 and V L2 Domain pair and V that forms a third antigen binding site that binds to tumor target proteins H3 and V L3 Domain pairs. In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain that forms a first antigen binding site that binds to a CD28 polypeptide. H1 and V L1 Domain pair, V domain that forms a second antigen binding site that binds to CD3 polypeptide H2 and V L2 The domain pair and the V domain that form the third antigen binding site that binds to the CD38 polypeptide H3 and V L3Domain pairs. In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain that forms a first antigen binding site that binds to a CD28 polypeptide. H1 and V L1 Domain pair, V domain that forms a second antigen binding site that binds to CD3 polypeptide H2 and V L2 The domain pair and the V domain that forms the third antigen binding site that binds to the HER2 polypeptide H3 and V L3 Domain pairs.

[0326] In some embodiments, the binding protein of the present disclosure binds to one or more tumor target proteins and one or more T cell target proteins. In some embodiments, the binding protein is capable of specifically binding to two different epitopes on a tumor target protein and a single T cell target protein. In some embodiments, the binding protein is capable of specifically binding to a tumor target protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the first and second polypeptide chains of the binding protein form two antigen binding sites that specifically target two T cell target proteins, and the third and fourth polypeptide chains of the binding protein form antigen binding sites that specifically bind to tumor target proteins. In some embodiments, the target protein is CD38 or HER2. Additional tumor target proteins are provided below. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28. Exemplary and non-limiting polypeptides that can be used in any trispecific binding protein described herein are provided in Table 1.

[0327] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 157; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 158; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 159.

[0328] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 162; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 163.

[0329] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 164; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 165; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 166; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 167.

[0330] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 168; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 171.

[0331] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 172; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 173; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 174; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 175.

[0332] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 176; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 177; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 178; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179.

[0333] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 181; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 182; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 183; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 184.

[0334] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 186; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 188.

[0335] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103.

[0336] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 107.

[0337] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 113; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.

[0338] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119.

[0339] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123.

[0340] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127.

[0341] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131.

[0342] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135.

[0343] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.

[0344] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143.

[0345] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147.

[0346] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151.

[0347] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155.

[0348] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289.

[0349] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293.

[0350] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297.

[0351] In some embodiments, the binding protein of the present disclosure comprises four polypeptide chains that form three antigen binding sites, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.

[0352] Anti-CD38 binding site

[0353] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a CD38 polypeptide. In some embodiments, the CD38 polypeptide is a human CD38 polypeptide, also known as ADPRC1. Human CD38 polypeptides are known in the art and include, but are not limited to, polypeptides represented by NCBI accession number NP_001766.2 or polypeptides produced from NCBI gene ID number 952. In some embodiments, the antigen binding site binds to a human CD38 polypeptide, a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide, or a human CD38 polypeptide and a non-human primate (e.g., cynomolgus monkey) CD38 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD38 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent.

[0354] In some embodiments, any of the CDRs and / or variable domains of the anti-CD38 binding sites described below can be used in a monospecific antibody.

[0355] In other embodiments, any of the CDRs and / or variable domains of the anti-CD38 binding sites described below can be used in any binding site of a trispecific binding protein (e.g., as described above) comprising four polypeptides forming three antigen binding sites. In certain embodiments, the binding protein comprising an antigen binding site that binds a CD38 polypeptide is a trispecific binding protein as described above comprising four polypeptides forming three antigen binding sites, wherein V H3 and V L3 The domains pair and form a third antigen-binding site that binds to the CD38 polypeptide.

[0356] Various features of exemplary binding sites and binding proteins are described herein. For example, in some embodiments, the anti-CD38 binding site cross-reacts with human CD38 (e.g., human CD38 subtype A and / or subtype E polypeptides) and cynomolgus monkey CD38. In some embodiments, the binding protein comprising the anti-CD38 binding site induces apoptosis of CD38+ cells. In some embodiments, the binding protein comprising the anti-CD38 binding site recruits T cells to CD38+ cells and optionally activates T cells (e.g., by TCR stimulation and / or co-stimulation).

[0357] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18).

[0358] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24). In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO: 20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO: 21); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO: 22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO: 24).

[0359] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30). In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30).

[0360] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36). In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHSNGNTY (SEQ ID NO:34), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:35), and a CDR-L3 sequence comprising the amino acid sequence of SQSTHVPLT (SEQ ID NO:36).

[0361] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO: 37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO: 38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO: 39); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO: 40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO: 41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO: 42). In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGISSY (SEQ ID NO:40), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:41), and a CDR-L3 sequence comprising the amino acid sequence of QQLNSFPYT (SEQ ID NO:42).

[0362] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48). In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QGIRND (SEQ ID NO:46), a CDR-L2 sequence comprising the amino acid sequence of AAS (SEQ ID NO:47), and a CDR-L3 sequence comprising the amino acid sequence of LQDYIYYPT (SEQ ID NO:48).

[0363] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO:79) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:79); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence of DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:79) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 79; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 79; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 80.

[0364] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO:81), an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO:82); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO:83); In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 81; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 81; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 82.

[0365] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO:83); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:83); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:83; In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 83; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 84. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 83; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 84.

[0366] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO:87), an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:87; and / or an antibody light chain variable (VL) domain comprising ... In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 87; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 87; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 88.

[0367] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO:89) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:89); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 89; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 89; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 90.

[0368] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO:85), an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:87); In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 85; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 85; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 86.

[0369] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWGQGTTLTVSS (SEQ ID NO:277); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (SEQ ID NO:278); In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 277; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 278. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 277; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 278. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 277; and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 278. In some embodiments, the VH domain and / or the VL domain are humanized.

[0370] In some embodiments, the binding site that binds CD38 comprises: an antibody heavy chain variable (VH) domain comprising the sequence of QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGFYYSMDYWGQGTSVTVSS (SEQ ID NO:279) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPK LLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPL TFGSGTQLEIK (SEQ ID NO:279); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence of DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPK LLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPL TFGSGTQLEIK (SEQ ID NO: In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 279; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 280. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 279; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 280. In some embodiments, the binding site that binds to CD38 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 279; and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 280. In some embodiments, the VH domain and / or the VL domain are humanized.

[0371] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen binding site that binds to a CD38 polypeptide, an antigen binding site that binds to a CD28 polypeptide, and an antigen binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides containing three antigen binding sites, wherein the polypeptide of Formula I and the polypeptide of Formula II form a cross-light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD38 antigen binding sites described above represent V H3 and V L3 and forms a third antigen binding site that binds to the CD38 polypeptide. H1 and V L1 Forming the first antigen binding site that binds to CD28 polypeptide, V H2 and V L2 The second antigen binding site that binds to the CD3 polypeptide is formed, and the VH and VL domains of any anti-CD38 antigen binding site described above and / or in Table 2 represent the V H3 and V L3 And form a third antigen binding site that binds to the CD38 polypeptide.

[0372] Sequences of exemplary anti-CD38 antigen binding sites are provided in Table 2. In some embodiments, the binding proteins of the present disclosure comprising an anti-CD38 antigen binding site comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-CD38 antibodies described in Table 2. In some embodiments, the binding proteins of the present disclosure comprising an anti-CD38 antigen binding site comprise a VH domain sequence and / or a VL domain sequence of the anti-CD38 antibodies described in Table 2.

[0373] Table 2. Anti-CD38 binding protein sequences.

[0374]

[0375]

[0376]

[0377]

[0378]

[0379] Further provided herein are antibodies (eg, monospecific antibodies) comprising any of the anti-CD38 CDRs and / or variable domains described above.

[0380] In some embodiments, the binding proteins of the present disclosure comprise an antigen binding site that binds to the extracellular domain of a human CD38 polypeptide and the extracellular domain of a cynomolgus monkey CD38 polypeptide. Exemplary assays for determining whether an antigen binding site binds an antigen are described herein and are known in the art, including, but not limited to, ELISA, SPR, and flow cytometry assays.

[0381] Anti-HER2 binding sites

[0382] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a HER2 polypeptide. In some embodiments, the HER2 polypeptide is a human HER2 polypeptide, also known as NEU, NGL, ERBB2, TKR1, CD340, HER-2, MLN19, and HER-2 / neu. Human HER2 polypeptides are known in the art and include, but are not limited to, polypeptides represented by NCBI accession numbers XP_024306411.1, XP_024306410.1, XP_024306409.1, NP_001276867.1, NP_001276866.1, NP_001276865.1, NP_001005862.1, or NP_004439.2, or polypeptides generated from NCBI gene ID number 2064. In some embodiments, the binding protein comprising an antigen binding site that binds to a HER2 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, the binding protein comprising an antigen binding site that binds to a HER2 polypeptide is a trispecific binding protein as described above comprising four polypeptides forming three antigen binding sites, wherein V H3 and V L3 The domains pair to form a third antigen binding site that binds the HER2 polypeptide.

[0383] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11). NO: 11) and a CDR-L2 sequence containing the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), SRWGGEGFYAMDY (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11). The CDR-L2 sequence comprises the amino acid sequence of SEQ ID NO: 11) and the CDR-L3 sequence comprises the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0384] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0385] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0386] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0387] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0388] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0389] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0390] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0391] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0392] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO: 8); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0393] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO: 5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO: 7); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12).

[0394] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the following sequences: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), NO:73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS(SEQ ID NO:74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS(SEQID NO:75) or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS(SEQ ID an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence ofand / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76; and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78.

[0395] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a residue corresponding to EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:72) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:72); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

[0396] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a residue corresponding to EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:73) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:73); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 73; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 73; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

[0397] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a residue corresponding to EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:75) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:75); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 75; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 75; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

[0398] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a residue corresponding to EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO:74) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:74); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 74; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 74; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

[0399] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a residue corresponding to EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:76) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:76); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 76; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 76; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 77.

[0400] In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising a residue corresponding to EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO:72) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:72); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the binding site that binds to HER2 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 78.

[0401] In some embodiments, the anti-HER2 antigen binding site of the present disclosure comprises 1, 2, 3, 4, 5 or all 6 CDR sequences of the anti-HER2 antibody trastuzumab, 30R / 55Q / 102E, 30R / 56A / 102S, 30R / 55Q / 102S, 30R / 56A / 102E or 30Q. In some embodiments, the anti-HER2 antigen binding site of the present disclosure comprises the VH domain sequence and / or VL domain sequence of the anti-HER2 antibody trastuzumab, 30R / 55Q / 102E, 30R / 56A / 102S, 30R / 55Q / 102S, 30R / 56A / 102E or 30Q.

[0402] Sequences of exemplary anti-HER2 antigen binding sites are provided in Table 3. In some embodiments, the anti-HER2 antigen binding sites of the present disclosure comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-HER2 antibodies described in Table 3. In some embodiments, the anti-HER2 antigen binding sites of the present disclosure comprise the VH domain sequences and / or VL domain sequences of the anti-HER2 antibodies described in Table 3.

[0403] Table 3. Anti-HER2 binding protein sequences.

[0404]

[0405]

[0406]

[0407] Other anti-tumor target binding sites

[0408] In some embodiments, the binding protein of the present disclosure comprises an antigen binding site that binds to a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, the tumor target proteins of the present disclosure include, but are not limited to, A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as MCP-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2 / eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2,Receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin White), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, ​​FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1 R, IL2Rβ, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as the receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as the receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as the co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments,One or more of the above antigenic targets are human antigenic targets.

[0409] Anti-CD28 binding site

[0410] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds a CD28 polypeptide. In some embodiments, the CD28 polypeptide is a human CD28 polypeptide, also known as Tp44. Human CD28 polypeptides are known in the art and include, but are not limited to, polypeptides represented by NCBI Accession Nos. XP_011510499.1, XP_011510497.1, XP_011510496.1, NP_001230007.1, NP_001230006.1, or NP_006130.1, or polypeptides derived from NCBI Gene ID No. 940. In some embodiments, the binding protein comprising an antigen binding site that binds a CD28 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, the binding protein comprising an antigen binding site that binds a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD28 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide and one antigen binding site binds to a CD3 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide, one antigen binding site binds to a CD3 polypeptide, and one antigen binding site binds to a CD38 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide, one antigen binding site binds to a CD3 polypeptide, and one antigen binding site binds to a HER2 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide, one antigen binding site binds to a CD3 polypeptide, and one antigen binding site binds to a tumor target protein.

[0411] In some embodiments, the binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54). In some embodiments, the binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY (SEQ ID NO:54).

[0412] In some embodiments, the binding site that binds CD28 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO:91) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:91); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ...) In some embodiments, the binding site that binds to CD28 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 91; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 92. In some embodiments, the binding site that binds to CD28 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 91; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 92.

[0413] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen binding site that binds to a tumor target protein (including but not limited to CD38 or HER2), an antigen binding site that binds to a CD28 polypeptide, and an antigen binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides containing three antigen binding sites, wherein the polypeptide of Formula I and the polypeptide of Formula II form a cross-light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any anti-CD28 antigen binding site described above represent V H1and V L1 and form a first antigen binding site that binds to the CD28 polypeptide. In some embodiments, the VH and VL domains of any anti-CD28 antigen binding site described above and / or in Table 4 represent V H1 and V L1 And form the first antigen binding site that binds to CD28 polypeptide, V H2 and V L2 Forming a second antigen binding site that binds to the CD3 polypeptide, and V H3 and V L3 A third antigen binding site is formed that binds to a tumor target protein, including but not limited to CD38 or HER2.

[0414] Sequences of exemplary anti-CD28 antigen binding sites are provided in Table 4. In some embodiments, the anti-CD28 antigen binding sites of the present disclosure comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-CD28 antibodies described in Table 4. In some embodiments, the anti-CD28 antigen binding sites of the present disclosure comprise the VH domain sequences and / or VL domain sequences of the anti-CD28 antibodies described in Table 4.

[0415] Table 4. Anti-CD28 binding protein sequences.

[0416]

[0417] Anti-CD3 binding site

[0418] Certain aspects of the present disclosure relate to binding proteins comprising an antigen binding site that binds to a CD3 polypeptide. In some embodiments, the CD3 polypeptide is a human CD3 polypeptide, including CD3-δ (delta) (also known as T3D, IMD19, and CD3-δ (DELTA)), CD3-ε (also known as T3E, IMD18, and TCRE), and CD3-γ (gamma) (also known as T3G, IMD17, and CD3-γ (GAMMA)). Human CD3 polypeptides are known in the art and include, but are not limited to, polypeptides represented by NCBI Accession No. XP_006510029.1 or NP_031674.1 or polypeptides generated from NCBI Gene ID No. 915, 916, or 917. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or multispecific and / or multivalent. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide and one antigen binding site binds to a CD3 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide, one antigen binding site binds to a CD3 polypeptide, and one antigen binding site binds to a CD38 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds to a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds to a CD28 polypeptide, one antigen binding site binds to a CD3 polypeptide, and one antigen binding site binds to a HER2 polypeptide. In some embodiments, the binding protein comprising an antigen binding site that binds a CD3 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen binding sites, wherein one antigen binding site binds a CD28 polypeptide, one antigen binding site binds a CD3 polypeptide, and one antigen binding site binds a tumor target protein.

[0419] In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F, a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the V L2 The CDR-L1 sequence of the domain comprises an amino acid sequence selected from the group consisting of: QSLVHQNAQTY (SEQ ID NO: 59), QSLVHENLQTY (SEQ ID NO: 60), QSLVHENLFTY (SEQ ID NO: 61), and QSLVHENLRTY (SEQ ID NO: 62).

[0420] In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO: 59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0421] In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO: 60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0422] In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLFTY (SEQ ID NO: 61), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0423] In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and / or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65). In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO: 56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO: 57); and an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLRTY (SEQ ID NO: 62), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO: 65).

[0424] In some embodiments, the binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:93); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: NO:95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO:96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO:97) and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK(SEQ ID NO:98) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence ofIn some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and an antibody light chain variable (VL) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0425] In some embodiments, the binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:93); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95.

[0426] In some embodiments, the binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:93); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 96. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 96.

[0427] In some embodiments, the binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:93); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 97.

[0428] In some embodiments, the binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:93) is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO:93); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that ... In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 93; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 98.

[0429] In some embodiments, the binding site that binds CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO:302); and / or an antibody light chain variable (VL) domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the binding site that binds to CD3 comprises: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 302; and / or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95.

[0430] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein comprises an antigen binding site that binds to a tumor target protein (including but not limited to CD38 or HER2), an antigen binding site that binds to a CD28 polypeptide, and an antigen binding site that binds to a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides containing three antigen binding sites, wherein the polypeptide of Formula I and the polypeptide of Formula II form a cross-light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any anti-CD3 antigen binding site described above represent VH2 and V L2 and forms a second antigen binding site that binds to the CD3 polypeptide. H1 and V L1 Forming the first antigen binding site that binds to the CD28 polypeptide, the VH and VL domains of any anti-CD3 antigen binding site described above and / or in Table 5 represent V H2 and V L2 and forms a second antigen binding site that binds to the CD3 polypeptide, and V H3 and V L3 A third antigen binding site is formed that binds to a tumor target protein, including but not limited to CD38 or HER2.

[0431] Sequences of exemplary anti-CD3 antigen binding sites are provided in Table 5. In some embodiments, the anti-CD3 antigen binding sites of the present disclosure comprise 1, 2, 3, 4, 5, or all 6 CDR sequences of the anti-CD3 antibodies described in Table 5. In some embodiments, the anti-CD3 antigen binding sites of the present disclosure comprise the VH domain sequences and / or VL domain sequences of the anti-CD3 antibodies described in Table 5.

[0432] Table 5. Anti-CD3 binding protein sequences.

[0433]

[0434]

[0435]

[0436] connector

[0437] In some embodiments, linkers L1, L2, L3, and L4 are in the range of no amino acids (length = 0) to about 100 amino acids in length, or less than 100, 50, 40, 30, 20, or 15 amino acids or less. Linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. L1, L2, L3, and L4 in a binding protein can all have the same amino acid sequence or can all have different amino acid sequences.

[0438] Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), GGSGSSGSGG (SEQ ID NO: 71), and DKTHT (SEQ ID NO: 66), as well as those disclosed in International Publication Nos. WO 2017 / 074878 and WO 2017 / 180913. The examples listed above are not intended to limit the scope of the present disclosure in any way, and linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, glycine, and proline have been shown to be suitable in binding proteins.

[0439] The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structure element that needs to be achieved in the linker. For example, glycine, serine, and alanine are optimal for linkers with maximum flexibility. If a more rigid and extended linker is desired, some combination of glycine, proline, threonine, and serine can be used. Depending on the desired properties, any amino acid residue can be considered as a linker in combination with other amino acid residues, to construct a larger peptide linker, if necessary.

[0440] In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3, and the length of L2 is at least twice the length of L4. In some embodiments, L1 is 3 to 12 amino acid residues in length, L2 is 3 to 14 amino acid residues in length, L3 is 1 to 8 amino acid residues in length, and L4 is 1 to 3 amino acid residues in length. In some embodiments, L1 is 5 to 10 amino acid residues in length, L2 is 5 to 8 amino acid residues in length, L3 is 1 to 5 amino acid residues in length, and L4 is 1 to 2 amino acid residues in length. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length.

[0441] In some embodiments, L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO: 68), L3 comprises the sequence S, and L4 comprises the sequence RT.

[0442] In some embodiments, at least one of L1, L2, L3, or L4 comprises the sequence DKTHT (SEQ ID NO: 66). In some embodiments, L1, L2, L3, and L4 comprise the sequence DKTHT (SEQ ID NO: 66).

[0443] Fc region and constant domains

[0444] In some embodiments, the binding protein of the present disclosure further comprises H1 In some embodiments, the binding protein of the present disclosure comprises a second polypeptide chain further comprising an Fc region connected to a C H1 In some embodiments, the binding protein of the present disclosure comprises a third polypeptide chain further comprising an Fc region connected to the immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains. H1 The second polypeptide chain of the Fc region connected to the Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 immunoglobulin heavy chain constant domain; and further comprising H1 A third polypeptide chain of the Fc region connected to the immunoglobulin hinge region and the C H2 and C H3 Immunoglobulin heavy chain constant domain.

[0445] In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or a polypeptide chain containing an Fc region. In some embodiments, the Fc region is a human Fc region, for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region includes the antibody hinge, C H1 、C H2 、C H3 and optional C H4 Domain. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more mutations described below. In some embodiments, the Fc region is an Fc region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding protein shown in Table 4. In some embodiments, the heavy chain constant region is a constant region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding protein shown in Table 4. In some embodiments, the light chain constant region is a constant region of one of the light chain polypeptides (e.g., polypeptide 1 or 4) of the binding protein shown in Table 4.

[0446] In some embodiments, the binding proteins of the present disclosure include one or two Fc variants. As used herein, the term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" may include a molecule or sequence that is humanized from a non-human native Fc. In addition, a native Fc contains regions that may be removed because they provide structural features or biological activities that are not required by the antibody-like binding proteins of the present invention. Thus, the term "Fc variant" includes a molecule or sequence that lacks one or more native Fc sites or residues or in which one or more Fc sites or residues have been modified, which sites or residues affect or participate in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0447] In some embodiments, a binding protein of the present disclosure (e.g., a trispecific binding protein) comprises a "knob" mutation on the second polypeptide chain and a "hole" mutation on the third polypeptide chain. In some embodiments, a binding protein of the present disclosure comprises a "knob" mutation on the third polypeptide chain and a "hole" mutation on the second polypeptide chain. In some embodiments, the "knob" mutation comprises one or more substitutions at positions corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "knob" mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the "hole" mutation comprises one or more substitutions at positions corresponding to positions 407 and optionally 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y407V or Y407T and optionally Y349C, T366S and / or L368A. In some embodiments, the "hole" mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is Y349C, T366S, L368A, and Y407V.

[0448] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises one or more amino acid substitutions at a position corresponding to human IgG1 or IgG4, and optionally 354, according to the EU index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises one or more amino acid substitutions at a position corresponding to human IgG1 or IgG4, and optionally 349, 366 and / or 368, according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and / or L368A.

[0449] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises one or more amino acid substitutions at positions corresponding to human IgG1 or IgG4, and optionally 349, 366 and / or 368, according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and optionally Y349C, T366S and / or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises one or more amino acid substitutions at positions corresponding to human IgG1 or IgG4, and optionally 354, according to the EU index, wherein the amino acid substitutions are T366W or T366Y and optionally S354C.

[0450] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises one or more amino acid substitutions at positions corresponding to positions 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366S, L368A and / or Y407V.

[0451] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises one or more amino acid substitutions at positions corresponding to positions 366, 368 and / or 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366S, L368A and / or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W.

[0452] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, the first Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to human IgG1 or IgG4 according to the EU index at positions 349, 366, 368, and 407, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, the second Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to human IgG1 or IgG4 according to the EU index at positions 354 and 366, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and / or second Fc region is a human IgG1 Fc region. In some embodiments, the first and / or second Fc region is a human IgG4 Fc region.

[0453] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region, wherein the human IgG4 Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region, wherein the human IgG4 The Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region, wherein the human IgG4 The Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, S354C, T366W, and R409K.

[0454] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region, wherein the human IgG4 Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region, wherein the human IgG4 The Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368 and 407 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region, wherein the human IgG4 The Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 234, 235, 354, and 366 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A, L235A, S354C, and T366W.

[0455] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to reduce effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a C H1 The first Fc region of the immunoglobulin comprises an immunoglobulin hinge region and a C H2 and CH3 Immunoglobulin heavy chain constant domain; wherein the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the first and the second Fc region are human IgG1 Fc regions; and wherein the first and the second Fc region each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc region of the second and the third polypeptide chains are human IgG1 Fc regions, and wherein the Fc region each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A. H1 The first Fc region of the immunoglobulin comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the third polypeptide chain further comprises a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the first and the second Fc region are human IgG1 Fc regions; and wherein the first and the second Fc region each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc region of the second and the third polypeptide chain is a human IgG1 Fc region, and wherein the Fc region each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc region of the second and the third polypeptide chain is a human IgG4 Fc region, and wherein the Fc region each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain, which further comprises a C H1 The first Fc region of the immunoglobulin comprises an immunoglobulin hinge region and a C H2 and C H3An immunoglobulin heavy chain constant domain; and a third polypeptide chain, said third polypeptide chain further comprising a C H1 The second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 An immunoglobulin heavy chain constant domain; and wherein the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A.

[0456] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region, wherein the human IgG4 The Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CHI, wherein the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K; and wherein the third polypeptide chain further comprises a second Fc region linked to CHI, wherein the second Fc region is a human IgG4 Fc region, wherein the human IgG4 The Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A, S354C, T366W and R409K.

[0457] In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising one or more mutations that reduce or eliminate FcγI and / or FcγII binding but do not affect FcRn binding. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are F234A and / or L235A. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S228P, F234A, L235A, and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index. In some embodiments, the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the Fc region is a human IgG4 Fc region comprising amino acid mutations at substitutions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutation is S228P; E233P, F234V, L235A, and deletion at 236; and / or R409K.

[0458] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate Fc receptor binding and / or effector functions (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)) of the Fc region.

[0459] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0460] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations to improve the stability of the hinge region and / or dimer interface of, for example, IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288: 26583-26593). In some embodiments, the mutations comprise substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein further comprises a C H1 A second polypeptide chain of a first Fc region connected to the first Fc region, wherein the first Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain, and further comprising H1 A third polypeptide chain of a second Fc region connected thereto, wherein the second Fc region comprises an immunoglobulin hinge region and a C H2 and C H3 Immunoglobulin heavy chain constant domain; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise an amino acid substitution at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding proteins of the present disclosure comprise knob and hole mutations and one or more mutations for improving stability. In some embodiments, the first and / or second Fc regions are human IgG4 Fc regions.

[0461] In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 234, 235, and / or 329 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are L234A, L235A, and / or P329A. In some embodiments, the Fc region is a human IgG1 Fc region comprising one or more amino acid substitutions at positions corresponding to positions 298, 299, and / or 300 of human IgG1 according to the EU index. In some embodiments, the amino acid substitutions are S298N, T299A, and / or Y300S.

[0462] Nucleic Acids

[0463] Other aspects of the present disclosure relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.

[0464] Other aspects of the disclosure relate to kits of polynucleotides, eg, encoding one or more polypeptides of a binding protein as described herein. In some embodiments, the kit of polynucleotides of the present disclosure comprises one, two, three or four polynucleotides of the kit of polynucleotides, the polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 200; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 200; (e) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 205, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 206, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 207, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 208; (f) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 209, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 210, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 211, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 212; (g) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 213, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 214, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 215, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: A fourth polynucleotide of the polynucleotide sequence of NO:216;(h) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 217, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 218, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 219, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 220; (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 221, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 222, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 223, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 224; (j) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 225, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 226, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 227, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 228; (k) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 229, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 230, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 231, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 233, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 234, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 235, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 236; (ii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 237, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 238, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 239, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 240; (iii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 241, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 242, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 243, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 244; (iv) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 246, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 247, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 248. a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 247 and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 248;(p) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 249, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 250, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 251, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 252; (q) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 253, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 254, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 255, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 256; (r) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 257, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 258, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 259, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 260; (s) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 261, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 262, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 263, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 264. (i) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 265, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 266, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 267, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 268; (ii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 269, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 270, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 271, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 272; or (iii) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 273, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 274, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 275, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 276.

[0465] Other aspects of the present disclosure relate to vector systems comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any binding protein described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein, as shown in the polynucleotides in Table 6. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system can be any vector described herein. In some embodiments, the one or more vectors are expression vectors. In some embodiments, the first, second, third, and fourth polynucleotides are present on one or more expression vectors, eg, one, two, three, or four expression vectors.

[0466] Standard recombinant DNA methods are used to construct polynucleotides encoding protein-binding polypeptides, these polynucleotides are incorporated into recombinant expression vectors, and such vectors are introduced into host cells. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd edition). Enzyme reactions and purification techniques can be carried out according to the manufacturer's instructions, as commonly achieved in the art or as described herein. Unless specifically defined, the terms used for analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein and the laboratory procedures and techniques for analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein are well known in the art and commonly used. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, drug preparation, preparation, delivery, and the treatment of patients.

[0467] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct the transcription of a nucleic acid sequence encoding a protein-binding protein. A promoter can refer to a nucleic acid control sequence that directs the transcription of a nucleic acid. When a first nucleic acid sequence is placed in a position that has a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence of the protein-binding protein. Examples of promoters can include, but are not limited to, promoters obtained from the genomes of viruses (such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), heterologous eukaryotic promoters (such as actin promoter, immunoglobulin promoter, heat shock promoter, etc.), CAG promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK) promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33:e43, 2005), lac system, trp system, tac system, trc system, the major operator and promoter region of bacteriophage λ, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast α mating factor. The polynucleotide encoding the binding protein of the present disclosure may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein, or other suitable promoters readily identified by those skilled in the art.

[0468] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. The expression vector may include one or more regulatory sequences operably connected to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Suitable enhancer examples may include, but are not limited to, enhancer sequences from mammalian genes (e.g., globulin, elastase, albumin, α-fetoprotein, insulin, etc.) and enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the rear side of the replication origin, adenovirus enhancer, etc.). Suitable vector examples may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus (Sindbis-viral), measles, herpes virus, slow virus, retrovirus, adeno-associated virus vector, etc.). Expression vectors may be used to transfect host cells, such as bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art and can be used to transfect any cell of interest.

[0469] host cells

[0470] Other aspects of the present disclosure relate to host cells (e.g., isolated host cells) comprising one or more isolated polynucleotides, vectors, and / or vector systems described herein. In some embodiments, the isolated host cells of the present disclosure are cultured in vitro. In some embodiments, the host cells are bacterial cells (e.g., E. coli cells). In some embodiments, the host cells are yeast cells (e.g., Saccharomyces cerevisiae cells). In some embodiments, the host cells are insect cells. Examples of insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five antagonists), and / or other similar host cells. TM cells) and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells can include, for example, human embryonic kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293 TMcells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical cancer cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., HepG2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2 / 0 cells).

[0471] Other aspects of the present disclosure relate to methods for producing any of the binding proteins described herein. In some embodiments, the methods include: a) culturing a host cell (e.g., any host cell described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any isolated nucleic acid, vector, and / or vector system described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods of culturing host cells under conditions that express the protein are well known to those of ordinary skill in the art. Methods of isolating proteins from cultured host cells are well known to those of ordinary skill in the art, including, for example, by affinity chromatography (e.g., two-step affinity chromatography, including protein A affinity chromatography, followed by size exclusion chromatography).

[0472] Pharmaceutical composition

[0473] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the present disclosure.Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a binding protein or binding protein-drug conjugate admixed with a pharmaceutically or physiologically acceptable formulation selected to be appropriate for the mode of administration.

[0474] Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.

[0475] Pharmaceutical compositions may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavorings, and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium),

[0014] The invention also includes, but is not limited to, preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronics; PEG; sorbitan esters; polysorbates (e.g., polysorbate 20 or polysorbate 80); triton; tromethamine; lecithin; cholesterol, or tyloxapal), stability enhancers (e.g., sucrose or sorbitol), tonicity adjusters (e.g., alkali metal halides (e.g., sodium chloride or potassium chloride) or mannitol sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical Sciences (18th ed., AR Gennaro, ed., Mack Publishing Company 1990) and subsequent editions thereof, which are incorporated herein by reference for any purpose).

[0476] The optimal pharmaceutical composition will be determined by the skilled artisan based on, for example, the intended route of administration, delivery form, and desired dosage.Such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0477] The main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, the vehicle or carrier suitable for injection can be water, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other materials commonly used in the composition for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. Other exemplary pharmaceutical compositions comprise a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, the conjugated protein composition can be prepared for storage by mixing the selected composition with the desired purity with an optional preparaton in the form of a lyophilized cake or an aqueous solution. In addition, suitable excipients such as sucrose can be used to formulate the conjugated protein into a lyophilized product.

[0478] The pharmaceutical compositions of the present disclosure can be selected for parenteral delivery or subcutaneous delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the technical scope of this area.

[0479] The formulation components are present in concentrations acceptable to the site of administration. For example, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within the pH range of about 5 to about 8.

[0480] When considering parenteral administration, therapeutic composition used can be in the form of pyrogen-free, parenteral acceptable aqueous solution, which is included in the required conjugated protein in a pharmaceutically acceptable vehicle. The vehicle particularly suitable for parenteral injection is sterile distilled water, in which the conjugated protein is formulated into a sterile isotonic solution of suitable preservation. Another preparation can relate to the preparation of the required molecule with a controlled or sustained-release reagent (such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), pearls or liposomes) providing the product, which can then be delivered via accumulation injection. Hyaluronic acid can also be used, and this can have the effect of promoting the lasting duration in circulation. Other suitable means for introducing the required molecule include implantable drug delivery devices.

[0481] In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein inhalation solution can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be atomized.

[0482] It is also contemplated that certain formulations can be administered orally. In one embodiment of the present disclosure, the bound protein administered in this manner can be formulated with or without those carriers commonly used in the compounding of solid dosage forms (such as tablets and capsules). For example, a capsule can be designed to release the active portion of the formulation at a certain point in the gastrointestinal tract, where bioavailability is maximized and pre-systemic degradation is minimized. Additional reagents can be included to facilitate the absorption of the bound protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and adhesives can also be used.

[0483] Another pharmaceutical composition can comprise an effective amount of the binding protein admixed with a nontoxic excipient suitable for tablet manufacture. Solutions can be prepared in unit dosage form by dissolving the tablet in sterile water or another appropriate vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0484] Other pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, and the pharmaceutical compositions are included in formulations comprising conjugated proteins in a sustained or controlled delivery formulation. The technology for preparing a variety of other sustained or controlled delivery means (such as liposome carriers, bioerodible microparticles or porous beads and accumulation injection) is also well known to those skilled in the art. Other examples of sustained release formulations include semipermeable polymer matrices in the form of molded articles (such as films) or microcapsules. Sustained release matrix can include copolymers of polyesters, hydrogels, polylactides, L-glutamic acid and γ ethyl-L-glutamate, poly-(2-hydroxyethyl-methacrylate), ethylene vinyl acetate or poly-D (-) -3-hydroxybutyric acid. Sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0485] Pharmaceutical compositions to be used for in vivo administration generally must be sterile. This can be achieved by filtration through a sterile filter membrane. In the case of freeze-dried compositions, this method can be used for sterilization before or after freeze-drying and reconstitution. Compositions for parenteral administration can be stored in a freeze-dried form or in solution. In addition, parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

[0486] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that requires reconstitution (e.g., lyophilization) before administration.

[0487] The present disclosure also encompasses kits for producing single-dose administration units. The kits can each contain both a first container with a dried protein and a second container with an aqueous formulation. Also included within the scope of the present disclosure are kits containing single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes).

[0488] The effective amount of the binding protein pharmaceutical composition used in treatment will depend on, for example, the treatment context and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will therefore vary in part according to the following: the delivered molecule, the indication for which the binding protein is used, the route of administration, and the patient's size (body weight, body surface or organ size) and condition (age and gen...

Claims

1. A binding protein, said binding protein comprising four polypeptide chains forming three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V L2 -L1-V L1 -L2-C L [I] and the second polypeptide chain comprises a structure represented by the following formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V H3 -C H1 -Hinge - C H2 -C H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V L3 -C L [IV] wherein: V L1 is the first immunoglobulin light chain variable domain; V L2 is the second immunoglobulin light chain variable domain; V L3 is the third immunoglobulin light chain variable domain; V H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; V H3 is the third immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 constant heavy chain domain; C H2 is immunoglobulin C H2 constant heavy chain domain; C H3 is the constant domain of the heavy chain of immunoglobulin C H3 constant domain; The hinge is the immunoglobulin hinge region that connects the said C H1 and C H2 structural domain; and L1, L2, L3 and L4 are amino acid linkers; wherein L1, L2, L3 and L4 are each independently of zero amino acid length or comprise a sequence selected from the group consisting of: GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), GGSGSSGSGG (SEQ ID NO: 71) and DKTHT (SEQ ID NO: 66); wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair; Wherein V H1 and V L1 form a first antigen-binding site that binds to a CD28 polypeptide, V H2 and V L2 form a second antigen-binding site that binds to a CD3 polypeptide, and V H3 and V L3 form a third antigen-binding site that binds to a HER2 polypeptide; and wherein: (a) The V H1 domain contains CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of the CDR-H1 consists of GYTFTSYY (SEQ ID NO: 49), the amino acid sequence of the CDR-H2 consists of IYPGNVNT (SEQ ID NO: 50), and the amino acid sequence of the CDR-H3 consists of TRSHYGLDWNFDV (SEQ ID NO: 51), and the V L1 domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR-L1 consists of QNIYVW (SEQ ID NO:52), the amino acid sequence of the CDR-L2 consists of KAS (SEQ ID NO: 53), and the amino acid sequence of the CDR-L3 consists of QQGQTYPY (SEQ ID NO: 54); the V H2 domain contains CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of the CDR-H1 consists of GFTFTKAW (SEQ ID NO: 55), the amino acid sequence of the CDR-H2 consists of IKDKSNSYAT (SEQ ID NO: 56), and the amino acid sequence of the CDR-H3 consists of RGVYYALSPFDY (SEQ IDNO: 57), and the V L2 domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR-L1 consists of QSLVHQNAQTY (SEQ ID NO: 59), the amino acid sequence of the CDR-L2 consists of KVS (SEQ ID NO:64), and the amino acid sequence of the CDR-L3 consists of GQGTQYPFT (SEQ ID NO: 65); and the V H3 domain contains CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of the CDR-H1 consists of GFNIKDTY (SEQ ID NO:1), the amino acid sequence of the CDR-H2 consists of IYPTNGYT (SEQ ID NO: 3), and the amino acid sequence of the CDR-H3 consists of SRWGGDGFYAMDY (SEQ ID NO: 6), and the V L3 The domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of CDR-L1 consists of QDVNTA (SEQ ID NO: 9), the amino acid sequence of CDR-L2 consists of SAS (SEQ ID NO: 11), and the amino acid sequence of CDR-L3 consists of QQHYTTP (SEQ ID NO: 12); or (b) said V H1 domain comprises CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of said CDR-H1 consists of GYTFTSYY (SEQ ID NO: 49), the amino acid sequence of said CDR-H2 consists of IYPGNVNT (SEQ ID NO: 50), and the amino acid sequence of said CDR-H3 consists of TRSHYGLDWNFDV (SEQ ID NO: 51), and said V L1 domain comprises CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of said CDR-L1 consists of QNIYVW (SEQ ID NO:52), the amino acid sequence of said CDR-L2 consists of KAS (SEQ ID NO: 53), and the amino acid sequence of said CDR-L3 consists of QQGQTYPY (SEQ ID NO: 54); said V H2 domain comprises CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of said CDR-H1 consists of GFTFTKAW (SEQ ID NO: 55), the amino acid sequence of said CDR-H2 consists of IKDKSNSYAT (SEQ ID NO: 56), and the amino acid sequence of said CDR-H3 consists of RGVYYALSPFDY (SEQ IDNO: 57), and said V L2 domain comprises CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of said CDR-L1 consists of QSLVHQNAQTY (SEQ ID NO: 59), the amino acid sequence of said CDR-L2 consists of KVS (SEQ ID NO:64), and the amino acid sequence of said CDR-L3 consists of GQGTQYPFT (SEQ ID NO: 65); and said V H3 domain comprises CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of said CDR-H1 consists of GFNIRDTY (SEQ ID NO:2), the amino acid sequence of said CDR-H2 consists of IYPTQGYT (SEQ ID NO: 4), and the amino acid sequence of said CDR-H3 consists of SRWGGEGFYAMDY (SEQ ID NO: 7), and said V L3 The domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR-L1 consists of QDVNTA (SEQ ID NO: 9), the amino acid sequence of the CDR-L2 consists of SAS (SEQ ID NO: 11), and the amino acid sequence of the CDR-L3 consists of QQHYTTP (SEQ ID NO: 12).

2. The binding protein according to claim 1, wherein the HER2 polypeptide is a human HER2 polypeptide.

3. The binding protein according to claim 1, wherein the V H1 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91), and / or the V L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92); the V H2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or the V L2 domain comprises the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95); and the V H3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ IDNO: 72) and / or the V L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77).

4. The binding protein according to claim 1, wherein the V H1 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91), and / or the V L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92); the V H2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) and / or the V L2 domain comprises the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95); and the V H3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73) and / or the V L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77).

5. The binding protein according to claim 1, wherein the V H1 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91), and / or the V L1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92); the V H2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302) and / or the V L2 domain comprises the amino acid sequence of DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95); and the V H3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73) and / or the V L3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77).

6. The binding protein according to claim 1, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 102; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103; (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107; (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 293; or (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

297.

7. The binding protein according to any one of claims 1-6, wherein at least one of L1, L2, L3 or L4 is independently of zero amino acid length.

8. The binding protein according to any one of claims 1-6, wherein L1, L2, L3, and L4 each independently comprise a sequence selected from the following: GGGGSGGGGS (SEQ ID NO: 69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71).

9. The binding protein according to any one of claims 1-6, wherein L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO: 68), L3 comprises the sequence S, and L4 comprises the sequence RT.

10. The binding protein according to any one of claims 1-6, wherein at least one of L1, L2, L3, or L4 comprises the sequence DKTHT (SEQ ID NO: 66).

11. The binding protein according to claim 10, wherein L1, L2, L3, and L4 each independently comprise the sequence DKTHT (SEQ ID NO: 66).

12. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 hinge-C H2 -C H3 domain, and wherein each of the hinge-C H2 -C H3 domains contains amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A.

13. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 hinge-C H2 -C H3 domain, and wherein each of the hinge-C H2 -C H3 domains comprises amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to the EU index, wherein the amino acid substitutions are E233P, F234V, L235A and a deletion at 236.

14. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 hinge-C H2 -C H3 domain, and wherein each of the hinge-C H2 -C H3 domains comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K.

15. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG1 hinge-C H2 -C H3 domain, and wherein each of the hinge-C H2 -C H3 domains comprises amino acid substitutions at positions corresponding to positions 234, 235 and 329 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A, L235A and P329A.

16. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG1 hinge-C H2 -C H3 domain, and wherein each of the hinge-C H2 -C H3 domains contains amino acid substitutions at positions corresponding to positions 298, 299 and 300 of human IgG1 according to the EU index, wherein the amino acid substitutions are S298N, T299A and Y300S.

17. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second polypeptide chain H2 -C H3 domain contains amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-C of the third polypeptide chain H2 -C H3 domain contains amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W.

18. The binding protein according to any one of claims 1-6, wherein the hinge-C of the second polypeptide chain H2 -C H3 domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-C of the third polypeptide chain H2 -C H3 domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V.

19. A binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein the first polypeptide chain comprises a structure represented by the following formula: V L2 -L1-V L1 -L2-C L [I] and the second polypeptide chain comprises a structure represented by the following formula: V H1 -L3-V H2 -L4-C H1 -Hinge-C H2 -C H3 [II] and the third polypeptide chain comprises a structure represented by the following formula: V H3 -C H1 -Hinge - C H2 -C H3 [III] and the fourth polypeptide chain comprises a structure represented by the following formula: V L3 -C L [IV] wherein: V L1 is the first immunoglobulin light chain variable domain; V L2 is the second immunoglobulin light chain variable domain; V L3 is the third immunoglobulin light chain variable domain; V H1 is the first immunoglobulin heavy chain variable domain; V H2 is the second immunoglobulin heavy chain variable domain; V H3 is the third immunoglobulin heavy chain variable domain; C L is the constant domain of the immunoglobulin light chain; C H1 is immunoglobulin C H1 constant heavy chain domain; C H2 is immunoglobulin C H2 constant domain of heavy chain; C H3 is immunoglobulin C H3 constant heavy chain domain; The hinge is connected to the C H1 and C H2 immunoglobulin hinge region of the domain; and L1, L2, L3, and L4 are amino acid linkers; wherein L1, L2, L3, and L4 each independently comprise the sequence DKTHT (SEQ ID NO: 66); wherein the polypeptide of formula I and the polypeptide of formula II form a cross light chain-heavy chain pair; Wherein V H1 and V L1 form a first antigen-binding site that binds to a CD28 polypeptide, V H2 and V L2 form a second antigen-binding site that binds to a CD3 polypeptide, and V H3 and V L3 form a third antigen-binding site that binds to a HER2 polypeptide; and wherein: The V H1 domain contains CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of CDR-H1 consists of GYTFTSYY (SEQ ID NO: 49), the amino acid sequence of CDR-H2 consists of IYPGNVNT (SEQ ID NO: 50), and the amino acid sequence of CDR-H3 consists of TRSHYGLDWNFDV (SEQ ID NO: 51); and the V L1 domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of CDR-L1 consists of QNIYVW (SEQ ID NO: 52), the amino acid sequence of CDR-H2 consists of KAS (SEQ ID NO: 53), and the amino acid sequence of CDR-L3 consists of QQGQTYPY (SEQ ID NO: 54); The V H2 domain contains CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of the CDR-H1 consists of GFTFTKAW (SEQ ID NO: 55), the amino acid sequence of the CDR-H2 consists of IKDKSNSYAT (SEQ ID NO: 56), and the amino acid sequence of the CDR-H3 consists of RGVYYALSPFDY (SEQ ID NO: 57), and the V L2 domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR-L1 consists of QSLVHQNAQTY (SEQ ID NO: 59), the amino acid sequence of the CDR-L2 consists of KVS (SEQ ID NO: 64), and the amino acid sequence of the CDR-L3 consists of GQGTQYPFT (SEQ ID NO: 65); and The said V H3 domain contains CDR-H1, CDR-H2 and CDR-H3, wherein the amino acid sequence of CDR-H1 consists of GFNIRDTY (SEQ ID NO: 2), the amino acid sequence of CDR-H2 consists of IYPTQGYT (SEQ ID NO: 4), and the amino acid sequence of CDR-H3 consists of SRWGGEGFYAMDY (SEQ ID NO: 7); and the said V L3 domain contains CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of CDR-L1 consists of QDVNTA (SEQ ID NO: 9), the amino acid sequence of CDR-L2 consists of SAS (SEQ ID NO: 11), and the amino acid sequence of CDR-L3 consists of QQHYTTP (SEQ ID NO: 12).

20. The binding protein according to claim 19, wherein: Wherein said V H1 domain contains the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91), and said V L1 domain contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92); wherein said V H2 domain comprises the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93), and said V L2 domain comprises an amino acid sequence selected from: DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95); and The said V H3 domain contains the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO:73), and the said V L3 domain contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77).

21. The binding protein according to claim 19 or 20, wherein the hinge-C H2 -C H3 domain of the second and the third polypeptide chains is a human IgG4 hinge-C H2 -C H3 domain, and wherein each of the hinge-C H2 -C H3 domains comprises amino acid substitutions at positions corresponding to positions 228, 234, 235 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P, F234A, L235A and R409K.

22. The binding protein according to claim 19 or 20, wherein the hinge-C of the second and the third polypeptide chains H2 -C H3 domain is a human IgG4 hinge-C H2 -C H3 domain, and wherein the hinge-C of the second polypeptide chain H2 -C H3 domain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the hinge-C of the third polypeptide chain H2 -C H3 domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W.

23. The binding protein according to claim 19, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

107.

24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein according to any one of claims 1-23.

25. An expression vector comprising the nucleic acid molecule according to claim 24.

26. An isolated host cell comprising the nucleic acid molecule according to claim 24 or the expression vector according to claim 25.

27. The isolated host cell according to claim 26, wherein the host cell is a mammalian cell or an insect cell.

28. A pharmaceutical composition comprising the binding protein according to any one of claims 1-23 and a pharmaceutically acceptable carrier.

29. Use of the binding protein according to any one of claims 1-23 or the pharmaceutical composition according to claim 28 in the preparation of a medicament for treating cancer in a patient, wherein the cancer is breast cancer, colorectal cancer, gastric cancer or non-small cell lung cancer (NSCLC).

30. Use according to claim 29, wherein the patient is a human.

31. Use according to claim 29 or 30, wherein the third antigen-binding site binds to a human HER2 polypeptide and wherein the cancer cells from the patient express HER2.

32. A vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of the binding protein according to any one of claims 1-23, wherein the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein and a fourth vector encoding the fourth polypeptide chain of the binding protein.

33. A kit of polynucleotides, the kit of polynucleotides comprising: (a) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 191 and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO: 192; or (b) a first polynucleotide containing the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide containing the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide containing the polynucleotide sequence of SEQ ID NO: 195 and a fourth polynucleotide containing the polynucleotide sequence of SEQ ID NO:

196.

34. The kit according to claim 33, wherein the first, second, third and fourth polynucleotides are present on one or more expression vectors.

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