Trispecific binding proteins, methods and uses thereof

Abstract

To provide trispecific and / or trivalent binding proteins.SOLUTION: Disclosed herein is a trispecific and / or trivalent binding protein comprising four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, where a first pair of the polypeptides that form the binding protein possesses double binding domains having crossover tropism, and a second pair of the polypeptides has a single variable domain that forms a single antigen binding site. In some embodiments, the binding protein comprises a binding site that binds to CD28 polypeptide, a binding site that binds to CD3 polypeptide, and a binding site that binds to a third polypeptide that binds to, for example, a tumor targeting protein.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 831,572, filed April 9, 2019, and European Patent Application No. EP19306311.2, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 183952032040SEQLIST.TXT, Recorded: April 6, 2020, Size: 526KB).

[0003] Field The present disclosure relates to 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 has dual variable domains with a crossover orientation. The present disclosure also relates to methods of making trispecific and / or trivalent binding proteins and uses of such binding proteins. [Background technology]

[0004] Monoclonal antibody-based biopharmaceuticals have become an important tool for new drug development. Monoclonal antibody technology provides specific targeting, precise signaling and / or payload to unique cell populations, and long-lasting biological effects through its Fc function. Efforts in antibody engineering have enabled the development of bispecific antibodies that combine the specificities of two monoclonal antibodies for various biological applications, expanding the scope of antibody drug development. Newly discovered neutralizing antibodies with improved breadth and potency may provide more options for developing biopharmaceuticals to treat complex diseases, such as cancer, arthritis, and / or inflammatory disorders.

[0005] Immuno-oncology is a promising and emerging therapeutic approach for disease management in cancer. The immune system is the first line of defense against cancer development and progression. There is now substantial evidence that T cells can control tumor growth and prolong the survival of cancer patients in both early and late stages of the disease. However, tumor-specific T cells are restricted in a number of ways that prevent them from controlling the disease.

[0006] As part of human adaptive immunity, T cell immunity plays a crucial role in controlling viral infections and cancer, and in some cases eliminates infected and malignant cells, resulting in the clearance of viral infections or the cure of cancer. In chronic infectious diseases, such as herpesvirus infections (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans through various mechanisms, including immunosuppression, T cell exhaustion, and establishment of latency. Nevertheless, viral infections generally induce 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.

[0007] Therefore, activation and / or expansion of viral antigen-specific T cells in vivo and / or ex vivo may provide a therapeutic strategy for chronic viral infections.

[0008] All references referred to in this specification, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein 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 [Problem to be solved by the invention]

[0009] To meet these and other needs, trispecific binding proteins (e.g., antibodies) that form three antigen binding sites are provided herein. These binding proteins can specifically bind 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 molecular subtypes of breast cancer, and also in gastric, ovarian, lung, and prostate cancer. Optimal activation of T cells requires two factors: 1. antigen recognition and 2. costimulation. Using the trispecific HER2 / CD28×CD3 trispecific binding proteins 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 proteins recruit T cells to the tumor via binding sites that recognize HER2, CD38, or another tumor target protein, and activate the engaged T cells via anti-CD3 and CD28. The resulting activation induces the killing potential of the immune cells against nearby tumor cells. Additionally, anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptide and with removed potential manufacturing disadvantages (e.g., deamidation sites).

[0010] Further provided herein are anti-CD38 / CD28×CD3 trispecific antibodies that have been developed and evaluated for their potential in T cell activation and subsequent proliferation and / or expansion of antigen-specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations in vitro, including antigen-specific CD8 T central and effector memory cells. In particular, in vitro expansion of CMV, EBV, HIV-1, and influenza-specific CD8 central and effector memory cells has been demonstrated. The anti-CD38 / CD28×CD3 trispecific antibodies described herein exhibit novel properties by engaging CD3 / CD28 / CD38 and providing a signaling pathway for stimulating and expanding T cells, which may provide an effective strategy for treating chronic infectious diseases, such as HSV, CMV, EBV, HIV-1, and HBV infections.

[0011] To meet these and other needs, binding proteins that bind to CD38 polypeptides (e.g., human and cynomolgus CD38 polypeptides) are provided herein, including monospecific, bispecific, or trispecific binding proteins that have at least one antigen-binding site that binds to CD38 polypeptides. Advantageously, these binding proteins have the ability to recruit T cells to the vicinity of cancer cells, subsequently activating T cells and promoting activated T cell killing of adjacent cancer cells through the granzyme / perforin mechanism, providing a different mode of action for anti-tumor activity than anti-CD38 antibodies, such as DARZALEX® (daratumumab). Furthermore, the ability to bind to both human and cynomolgus CD38 polypeptides allows the binding proteins to be easily tested in preclinical toxicology studies, for example, to evaluate their safety profile for later clinical use. [Means for solving the problem]

[0012] In some embodiments, a binding protein comprising four polypeptide chains forming three antigen binding sites, wherein the first polypeptide chain has the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site; V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The 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), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; and V H3 and V L3 forms a third antigen-binding site, Said binding proteins are provided herein.

[0013] In some embodiments, the first binding site binds to a CD28 polypeptide. H1The 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), 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). H1 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); L1 The domain is It contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92).

[0014] In some embodiments, 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 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 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 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, a binding protein of the 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 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). H2 The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93); L2 The domain is DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSL In some embodiments, the V comprises an amino acid sequence selected from the group consisting of: IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). H2 The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302); L2 The domain is DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLS VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, a binding protein of the 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: 95. In some embodiments, a binding protein of the disclosure comprises 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, a binding protein of the 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: 96. In some embodiments, a binding protein of the 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: 97.In some embodiments, a binding protein of the 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.

[0015] In some embodiments, the third antigen-binding site binds to a tumor-targeting protein. In some embodiments, the tumor-targeting protein is a CD38 polypeptide (e.g., human CD38 polypeptide). 38 polypeptide). In some embodiments, the tumor targeting protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, tumor targeting proteins of the present disclosure include 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 ( 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), CD277 (also known as B7H4), CD278 (also known as B7H5), CD279 (also known as B7H6), CD280 (also known as B7H7), CD281 (also known as B7H8), CD282 (also known as B7H9), CD283 (also known as B7H1), CD284 (also known as B7H2), CD285 (also known as B7H1), CD286 (also known as B7H2), CD287 (also known as B7H1), CD288 (also known as B7H1), CD289 (also known as B7H1), CD290 (also known as B7H1), CD291 (also known as B7H1), CD292 (also known as B7H1), CD293 (also known as B7H2), CD294 (also known as B7H1), CD295 (also known as B7H1), CD296 (also known as B7H1), CD297 (also known as B7H1), CD298 (also known as B7H1), CD299 (also known as B7H1), CD300 (also known as B7H1), CD301 7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), 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, IGF1R, IL2Rbeta, 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, LA G3, 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 coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0016] In some embodiments, the third antigen-binding site binds to a 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), L3 The domain comprises a CDR-L1 sequence containing the amino acid sequence QSVSSYGQGF (SEQ ID NO: 16), an amino acid sequence GAS (SEQ ID NO: 17), In some embodiments, the CDR-L2 sequence comprises the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). 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), L3 The 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). 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), L3 The 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). H3 The 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), 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).

[0017] In some embodiments, V H3The 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), L3 The 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 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), L3 The domain comprises 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). H3 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79); L3 The domain is In some embodiments, the amino acid sequence of V is: DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80). H3 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81); L3 The domain is EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQ In some embodiments, the amino acid sequence of V is H3 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83); L3 The domain is In some embodiments, the amino acid sequence of V is: DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84). H3 The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87); L3 The domain is In some embodiments, the amino acid sequence of V is: DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88). H3 The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89); L3 The domain is In some embodiments, the amino acid sequence of V is: AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90). H3 The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); L3 The domain is It contains the amino acid sequence of DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO: 86).

[0018] 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 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 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 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 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.

[0019] In some embodiments, the third antigen-binding site binds to a human HER2 polypeptide. H3The 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), L3 Domain In some embodiments, the VDV 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). 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), 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). 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), 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). H3The 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), 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). 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 SRWGGSGFYAMDY (SEQ ID NO: 8), 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). 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), 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). 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), L3The 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). H3 The domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGS and / or comprising the amino acid sequence of V L3 The domain is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78). H3 The domain is and / or comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72); L3 The domain is In some embodiments, the amino acid sequence of V is: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). H3 The domain is and / or comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73); L3 The domain is In some embodiments, the amino acid sequence of V is: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). H3 The domain is and / or comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75); L3 The domain is In some embodiments, the amino acid sequence of V is: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). H3 The domain is and / or comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74); L3 The domain is In some embodiments, the amino acid sequence of V is: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). H3 The domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS and / or comprising the amino acid sequence of (SEQ ID NO: 76), L3 The domain is In some embodiments, the amino acid sequence of V is: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). H3 The domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), and / or L3 The domain is It contains the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78).

[0020] 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 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 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 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 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: 120 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120. 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 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 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 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 at least 95% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the first polypeptide chain comprises 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.

[0021] In some embodiments that can 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 are each independently 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 (sequence number 66).

[0022] In some embodiments that may be combined with any other embodiments described herein, the hinge-C of the second and third polypeptide chains H2 -C H3 Domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains each comprise an amino acid substitution at a position corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A. H2 -C H3 Domain is human IgG4 hinge-C H2 -C H3domain, hinge-C H2 -C H3 Each domain comprises an amino acid substitution at a position corresponding to 233-236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-C of the second and third polypeptide chains H2 -C H3 Domain is human IgG4 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains each comprise an amino acid substitution at a position corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K. H2 -C H3 Domain is human IgG1 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 In some embodiments, the domains comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, and the amino acid substitutions are L234A, L235A, and P329A. H2 -C H3 Domain is human IgG1 hinge-C H2 -C H3 domain, hinge-C H2 -C H3 The domains each comprise an amino acid substitution at a position corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, the amino acid substitutions being S298N, T299A, and Y300S. H2 -C H3The 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, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the hinge-C of the third polypeptide chain H2 -C H3 In some embodiments, the domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W. 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, the amino acid substitutions being S354C and T366W; the hinge-C of the third polypeptide chain H2 -C H3 The 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, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

[0023] In some embodiments, provided herein is an isolated nucleic acid molecule comprising the nucleotide sequence encoding the binding protein of any one of the above embodiments.In some embodiments, provided herein is an expression vector comprising the nucleic acid molecule of any one of the above embodiments.In some embodiments, provided herein is an isolated host cell comprising the nucleic acid molecule of any one of the above embodiments or the expression vector of any one of the above embodiments.In some embodiments, the host cell is a mammalian or insect cell.

[0024] In some embodiments, provided herein is a pharmaceutical composition comprising a binding protein of any one of the above embodiments and a pharma- ceutically acceptable carrier.

[0025] In some embodiments, the method includes the steps of: Provided herein is a method for preventing and / or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of at least one of the binding proteins or pharmaceutical compositions of any one of the above embodiments. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in a method for preventing and / or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition. In some embodiments, provided herein is a binding protein or pharmaceutical composition 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.

[0026] In some embodiments, the at least one binding protein is administered in combination with a chemotherapeutic agent.In some embodiments, the patient is a human.

[0027] In some embodiments, the third antigen-binding site binds to a human CD38 polypeptide, and 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, before administration of the binding protein, the patient has been treated with daratumumab without a washout period.

[0028] In some embodiments, the third antigen-binding site binds to a human HER2 polypeptide, and 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).

[0029] In some embodiments, a method of expanding virus-specific memory T cells comprises contacting a virus-specific memory T cell with a binding protein, the binding protein comprising four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The 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), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; H3 and V L3 forms a third antigen-binding site which binds to the CD38 polypeptide; Such methods are provided herein.

[0030] In some embodiments, a binding protein for use in the expansion of virus-specific memory T cells comprises four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The domain is a CDR-H1 sequence containing the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence containing 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), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; H3 and V L3 Provided herein are binding proteins for said uses, wherein said binding protein forms a third antigen-binding site which binds to the CD38 polypeptide.

[0031] 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.

[0032] In some embodiments, a method of expanding T cells comprises contacting a T cell in vitro or ex vivo with a binding protein, the binding protein comprising four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The 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), L2 The domain is QSLVHX1NX2X3TY, where X X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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); H3 and VL3 forms a third antigen-binding site which binds to the CD38 polypeptide; Such methods are provided herein.

[0033] In some embodiments, a binding protein for use in a method of expanding T cells comprises four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The 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), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; H3 and V L3 forms a third antigen-binding site which binds to the CD38 polypeptide; Binding proteins for said uses are provided herein.

[0034] In some embodiments, the T cells are memory T cells or effector T cells. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.

[0035] In some embodiments, a method of treating a chronic viral infection comprises administering to an individual or patient in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The 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), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F;H3 and V L3 forms a third antigen-binding site which binds to the CD38 polypeptide; Such methods are provided herein.

[0036] In some embodiments, a binding protein for use in a method of treating a chronic viral infection comprises four polypeptide chains that form three antigen binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H2 The 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), L2 The domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY (SEQ ID NO: 180), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F; H3 and V L3 forms a third antigen-binding site which binds to the CD38 polypeptide; The method comprises administering to an individual or patient in need thereof an effective amount of a binding protein. Binding proteins for said uses are provided herein.

[0037] In some embodiments, the individual or patient is human.In some embodiments, the binding protein is administered to the individual or patient in a pharmaceutical preparation comprising the binding protein and a pharmaceutically acceptable carrier.In some embodiments, the administration of the binding protein results in the activation and / or proliferation of virus-specific memory T cells in the individual or patient.

[0038] In some embodiments that may be combined with any other embodiments 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 ).

[0039] In some embodiments that may be combined with any other embodiments 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).

[0040] In some embodiments that may be combined with any other embodiments described herein, the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide.

[0041] In some embodiments, a vector system is provided herein, comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of the binding protein of any one of the above embodiments. 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.

[0042] In some embodiments, a kit is provided herein, comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above embodiments. In some embodiments, the kit further comprises instructions for using the polypeptide chains or binding protein according to any of the methods or uses described herein, e.g., above.

[0043] In some embodiments, a kit is provided herein, the kit comprising one, two, three or four polynucleotides according to any one of the above embodiments. In some embodiments, a kit of polynucleotides, the kit comprising: (a) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 200. (d) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:204; (e) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising a 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: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, (k) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:229, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:230, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:231, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:232; (l) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:236; (m) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:240;(n) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:244; (o) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:246, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:247, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:248; (p) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:249, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:250, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:251, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:252; (q) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:253, a second polynucleotide comprising a 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; (t) 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;(u) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 269, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 270; Provided herein are kits comprising one, two, three, or four of the following polynucleotides: (i) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:276; or (ii) a kit of polynucleotides comprising a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:273, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:274, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:275, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:276.

[0044] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief description of the drawings]

[0045] [Figure 1A] FIG. 1A provides a schematic diagram of a trispecific binding protein that includes four polypeptide chains that form three antigen-binding sites that bind to three target proteins: CD28, CD3, and HER2. The first pair of polypeptides has dual variable domains with crossover orientations (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes HER2. The trispecific binding protein shown in FIG. 1A uses a constant region with a "knob-into-hole" mutation, where the knob is in the second pair of polypeptides with the single variable domain. [Figure 1B]FIG. 1B provides the fold change in binding affinity (relative to the parent) of anti-CD28 / CD3 / HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains. Mutations 3233QQ-QEQ (from top to bottom) refer to mutations introduced at residues 32-35 of the VL domain of the anti-CD3 binding site (indicated by *); the remaining mutations were introduced in the VH or VL domains of the trastuzumab anti-HER2 binding site (indicated by #; numbering follows Kabat). For mutations in the anti-HER2 binding site, mutation 30Q was introduced in the VL domain and the remaining mutations were introduced in the VH domain. Binding affinity was measured by ELISA and the values ​​provided are relative to the parent trispecific antibody. [Figure 1C] FIG. 1C provides binding curves of the trispecific antibodies that bind to the indicated human HER2, human CD28, and CD3 as determined by ELISA. [Figure 1D] FIG. 1D provides a proposed mechanism of action of HER2 / CD28×CD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing. [Figure 2-1]Figure 2A provides a schematic diagram of a trispecific binding protein that includes four polypeptide chains that form three antigen-binding sites that bind three target proteins: CD28, CD3, and CD38. The first pair of polypeptides has dual variable domains with crossover orientations (VH1-VH2 and VL2-VL1) that form two antigen-binding sites that recognize CD3 and CD28, and the second pair of polypeptides has a single variable domain (VH3 and VL3) that forms a single antigen-binding site that recognizes CD38. The trispecific binding protein shown in Figure 2A uses an IgG4 constant region with a "knob-into-hole" mutation, where the knob is in the second pair of polypeptides with a single variable domain. Figures 2B-2E show the binding affinity of 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 monkey CD38 (Figure 2C), human CD3 (Figure 2D), and human CD28 (Figure 2E) as measured by ELISA. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Diagram 3] Figure 3 shows an SPR competition assay for binding to CD38 by daratumumab and an anti-CD38 monospecific antibody with the indicated anti-CD38 binding domains. If the antibody recognized an epitope on CD38 different from that of daratumumab, injection of the antibody resulted in an increase in the SPR signal. If the antibody recognized an overlapping epitope as daratumumab, injection of the antibody did not increase the SPR signal. [Figure 4]Figures 4A-4B show the in vitro cell killing activity of CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human multiple myeloma NCI-H929 cells (CD38+ / CD28+). Assays were 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. [Diagram 5] Figures 5A-5B show the in vitro cell killing activity of CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains against human lymphoma OCI-Ly19 cells (CD38+ / CD28-). Assays were performed in the presence of 5 nM isotype control antibody (Figure 5A) or daratumumab (Figure 5B). Daratumumab caused a decrease in the cell killing activity of the anti-CD38 / CD28 x CD3 trispecific antibody. [Figure 6-1]Figures 6A-6J show characterization of in vitro T cell subset expansion proliferation in PBMCs collected from CMV-infected donor D in response to CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantitate CMV-specific CD8+ T cells (Figures 6A-6B), CMV-specific Tcm CD8+ cells (Figures 6C-6D), and CMV-specific Tem CD8+ cells (Figures 6E-6F). In addition, the percentages of CMV-specific Tcm (Figures 6G-6H) and Tem (Figures 6I-6J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of CMV-specific memory CD8+ T cells in a dose-responsive manner with different potencies and kinetics. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 6-5] Continued from Figure 6-4. [Figure 7-1]Figures 7A-7J show characterization of in vitro T cell subset expansion in PBMCs collected from CMV-infected donor E in response to CD38 / CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup x ΔCD3mid IgG4 FALA). Antibodies indicated from top to bottom in the legend are indicated from left to right in the graph. T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM, 1 nM, and 2 nM. Flow cytometry was used to quantitate CMV-specific CD8+ T cells (Figures 7A-7B), CMV-specific Tcm CD8+ cells (Figures 7C-7D), and CMV-specific Tem CD8+ cells (Figures 7E-7F). In addition, the percentages of CMV-specific Tcm (Figures 7G-7H) and Tem (Figures 7I-7J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 7-4] Continued from Figure 7-3. [Figure 7-5] Continued from Figure 7-4. [Figure 8-1]Figures 8A-8J show characterization of in vitro T cell subset expansion proliferation in PBMCs collected from EBV-infected donor C in response to CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 8A-8B), CMV-specific Tcm CD8+ cells (Figures 8C-8D), and CMV-specific Tem CD8+ cells (Figures 8E-8F). In addition, the percentages of EBV-specific Tcm (Figures 8G-8H) and Tem (Figures 8I-8J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the expansion of CMV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 8-4] Continued from Figure 8-3. [Figure 8-5] Continued from Figure 8-4. [Figure 9-1]Figures 9A-12 show the characterization of in vitro T cell subset expansion proliferation in PBMCs collected from EBV-infected donor D in response to CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A-9B), EBV-specific Tcm CD8+ cells (Figures 9C-9D), and EBV-specific Tem CD8+ cells (Figures 9E-9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 9-4] Continued from Figure 9-3. [Figure 10]Figures 9A-12 show the characterization of in vitro T cell subset expansion proliferation in PBMCs collected from EBV-infected donor D in response to CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A-9B), EBV-specific Tcm CD8+ cells (Figures 9C-9D), and EBV-specific Tem CD8+ cells (Figures 9E-9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 11]Figures 9A-12 show the characterization of in vitro T cell subset expansion proliferation in PBMCs collected from EBV-infected donor D in response to CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A-9B), EBV-specific Tcm CD8+ cells (Figures 9C-9D), and EBV-specific Tem CD8+ cells (Figures 9E-9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 12]Figures 9A-12 show the characterization of in vitro T cell subset expansion proliferation in PBMCs collected from EBV-infected donor D in response to CD38 / CD28sup×CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibody with the indicated alternative anti-CD38 binding domains. A trispecific antibody with the CD38VH1 anti-CD38 binding domain deleted was used as a negative control (ΔCD38VH1 / ΔCD28sup×ΔCD3mid IgG4 FALA). T cell populations were measured at the indicated time points (D3 refers to day 3; D7 refers to day 7). The indicated trispecific antibodies were tested at the indicated concentrations of 0.2 nM and 1 nM. Flow cytometry was used to quantify EBV-specific CD8+ T cells (Figures 9A-9B), EBV-specific Tcm CD8+ cells (Figures 9C-9D), and EBV-specific Tem CD8+ cells (Figures 9E-9F). In addition, the percentages of EBV-specific Tcm (Figures 9G-10) and Tem (Figures 11-12) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of EBV-specific memory CD8+ T cells in a dose-response manner with different potencies and kinetics. [Figure 13-1] Figures 13A-13D show the change (in days) in tumor volume (Figure 13A) and body weight (Figure 13B) over time in NSG mice bearing ZR-75-1 tumors engrafted with in vitro expanded human CD3+ T cells. Groups of 10 mice were treated with either vehicle or Her2 / CD28xCD3 trispecific antibody at the indicated dosages. Arrowheads indicate the day of administration. Tumor volume is depicted as mean ± SEM, mm3. Body weight change is depicted as % change, mean ± SEM. X-axis shows days after ZR-75-1 cell implantation. Figure 13C shows tumor volume (mm3) over time per individual mouse in each treatment group. Figure 13D shows tumor weight (mg) for each treatment group. **=p<0.001; ***=p<0.0003 (2-way ANOVA, control vs. 100 and 10ug / kg). [Figure 13-2] Continued from Figure 13-1. [Figure 13-3] Continued from Figure 13-2. [Figure 13-4] Continued from Figure 13-3. [Figure 14-1] Figures 14A-14C show the effect of Her2 / CD28xCD3 trispecific antibody treatment on T cells from whole blood. Figure 14A shows analysis of hCD45+, CD8+, CD4+, and mCD45+ cells by flow cytometry. Figure 14B shows the effect of control or Her2 / CD28xCD3 trispecific antibody treatment (at the doses shown) on hCD45+, CD8+, CD4+, and mCD45+ cell counts. Figure 14C shows the effect of control or Her2 / CD28xCD3 trispecific antibody treatment (at the doses shown) 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 Figures 14B and 14C are based on the control sample versus 100ug / kg. [Figure 14-2] Continued from Figure 14-1. [Figure 15-1] Figures 15A-15C show the effect of Her2 / CD28xCD3 trispecific antibody treatment on tumor infiltrating lymphocytes (TILs) as examined by immunohistochemistry (IHC). Arrows indicate tumor infiltrating T cells identified in ZR-75-1 breast tumors. Top image is 1x magnification; bottom image is 20x magnification. In both sets of images, staining for human CD45, human CD4, and human CD8 is shown from left to right. Tumors from mice treated with vehicle control (Figure 15A), 100ug / kg trispecific antibody (Figure 15B), or 0.1ug / kg trispecific antibody (Figure 15C) are shown. [Figure 15-2] Continued from Figure 15-1. [Figure 15-3] Continued from Figure 15-2. [Figure 16-1]Figures 16A-16C show quantification of the effect of Her2 / CD28xCD3 trispecific antibody treatment on TILs as measured by IHC. Each dot represents one tumor from an individual mouse; rectangles represent group means; error bars show standard deviation. *=p<0.05 (ANOVA) compared to vehicle control group. Numbers of CD45+ (Figure 16A), CD4+ (Figure 16B), or CD8+ (Figure 16C) cells are shown. In Figure 16C, due to excess nonspecific signal in the IHC slides for CD8, a $area quantification approach was used for CD8+ cells instead of a cell counting algorithm. [Figure 16-2] Continued from Figure 16-1. [Figure 16-3] Continued from Figure 16-2. [Figure 17-1] Figures 17A-17F show in vitro lysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by a Her2 / CD28xCD3 trispecific antibody with wild-type trastuzumab and anti-CD3 antigen binding domains and no 32 / 35QQ mutation in the VL domain ("ctl") compared to a Her2 / CD28xCD3 trispecific antibody with mutations in the VL domains of the anti-HER2 and anti-CD3 arms (numbering as shown in Table 1). Cell killing activity against cell lines with different expression of HER2 is depicted: HCC1954 for high HER2 expression (Figure 17A), BT20 for medium HER2 expression (Figure 17C), and MDA-MD-231 for low HER2 expression (Figure 17E). Graphs are shown depicting cell killing as a function of antibody concentration for target cells HCC1954 (Figure 17B), BT20 (Figure 17D), and MDA-MD-231 (Figure 17F), comparing binding protein #2 to a control or binding proteins #1 and #5 to a control. [Figure 17-2] Continued from Figure 17-1. [Figure 17-3] Continued from Figure 17-2. [Figure 17-4] Continued from Figure 17-3. [Figure 17-5] Continued from Figure 17-4. [Figure 17-6]Continued from Figure 17-5. [Figure 18-1] Figures 18A and 18B summarize the mean EC50 (pM) of in vitro cell killing by experimental or control Her2 / CD28xCD3 trispecific antibodies against the indicated breast (Figure 18A) or gastric cancer cell lines (Figure 18B). The amino acid sequences of the indicated trispecific antibodies are provided in Table 1. [Figure 18-2] Continued from Figure 18-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] 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 have dual variable domains with crossover orientation.

[0047] general definition As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: Unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0048] It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that "comprise," "consist," and "consist essentially of."

[0049] The term "polynucleotide" as used herein refers to a single-stranded or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide. Such modifications can include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroaniladate, and phosphoroamidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[0050] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or any combination thereof, which (1) is not associated with all or a portion of a polynucleotide with which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide with which it is not linked in nature, or (3) is not found in nature as part of a larger sequence.

[0051] An "isolated polypeptide" is a polypeptide that is (1) free from at least some other polypeptides with which it is normally found, (2) essentially free from other polypeptides from the same source, e.g., the same species, (3) expressed by cells from a different species, (4) separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) not associated (by covalent or non-covalent interactions) with portions of the polypeptide with which it is naturally associated, (6) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) not naturally occurring. Such isolated polypeptides can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination of these. Preferably, an isolated polypeptide is substantially free from polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0052] Naturally occurring antibodies typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, with 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). The terms "heavy chain" and "light chain", as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically contains a variable domain of about 100-110 or more amino acids that are typically responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain that is responsible for effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide contains a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3), including V H The domain is located at the amino terminus of the polypeptide and is C H3 The domain is located at the carboxyl terminus and contains the full-length light chain immunoglobulin polypeptide. The peptide is a variable domain (V L ) and the constant domain (C L ), including V L The domain is located at the amino terminus of the polypeptide and is C L The domain is at the carboxyl terminus.

[0053] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA is similarly further divided into subclasses, including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, and heavy chains also include a "D" region of about 10 additional amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989) (incorporated in its entirety by reference for all purposes). The variable regions of each light / heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same overall structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may allow binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both the light and heavy chains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0054] The term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17; Chothia et al., J. Immunol. 1999:143-144; Chothia et al., J. Immunol. 1999:145-146; Chothia et al., J. Immunol. 1999:146-147; Chothia et al., J. Immunol. 1999:147-148; Chothia et al., J. Immunol. 1999:147-148; Chothia et al., J. Immunol. 1999:147-148; Chothia et al., J. Immunol. 20 ... (E. al., 1989, Nature 342:877-83) found that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3 or H1, H2, and H3, with "L" and "H" referring to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are 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 may nevertheless overlap with the Kabat CDRs, but may be shortened or lengthened in light of predictions or experimental findings that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs. Identification of predicted CDRs using amino acid sequences may be performed using methods such as those described by Martin, A. The method is well known in the art, for example, in "Protein sequence and structure analysis of antibody variable domains", Antibody Engineering, Vol. 2. Kontermann R., Dubel S. eds., Springer-Verlag, Berlin, pp. 33-51 (2010). The amino acid sequence of the heavy and / or light chain variable domains can be inspected to identify the sequences of the CDRs by other conventional methods, for example, by determining regions of sequence hypervariability by comparison with known amino acid sequences of other heavy and light chain variable regions. The numbered sequences can be aligned by eye or by using an alignment program, for example, one of the programs in the CLUSTAL suite, as described in Thompson, 1994, Nucleic Acids Res. 22:4673-80. Molecular models are conventionally used to accurately delineate the framework and CDR regions, and therefore the accurate sequence-based assignment.

[0055] The term "Fc" as used herein refers to a molecule comprising a sequence of non-antigen-binding fragments resulting from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins, with IgG1 and IgG2 being preferred. Fc molecules are composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent associations into dimeric or multimeric forms. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of an Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. The term "native Fc" as used herein is inclusive to monomeric, dimeric and multimeric forms.

[0056] F(ab) fragments typically contain one light chain and one heavy chain V H and C H1 domain, and the V of the F(ab) fragment H -C H1 The heavy chain portion cannot form disulfide bonds with another heavy chain polypeptide. As used herein, a F(ab) fragment also refers to one light chain containing two variable domains separated by an amino acid linker, and one light chain containing two variable domains and a C separated by an amino acid linker. H1 The antibody may comprise one heavy chain containing the domain.

[0057] F(ab') fragments typically contain one light chain and one H1 and C H2 The F(ab')2 molecule comprises a portion of one heavy chain that contains an additional constant region (between the F(ab')2 domains) such that interchain disulfide bonds can form between the two heavy chains to form the F(ab')2 molecule.

[0058] The term "binding protein," as used herein, refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen. Trispecific binding proteins of the present disclosure, unless otherwise specified, typically comprise four polypeptide chains that form at least three antigen-binding sites, with the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] having a structure represented by The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] having a structure represented by The third polypeptide chain has the formula: V H3 -C H1 [III] having a structure represented by The fourth polypeptide chain has the formula: V L3 -C L [IV] having a structure represented by Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C Lis an immunoglobulin light chain constant domain; C H1 is immunoglobulin C H1 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair.

[0059] A "recombinant" molecule is one that is produced, expressed, created, or isolated by recombinant means.

[0060] One embodiment of the disclosure provides a binding protein having biological and immunological specificity for one to three target antigens. Another embodiment of the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Another embodiment of the disclosure provides an expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide chain forming such a binding protein. Yet another embodiment of the disclosure provides a host cell that expresses such a binding protein (i.e., comprises a nucleic acid molecule or vector encoding a polypeptide chain forming such a binding protein).

[0061] The term "swappability" as used herein refers to the interchangeability of variable domains within a binding protein format and with retention of folding and ultimately binding affinity. "Full swappability" refers to the interchangeability of variable 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 retention of binding affinity. H1 and V H2 The order of both domains, and therefore V L1 and V L2 It refers to the ability to swap (i.e. reverse) the order of domains.H and V L It should be noted that the designation simply refers to the location of the domain on a particular protein chain in the final format. For example, V H1 and V H2 is the V in the parent antibody L1 and V L2 domains, V in binding proteins H1 and V H2 Similarly, V L1 and V L2 is the V in the parent antibody H1 and V H2 domains, V in binding proteins H1 and V H2 Therefore, V H and V L The designation refers to the current location, not the original location in the parent antibody. H and V L Domains are therefore "swappable".

[0062] The term "antigen" or "target antigen" or "antigen target" as used herein refers to a molecule or portion of a molecule to which a binding protein can bind and which can additionally be used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. Binding For each target antigen recognized by a protein, the binding protein can compete with intact antibodies that recognize the target antigen.

[0063] The term "Her2" refers to human epidermal growth factor receptor 2, a member of the epidermal growth factor receptor family.

[0064] "CD3" is cluster of differentiation factor 3 polypeptide, typically a T cell surface protein that is part of the T cell receptor (TCR) complex.

[0065] "CD28" is cluster of differentiation 28 polypeptide, a T cell surface protein that provides a costimulatory signal for T cell activation and survival.

[0066] "CD38" is cluster of differentiation 38 polypeptide, a glycoprotein found on the surface of many immune cells.

[0067] The term "T cell engager" refers to a binding protein that is directed to a tumor targeting protein in addition to being directed to the host's immune system, and more specifically, the cytotoxic activity of T cells.

[0068] The term "monospecific binding protein" refers to a binding protein that specifically binds to one antigen target.

[0069] The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

[0070] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigen targets.

[0071] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[0072] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigen targets.

[0073] The term "trivalent binding protein" refers to a binding protein having three binding sites. In certain embodiments, the trivalent binding protein can bind to one antigen target. In other embodiments, the trivalent binding protein can bind to two antigen targets. In other embodiments, the trivalent binding protein can bind to three antigen targets.

[0074] An "isolated" binding protein is a binding protein that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the binding protein, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence 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 is one that is free of at least one component of the binding protein's natural environment. Examples include binding proteins in situ within recombinant cells.

[0075] The terms "substantially pure" or "substantially purified," as used herein, refer to a compound or species that is the predominant species present (i.e., it is more abundant than any other individual species in the composition on a molar basis). In some embodiments, a substantially purified fraction is a composition in which the species constitutes at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition is a composition in which all macromolecular species present in the composition constitute greater than about 80%, 85%, 90%, 95%, or 99%. In still other embodiments, the species is purified to an essential homogeneity state in which the composition consists essentially of a single macromolecular species (contaminant species cannot be detected in the composition by conventional detection methods).

[0076] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have distinctive three-dimensional structural features and / or distinctive charge characteristics. An epitope is the 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, the equilibrium dissociation constant is ≦10 -8 M, more preferably the equilibrium dissociation constant is ≦10 -9 M, most preferably the dissociation constant is ≦10 -10 When it is M, the binding protein is said to specifically bind to the antigen.

[0077] Dissociation constant of the binding protein (K D ) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis measures real-time binding interactions 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, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). "K D The term "dissociation constant" as used herein refers to the dissociation constant of the interaction between a particular binding protein and a target antigen.

[0078] The term "specifically binds" as used herein means a concentration of about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10-9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 It refers to the ability of a binding protein or antigen-binding fragment thereof to bind to an antigen containing the epitope with a Kd of M or less and / or to bind to the epitope with an affinity at least two-fold higher than its affinity for a nonspecific antigen.

[0079] In some embodiments, the antigen-binding domains and / or binding proteins of the present disclosure are "cross-reactive" with human and cynomolgus CD38 polypeptides, e.g., CD38 extracellular domain, human CD38 isoform A, human CD38 isoform E, and cynomolgus CD38. 50 The binding of a binding protein to antigen 1 (Ag1) is "cross-reactive" to antigen 2 (Ag2) if the affinity of Ag1 is within a similar range for both antigens. In this application, the binding of a binding protein to Ag1 is cross-reactive to Ag2 if the ratio of the affinity of Ag2 to the affinity of Ag1 is equal to or less than 20, where affinity is measured using the same method for both antigens.

[0080] The term "linker" as used herein refers to an immunoglobulin domain. Linker refers to one or more amino acid residues inserted between the V and H domains to provide sufficient mobility to the light and heavy domains to fold into a crossover dual variable region immunoglobulin. The linker is inserted at the transition between the variable domains or between the variable and constant domains at the sequence level. The approximate sizes of immunoglobulin domains are well understood, allowing the transition between domains to be identified. The precise location of the domain transition can be determined by locating secondary structure elements, such as peptide stretches that do not form beta sheets or alpha-helices, as demonstrated by experimental data or as can be predicted by modeling or secondary structure prediction techniques. The linkers described herein are V L2C-terminus and V-terminus of the domain L1 L1, located on the light chain between the N-terminus of the domain; and V L1 The C-terminus and C L Located on the light chain between the N-terminus of the domains is L2. The heavy chain linker is V H1 C-terminus and V-terminus of the domain H2 L3, located between the N-terminus of the domain; and V H2 The C-terminus and C H1 Located between the N-terminus of the domain is known as L4.

[0081] The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are sometimes used interchangeably herein, as plasmids are the most commonly used form of vector. However, the present disclosure is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0082] The phrase "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, since certain modifications may occur in successive generations, either due to mutations or environmental influences, but such cells are still included within the scope of the term "host cell" as used herein. A variety of 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. To recombinantly express the binding protein, the host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chain of the binding protein, so that the polypeptide chain is expressed in the host cell and is preferably secreted into the medium in which the host cell is cultured, from which the binding protein can be recovered.

[0083] The term "transformation" as used herein refers to the alteration of the genetic characteristics of a cell. "Transfection" refers to a change in the DNA of a cell, and a cell is transformed when it is modified to contain new DNA. For example, a cell is transformed and genetically modified from its native state. After transformation, the transforming DNA can recombine with the cell's DNA by being physically integrated into the cell's chromosome, or can be maintained transiently as an episomal element without being replicated, or can be replicated independently as a plasmid. A cell is considered stably transformed when the DNA is replicated with the division of the cell. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell is "transfected" when the exogenous DNA is introduced inside the cell membrane. Numerous transfection techniques are well known in the art. Using such techniques, one or more exogenous DNA molecules can be introduced into a suitable host cell.

[0084] The term "naturally occurring" as used herein, when applied to an object, 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 that can be isolated from a source in nature and exists in an organism (including viruses) that has not been intentionally modified by humans is naturally occurring. Similarly, "non-naturally occurring" as used herein refers to an object that is not found in nature or that has been structurally modified or synthesized by humans.

[0085] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids); non-natural amino acids and analogs, such as α-, α-disubstituted amino acids, N-alkylamino acids, lactic acid, and other non-conventional amino acids, may also be suitable components for the polypeptide chain of binding proteins. Examples of non-conventional 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, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction, in accordance with standard usage and convention.

[0086] Naturally occurring residues can be divided into classes based on common side chain properties: (1) Hydrophobicity: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) Polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) Aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) Aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; (5) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) Acidic: Asp, Glu; (7) Basic: His, Lys, Arg; (8) Residues affecting chain orientation: Gly, Pro; (9) Aromatic: His, Trp, Tyr, Phe; and (10) Aromatic hydrophobic: Phe, Trp, Tyr.

[0087] Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.

[0088] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptide chain of binding protein.For example, those skilled in the art can identify suitable sections of the polypeptide chain that can be changed without destroying activity by targeting regions that do not seem to be important for activity.Alternatively, those skilled in the art can identify residues and parts of molecules that are conserved between similar polypeptides.In addition, even sections that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0089] The term "patient", as used herein, includes human and animal subjects.

[0090] The term "treatment" or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those with a disorder as well as those predisposed to having a disorder or those in whom a disorder should be prevented. In certain embodiments, the binding proteins can be used to treat humans with or susceptible to cancer, or to ameliorate cancer in human subjects. The binding proteins can also be used to prevent cancer in human patients. In certain 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, and in other embodiments, the binding proteins can be used to treat humans with or susceptible to an inflammatory disorder, or to ameliorate an inflammatory disorder in human subjects.

[0091] The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0092] The term "pharmacologically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for achieving or enhancing delivery of a binding protein.

[0093] The terms "effective amount" and "therapeutically effective amount", when used in reference to a pharmaceutical composition comprising one or more binding proteins, refer to an amount or dosage sufficient to produce a 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 over a period of time. The effective amount may vary depending on the specific binding protein being used, as well as on various factors and conditions related to the patient being treated and the severity of the disorder. For example, when the binding protein is administered in vivo, factors such as the age, weight, and health of the patient, as well as dose-response curves and toxicity data obtained in preclinical animal studies, are among the factors to be considered. The determination of the effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capabilities of one of ordinary skill in the art.

[0094] One embodiment of the disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of a binding protein.

[0095] Trispecific and / or trivalent binding proteins Certain embodiments of the present disclosure relate to trispecific and / or trivalent binding proteins that include four polypeptide chains that form three antigen binding sites that specifically bind to one or more target proteins, the first of the polypeptides that form the binding protein comprising The pair relates to binding proteins having dual variable domains with crossover orientation and a second pair of polypeptides forming the binding protein having a single variable domain. Any CDR or variable domain of any of the antigen binding proteins described herein may have use in the trispecific binding proteins of the disclosure.

[0096] 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 that form the three antigen-binding sites, the first polypeptide chain having the formula: V L2 -L1-V L1 -L2-C L [I] The structure includes: The second polypeptide chain has the formula: V H1 -L3-V H2 -L4-C H1 - Hinge-C H2 -C H3 [II] The structure includes: The third polypeptide chain has the formula: V H3 -C H1 - Hinge-C H2 -C H3 [III] The structure includes: The fourth polypeptide chain has the formula: V L3 -C L [IV] The structure includes: Where: V L1 is a first immunoglobulin light chain variable domain; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; V H3 is a third immunoglobulin heavy chain variable domain; C L is an immunoglobulin light chain constant domain; C H1is immunoglobulin C H1 a heavy chain constant domain; C H2 is immunoglobulin C H2 a heavy chain constant domain; C H3 is immunoglobulin C H3 a heavy chain constant domain; The hinge is C H1 and C H2 an immunoglobulin hinge region connecting the domains; L1, L2, L3 and L4 are amino acid linkers; The polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair.

[0097] It is contemplated that any of the antigen-binding sites described herein may have use in the trispecific binding proteins of the present disclosure, e.g., comprising four polypeptide chains having the structures described above. For example, in some embodiments, the trispecific binding proteins of the present disclosure comprise a V H1 and V L1 A domain pair, V, that forms a second antigen-binding site that binds to the CD3 polypeptide H2 and V L2 The domain pair, as well as the V domain, which forms the third antigen-binding site H3 and V L3 In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide. H1 and V L1 A domain pair, V, that forms a second antigen-binding site that binds to the CD3 polypeptide H2 and V L2 The domain pair, as well as the V domain, which forms the third antigen-binding site H3 and V L3 In some embodiments, the trispecific binding protein of the present disclosure comprises a V domain pair that forms a first antigen-binding site. H1 and V L1A domain pair, V, that forms a second antigen-binding site that binds to the CD3 polypeptide H2 and V L2 The V domain pairs together with the V domain to form a third antigen-binding site that binds to tumor-targeting proteins. H3 and V L3 In some embodiments, the domain pair The trispecific binding protein of the present disclosure comprises a V that forms a first antigen-binding site that binds to a CD28 polypeptide. H1 and V L1 A domain pair, V, that forms a second antigen-binding site that binds to the CD3 polypeptide H2 and V L2 The V domain pairs together with the V domain to form a third antigen-binding site that binds to tumor-targeting proteins. H3 and V L3 In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide. H1 and V L1 A domain pair, V, that forms a second antigen-binding site that binds to the CD3 polypeptide H2 and V L2 The domain pair, as well as the V domain, form a third antigen-binding site that binds to the CD38 polypeptide. H3 and V L3 In some embodiments, the trispecific binding proteins of the present disclosure comprise a V domain pair that forms a first antigen-binding site that binds to a CD28 polypeptide. H1 and V L1 A domain pair, V, that forms a second antigen-binding site that binds to the CD3 polypeptide H2 and V L2 The domain pair, as well as the V domain, which forms a third antigen-binding site that binds to the HER2 polypeptide. H3 and V L3 Contains domain pairs.

[0098] In some embodiments, the binding proteins of the present disclosure bind to one or more tumor targeting proteins and one or more T cell targeting proteins. In some embodiments, the binding proteins can specifically bind to one tumor targeting protein and two different epitopes on a single T cell targeting protein. In some embodiments, the binding proteins can specifically bind to one tumor targeting protein and two different T cell targeting 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 targeting proteins, and the third and fourth polypeptide chains of the binding protein form an antigen binding site that specifically binds to a tumor targeting protein. In some embodiments, the targeting protein is CD38 or HER2. Additional tumor targeting proteins are provided below. In some embodiments, the one or more T cell targeting proteins are one or more of CD3 and CD28. Exemplary, non-limiting polypeptides that may have use in any of the trispecific binding proteins described herein are provided in Table 1.

[0099] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0100] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0101] In some embodiments, the binding proteins of the present disclosure form three antigen-binding sites. the first polypeptide chain comprises an 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 an 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 an 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 an 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.

[0102] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0103] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0104] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0105] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0106] In some embodiments, the binding proteins of the present disclosure form three antigen-binding sites. the first polypeptide chain comprises an 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 an 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 an 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 an 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.

[0107] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0108] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0109] In some embodiments, a binding protein of the 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.

[0110] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0111] In some embodiments, the binding proteins of the present disclosure form three antigen-binding sites. the first polypeptide chain comprises an 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 an 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 an 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 an 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.

[0112] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0113] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0114] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0115] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0116] In some embodiments, the binding proteins of the present disclosure form three antigen-binding sites. the first polypeptide chain comprises an 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 an 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 an 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 an 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.

[0117] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0118] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0119] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0120] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0121] In some embodiments, the binding proteins of the present disclosure form three antigen-binding sites. the first polypeptide chain comprises an 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 an 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 an 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 an 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.

[0122] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0123] In some embodiments, a binding protein of the disclosure comprises four polypeptide chains that form three antigen-binding sites, wherein a 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; a 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; a 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 a 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.

[0124] Anti-CD38 binding site 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, including, but not limited to, the polypeptide represented by NCBI Accession No. NP_001766.2, or the polypeptide produced from NCBI Gene Identification No. 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.

[0125] In some embodiments, any of the CDRs and / or variable domains of the anti-CD38 binding sites described below may be used in a monospecific antibody.

[0126] In other embodiments, any of the CDRs and / or variable domains of the anti-CD38 binding sites described below may be used in any binding site of a trispecific binding protein comprising, for example, four polypeptides forming the three antigen-binding sites described above. In certain embodiments, an antigen-binding site that binds to a CD38 polypeptide may be used in any binding site of a trispecific binding protein comprising, for example, four polypeptides forming the three antigen-binding sites described above. The binding protein is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites as described above, H3 and V L3 The pair of domains forms a third antigen-binding site that binds to the CD38 polypeptide.

[0127] Various features of exemplary binding sites and binding proteins are described herein. For example, in some embodiments, anti-CD38 binding sites cross-react with human CD38 (e.g., human CD38 isoform A and / or isoform E polypeptide) and cynomolgus monkey CD38. In some embodiments, binding proteins comprising anti-CD38 binding sites induce apoptosis of CD38+ cells. In some embodiments, binding proteins comprising anti-CD38 binding sites recruit T cells to CD38+ cells and optionally activate T cells (e.g., via TCR stimulation and / or co-stimulation).

[0128] In some embodiments, the binding site that binds to 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 to 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).

[0129] In some embodiments, the binding site that binds to 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 to 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).

[0130] In some embodiments, the binding site that binds to 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 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-H3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO: 21). In some embodiments, the binding site that binds to 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).

[0131] In some embodiments, the binding site that binds to 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 to 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).

[0132] In some embodiments, the binding site that binds to 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 to 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).

[0133] 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); an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of YG (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).

[0134] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79); and / or DIVLTQSPATLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO: 80). In some embodiments, the binding site that binds CD38 comprises an antibody heavy chain variable (VH) domain comprising an amino acid sequence 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 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:80.

[0135] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSTDTAYMEMNSLRSEDTAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81); and / or EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO: 82). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:82.

[0136] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83); and / or The antibody light chain variable (VL) domain comprises an amino acid sequence 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 SEQ ID NO: 83. DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO: 84). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:84.

[0137] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87); and / or DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO: 88). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:88.

[0138] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89); and / or The antibody light chain variable (VL) domain comprises an amino acid sequence 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 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:90.

[0139] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEGFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); and / or DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO: 86). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:86.

[0140] In some embodiments, the binding site that binds to CD38 is QVQLQQSGPELVRPGTSVKVSCKASGYAFTTYLVEWIKQRPGQGLEWIGVINPGSGSTNYNEKFKGKATLTVDRSSTTAYMHLSGLTSDDSAVYFCARYAYGYWGQGTTLTVSS (Sequence number 2 77), and / or DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQQPGHSPKQLIYSASNRYTGVPDRFTGSGAGTDFTLTISNIQSEDLADYFCQQYSTYPFTFGSGTKLEIK (SEQ ID NO: 278). In some embodiments, the binding site that binds 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 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 and / or VL domains are humanized.

[0141] In some embodiments, the binding site that binds to CD38 is an antibody heavy chain variable (VH) 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 QVQLLQSGAELVRPGVSVKISCTGSGYSFTNYAVHWVKQSHVKSLEWIGVISPYYGDTTYNQKFTGKATMTVDKSSSTAYMELARLTSEDSAIYFCARRFEGFYYSMDYWGQGTSVTVSS (SEQ ID NO: 279); and / or DVVMIQTPLSLPVSLGDQASISCRPSQSLVHSNGNTYLNWYLQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYLCSQSTHVPLTFGSGTQLEIK (SEQ ID NO: 280). In some embodiments, the binding site that binds 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 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 and / or VL domains are humanized.

[0142] 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 comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II are , forming a crossover 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 are H3 and V L3 and forms a third antigen-binding site that binds to the CD38 polypeptide.H1 and V L1 forms a first antigen-binding site that binds to the CD28 polypeptide, and V H2 and V L2 form a second antigen-binding site that binds to a CD3 polypeptide, and the VH and VL domains of any of the anti-CD38 antigen-binding sites described above and / or in Table 2 are H3 and V L3 which forms a third antigen-binding site that binds to the CD38 polypeptide.

[0143] Exemplary anti-CD38 antigen binding site sequences are provided in Table 2. In some embodiments, a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD38 antibody set forth in Table 2. In some embodiments, a binding protein comprising an anti-CD38 antigen binding site of the present disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-CD38 antibody set forth in Table 2.

[0144] [Table 1] [Table 2] [Table 3]

[0145] Further provided herein are antibodies (eg, monospecific antibodies) comprising any of the above-described anti-CD38 CDRs and / or variable domains.

[0146] 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 to an antigen are described herein and known in the art, including (but not limited to) ELISA, SPR, and flow cytometry assays.

[0147] Anti-HER2 binding site Certain aspects of the present disclosure relate to binding proteins that comprise an antigen-binding site that binds to HER2 polypeptide.In some embodiments, the HER2 polypeptide is the 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, the 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 the polypeptides produced by NCBI gene identification number 2064. In some embodiments, a 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, a binding protein comprising an antigen-binding site that binds to a HER2 polypeptide is a trispecific binding protein comprising four polypeptides forming three antigen-binding sites as described above, V H3 and V L3 The pair of domains forms a third antigen-binding site that binds to the HER2 polypeptide.

[0148] In some embodiments, the binding site that binds to HER2 comprises the CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1) or GFNIRDTY (SEQ ID NO: 2), IYPTNGYT (SEQ ID NO: 3), IYPTQGYT (SEQ ID NO: 4), or IYPT an antibody heavy chain variable (VH) domain comprising a CDR-H2 sequence comprising the amino acid sequence of NAYT (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) 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 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) 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).

[0149] 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).

[0150] 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).

[0151] 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).

[0152] 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).

[0153] 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).

[0154] In some embodiments, the binding site that binds to HER2 comprises the CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: 1), the amino acid sequence of IYPTNGYT (SEQ ID NO: 3), and / or an antibody heavy chain variable (VH) domain comprising a CDR-H2 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO: 6), 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).

[0155] 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).

[0156] 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).

[0157] In some embodiments, the binding site that binds to HER2 comprises the CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: 2), the amino acid sequence of IYPTQGYT (SEQ ID NO: 4), an antibody heavy chain variable (VH) domain comprising a CDR-H2 sequence comprising the amino acid sequence of SRWGGSGFAMDY (SEQ ID NO: 8), 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).

[0158] 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).

[0159] In some embodiments, the binding site that binds to HER2 is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74) 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least 145%, at least 146%, at least 147%, at least 148%, at least 149 ... an antibody heavy chain variable (VH) domain comprising an amino acid sequence that is at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the The antibody comprises 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 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 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.

[0160] In some embodiments, the binding site that binds to HER2 is an antibody heavy chain variable (VH) 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 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72); and / or The antibody light chain variable (VL) domain comprises an amino acid sequence 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 SEQ ID NO: 72. DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.

[0161] In some embodiments, the binding site that binds to HER2 is At least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% or more of the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73). %, 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 amino acid sequence to the antibody heavy chain variable (VH) domain; and / or The antibody light chain variable (VL) domain comprises an amino acid sequence 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 SEQ ID NO: 73. DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.

[0162] In some embodiments, the binding site that binds to HER2 is an antibody heavy chain variable (VH) 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 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 75); and / or The antibody light chain variable (VL) domain comprises an amino acid sequence 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 SEQ ID NO: 75. DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.

[0163] In some embodiments, the binding site that binds to HER2 is an antibody heavy chain variable (VH) 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 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74); and / or DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising an amino acid sequence 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 SEQ ID NO: 74. In some embodiments, the binding site that binds to HER2 comprises an antibody heavy chain variable (VH) domain comprising an amino acid sequence of SEQ ID NO: 74, and / or an antibody light chain variable (VL) domain comprising an 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 an amino acid sequence of SEQ ID NO: 74, and an antibody light chain variable (VL) domain comprising an amino acid sequence of SEQ ID NO: 77.

[0164] In some embodiments, the binding site that binds to HER2 is an antibody heavy chain variable (VH) 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 EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76); and / or The antibody light chain variable (VL) domain comprises an amino acid sequence 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 SEQ ID NO: 76. DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:77.

[0165] In some embodiments, the binding site that binds to HER2 is an antibody heavy chain variable (VH) 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 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72); and / or 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 200%, at least 201%, at least 202%, at least 203%, at least 204%, at least 205%, at least 206%, at least 207%, at least 208%, at least 209%, at least 300%, at least 301%, at least 302%, at least 3 In some embodiments, the binding site that binds 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 HER2 comprises an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO: 72, and an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 78.

[0166] In some embodiments, an anti-HER2 antigen-binding site of the present disclosure comprises one, two, three, four, five, or all six 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, an anti-HER2 antigen-binding site of the present disclosure comprises the VH and / or VL domain sequences of the anti-HER2 antibody trastuzumab, 30R / 55Q / 102E, 30R / 56A / 102S, 30R / 55Q / 102S, 30R / 56A / 102E, or 30Q.

[0167] Exemplary anti-HER2 antigen binding site sequences are provided in Table 3. In some embodiments, an anti-HER2 antigen binding site of the disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-HER2 antibody set forth in Table 3. In some embodiments, an anti-HER2 antigen binding site of the disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-HER2 antibody set forth in Table 3.

[0168] [Table 4] [Table 5]

[0169] Other anti-tumor target binding sites In some embodiments, the binding protein of the present disclosure comprises an antigen-binding site that binds to a tumor targeting protein. In some embodiments, the tumor targeting protein is a CD38 polypeptide (e.g., a human CD38 polypeptide). In some embodiments, the tumor targeting protein is a HER2 polypeptide (e.g., a human HER2 polypeptide).In some embodiments, tumor targeting proteins of the present disclosure include 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-9), CCL9 (also known as MCP-1b), CCL10 (also known as MCP-1b), CCL11 (also known as MCP-1b), CCL12 (also known as MCP-1c), CCL13 (also known as MCP-1b), CCL14 (also known as MCP-1c), CCL15 (also known as MCP-1b), CCL16 (also known as MCP-1c), CCL17 (also known as MCP-1b), CCL18 (also known as MCP-1c), CCL19 (also known as MCP-1b), CCL20 (also known as MCP-1c), CCL21 (also known as MCP-1b), CCL22 (also known as MCP-1c), CCL23 (also known as MIP-1a), CCL24 (also known as MIP-1b), CCL25 (also known as MCP-1c), CCL26 (also known as MCP-1c), CCL27 (also known as MCP-1c), CCL28 (also known as MCP-1c), CCL29 (also known as MCP-1c), CCL210 (also known as MCP-1 -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 (FCER2, a receptor for IgE) Also known as 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), (also known as PD-1), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), 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, IGF1R, IL2Rbeta, 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, L These include, but are not limited to, PFS2, 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 coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5 / CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0170] Anti-CD28 binding site Certain aspects of the present disclosure relate to binding proteins comprising an antigen-binding site that binds to 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, including, but not limited to, the polypeptides represented by NCBI accession numbers XP_011510499.1, XP_011510497.1, XP_011510496.1, NP_001230007.1, NP_001230006.1, or NP_006130.1, or the polypeptides produced from NCBI gene identification number 940. In some embodiments, the binding proteins comprising an antigen-binding site that binds to a CD28 polypeptide are 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 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, one of the three antigen-binding sites binds to a CD28 polypeptide and one of the three antigen-binding sites 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, one of the three antigen-binding sites binds to a CD28 polypeptide, one of the three antigen-binding sites binds to a CD3 polypeptide and one of the three antigen-binding sites 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, one of the three antigen-binding sites binds to a CD28 polypeptide, one of the three antigen-binding sites binds to a CD3 polypeptide and one of the three antigen-binding sites binds to a HER2 polypeptide.In some embodiments, a 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, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a tumor targeting protein.

[0171] In some embodiments, the binding site that binds to CD28 is GYTFTSYY (sequence 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 to 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).

[0172] In some embodiments, the binding site that binds to CD28 is an antibody heavy chain variable (VH) 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 QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); and / or DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:92.

[0173] 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 targeting protein (such as, 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 comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD28 antigen-binding sites described above are VH and VL domains of ... H1 and V L1 and forms a first antigen-binding site that binds to a CD28 polypeptide. In some embodiments, the VH and VL domains of any of the anti-CD28 antigen-binding sites described above and / or in Table 4 represent V H1 and V L1 represents the CD28 polypeptide. The first antigen-binding site binds to the V H2 and V L2 forms a second antigen-binding site that binds to the CD3 polypeptide, and V H3 and V L3 forms a third antigen-binding site that binds to a tumor targeting protein (such as, but not limited to, CD38 or HER2).

[0174] Exemplary anti-CD28 antigen-binding site sequences are provided in Table 4. In some embodiments, an anti-CD28 antigen-binding site of the disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD28 antibody set forth in Table 4. In some embodiments, an anti-CD28 antigen-binding site of the disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-CD28 antibody set forth in Table 4.

[0175] [Table 6]

[0176] Anti-CD3 binding site 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, such as CD3-delta (also known as T3D, IMD19, and CD3-delta), CD3-epsilon (also known as T3E, IMD18, and TCRE), and CD3-gamma (also known as T3G, IMD17, and CD3-gamma). Human CD3 polypeptides are known in the art, including, but not limited to, the polypeptides represented by NCBI accession numbers XP_006510029.1 or NP_031674.1, or the polypeptides produced from NCBI gene identification numbers 915, 916, or 917. In some embodiments, binding proteins comprising an antigen-binding site that binds to a CD3 polypeptide are monospecific and / or monovalent, bispecific and / or bivalent, trispecific and / or trivalent, or is 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, one of the three antigen-binding sites binds to a CD28 polypeptide and one of the antigen-binding sites 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, one of the three antigen-binding sites binds to a CD28 polypeptide, one of the three antigen-binding sites binds to a CD3 polypeptide and one of the antigen-binding sites 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, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which 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, one of which binds to a CD28 polypeptide, one of which binds to a CD3 polypeptide, and one of which binds to a tumor targeting protein.

[0177] 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), 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), where X1 is E or Q, X2 is A or L, and X3 is Q, R, or F. 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).

[0178] 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).

[0179] 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).

[0180] 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).

[0181] 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).

[0182] In some embodiments, the binding site that binds to CD3 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQ The antibody comprises an antibody light chain variable (VL) domain comprising an amino acid sequence 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 selected from the group consisting of: YPFTFGSGTKVEIK (SEQ ID NO:97), and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98). In some embodiments, the binding site that binds 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 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.

[0183] In some embodiments, the binding site that binds to CD3 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95). The binding site that binds 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 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 the amino acid sequence of SEQ ID NO: 95.

[0184] In some embodiments, the binding site that binds to CD3 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:96.

[0185] In some embodiments, the binding site that binds to CD3 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97). In some embodiments, the binding site that binds 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 an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:97. Contains an antibody light chain variable (VL) domain.

[0186] In some embodiments, the binding site that binds to CD3 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 98). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.

[0187] In some embodiments, the binding site that binds to CD3 is an antibody heavy chain variable (VH) 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 302); and / or DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95). In some embodiments, the binding site that binds 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 an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO: 95.

[0188] In some embodiments of any of the above embodiments, the binding protein is a trispecific binding protein. In some embodiments, the trispecific binding protein is a In some embodiments, the binding protein comprises an antigen-binding site that binds a tumor targeting protein (such as, but not limited to, CD38 or HER2), an antigen-binding site that binds a CD28 polypeptide, and an antigen-binding site that binds a CD3 polypeptide. In some embodiments, the binding protein is a trispecific binding protein comprising four polypeptides comprising three antigen-binding sites, wherein the polypeptide of formula I and the polypeptide of formula II form a crossover light chain-heavy chain pair (e.g., as described herein). In some embodiments, the VH and VL domains of any of the anti-CD3 antigen-binding sites described above are VH and VL domains of any of the anti-CD3 antigen-binding sites described above. H2 and V L2 and forms a second antigen-binding site that binds to the CD3 polypeptide. H1 and V L1 The VH and VL domains of any of the anti-CD3 antigen-binding sites described above and / or in Table 5 form a first antigen-binding site that binds to a CD28 polypeptide. H2 and V L2 which forms a second antigen-binding site which binds to the CD3 polypeptide, and V H3 and V L3 forms a third antigen-binding site that binds to a tumor targeting protein (such as, but not limited to, CD38 or HER2).

[0189] Exemplary anti-CD3 antigen-binding site sequences are provided in Table 5. In some embodiments, an anti-CD3 antigen-binding site of the disclosure comprises one, two, three, four, five, or all six CDR sequences of an anti-CD3 antibody set forth in Table 5. In some embodiments, an anti-CD3 antigen-binding site of the disclosure comprises a VH domain sequence and / or a VL domain sequence of an anti-CD3 antibody set forth in Table 5.

[0190] [Table 7] [Table 8]

[0191] Linker In some embodiments, linkers L1, L2, L3, and L4 range in length from no amino acids (length=0) to about 100 amino acids, or are less than 100, 50, 40, 30, 20, or 15 amino acids, or fewer. Linkers may be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long. L1, L2, L3, and L4 in one binding protein may all have the same amino acid sequence or may all have different amino acid sequences.

[0192] Examples of suitable linkers include, for example, GGGGSGGGGS (SEQ ID NO: 69), GGGSGGGGSGGGGGS (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 Publications WO2017 / 074878 and WO2017 / 180913. The above-listed examples are not intended to limit the scope of the 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.

[0193] The identity and sequence of the amino acid residues in the linker can be changed depending on the type of secondary structure element that needs to be achieved in the linker.For example, glycine, serine, and alanine are best for linkers with maximum flexibility.If a more rigid and long linker is required, some combinations of glycine, proline, threonine, and serine are useful.Any amino acid residue can be considered as a linker in combination with other amino acid residues to construct larger peptide linkers that are required depending on the desired properties.

[0194] In some embodiments, the length of L1 is at least twice the length of L3. In an embodiment, the length of L2 is at least twice as long as L4. In some embodiments, the length of L1 is at least twice as long as L3, and the length of L2 is at least twice as long as L4. In some embodiments, L1 is 3-12 amino acid residues long, L2 is 3-14 amino acid residues long, L3 is 1-8 amino acid residues long, and L4 is 1-3 amino acid residues long. In some embodiments, L1 is 5-10 amino acid residues long, L2 is 5-8 amino acid residues long, L3 is 1-5 amino acid residues long, and L4 is 1-2 amino acid residues long. In some embodiments, L1 is 7 amino acid residues long, L2 is 5 amino acid residues long, L3 is 1 amino acid residue long, and L4 is 2 amino acid residues long.

[0195] 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), GGGSGGGGSGGGGGS (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 are each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGSGGGGSGGGGGS (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.

[0196] 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).

[0197] Fc Region and Constant Domains In some embodiments, the binding protein of the present disclosure is H1 and a second polypeptide chain further comprising an Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 In some embodiments, the binding proteins of the present disclosure comprise an immunoglobulin heavy chain constant domain. H1 and a third polypeptide chain further comprising an Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 In some embodiments, the binding proteins of the present disclosure comprise an immunoglobulin heavy chain constant domain. H1 a second polypeptide chain further comprising an Fc region linked to said second polypeptide chain, said Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain, and H1 a third polypeptide chain further comprising an Fc region linked to the Fc region, the Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain.

[0198] In some embodiments, the binding proteins of the present disclosure comprise a full-length antibody heavy chain or polypeptide chain comprising an Fc region. In some embodiments, the Fc region is a human Fc region, e.g., an Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region comprises an antibody hinge, C H1 , C H2 , C H3 , and optionally C H4In some embodiments, the Fc region comprises 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 of the 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 proteins shown in Table 4. In some embodiments, the Fc region comprises a human IgG1 Fc region. In some embodiments, the Fc region comprises a human IgG4 Fc region. In some embodiments, the Fc region comprises one or more of the 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 proteins shown in Table 4. In some embodiments, the heavy chain constant region is the constant region of one of the heavy chain polypeptides (e.g., polypeptide 2 or 3) of the binding proteins shown in Table 4. In some embodiments, the light chain constant region is the constant region of one of the light chain polypeptides (e.g., polypeptide 1 or 4) of the binding proteins shown in Table 4.

[0199] In some embodiments, the binding protein of the present disclosure comprises one or two Fc variants. The term "Fc variant" as used herein 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 the salvage receptor 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. Additionally, the native Fc contains regions that can be removed to provide structural features or biological activities that are not required for the antibody-like binding protein of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues, or have one or more altered Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0200] In some embodiments, a binding protein of the disclosure (e.g., a trispecific binding protein) comprises a "knob" mutation in the second polypeptide chain and a "hole" mutation in the third polypeptide chain. In some embodiments, a binding protein of the disclosure comprises a "knob" mutation in the third polypeptide chain and a "hole" mutation in the second polypeptide chain. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and / or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the "knob" mutation comprises a substitution at a position corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitution is S354C and T366W. In some embodiments, the "hole" mutation comprises a substitution at a position corresponding to position 407, and optionally positions 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 a substitution at a position 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.

[0201] 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 a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 and optionally position 354 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W or T366Y and optionally S354C; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to position 407 and optionally positions 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being Y40 7V or Y407T and optionally Y349C, T366S, and / or L368A.

[0202] 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 a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 407 and optionally 349, 366, and / or 368 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y407V or Y407T and optionally Y349. and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising an amino acid substitution at a position corresponding to 366 and optionally 354 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W or T366Y and optionally S354C.

[0203] 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 a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to positions 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V.

[0204] 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 a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising an amino acid substitution at a position corresponding to position 366, 368, and / or 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366S, L368A, and / or Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitution being T366W.

[0205] 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 a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the second Fc region comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V. 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 a CH2 and CH3 immunoglobulin hinge region. The first polypeptide chain comprises an immunoglobulin heavy chain constant domain, the first 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, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprises an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being 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.

[0206] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K; The polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, Y349C, T366S, L368A, Y4 07V, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to 228, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, S354C, T366W, and R409K.

[0207] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 234, 235, 354, and 366 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, S354C, and T366W; In some embodiments, the polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, the amino acid substitutions being F234A, L235A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains. and the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region being an Fc region of human IgG4 comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the second Fc region comprising amino acid substitutions at positions corresponding to positions 234, 235, 349, 366, 368, and 407 of human IgG4 according to the EU index, and the amino acid substitutions are F234A, L235A, Y349C, T366S, L368A, and Y407V.

[0208] In some embodiments, the binding proteins of the present disclosure comprise one or more mutations that can reduce effector function, such as Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). H1 and a first Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 the third polypeptide chain comprises an immunoglobulin heavy chain constant domain; H1 and a second Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 The polypeptide chains each comprise an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG1 Fc regions; the first and second Fc regions each comprise an amino acid substitution at a position corresponding to 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions; the Fc regions each comprise an amino acid substitution at a position corresponding to 234 and 235 of human IgG1 according to the EU index, the amino acid substitutions being L234A and L235A. In some embodiments, the second polypeptide chain comprises a C H1 and a first Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3 the third polypeptide chain comprises an immunoglobulin heavy chain constant domain; H1 and a second Fc region linked to the hinge region of an immunoglobulin as well as a C H2 and C H3the first and second Fc regions are human IgG1 Fc regions; and the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, the amino acid substitutions being L234A, L235A, and P329A. In some embodiments, the Fc region of the second and third polypeptide chains is a human IgG4 Fc region, and the Fc region comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, the amino acid substitutions being F234A and L235A. H1 a second polypeptide chain further comprising a first Fc region linked to said first polypeptide chain, said first Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain, and H1 a third polypeptide chain further comprising a second Fc region linked to said third polypeptide chain, said second Fc region being linked to a hinge region and a C region of an immunoglobulin; H2 and C H3 a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, respectively, the amino acid substitutions being F234A and L235A.

[0209] In some embodiments, the second polypeptide chain comprises a first Fc region linked to CH1. the first Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, the first Fc region comprising amino acid substitutions at positions corresponding to positions 228, 234, 235, 354, 366, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, S354C, T366W, and R409K; the third polypeptide chain further comprises a second Fc region linked to CH1. wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains, and the second Fc region comprises amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, and R409K. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region being a human IgG4 Fc region comprising an immunoglobulin hinge region and a CH2 and CH3 immunoglobulin heavy chain constant domain, the first Fc region comprising amino acid substitutions at positions corresponding to 228, 234, 235, 349, 366, 368, 407, and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P, F234A, L235A, Y349C, T366S, L368A, Y4 07V, and R409K; and the third polypeptide chain further comprises a second Fc region linked to CH1, wherein the second Fc region is a human IgG4 Fc region comprising an immunoglobulin hinge region and the CH2 and CH3 immunoglobulin heavy chain constant domains, wherein the second Fc region comprises amino acid substitutions at positions corresponding to 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.

[0210] 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 an amino acid substitution at a position corresponding to 228 and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is S228P and / or R409K. In some embodiments, the Fc region is a human IgG4 Fc region comprising an amino acid substitution at a position corresponding to 234 and / or 235 of human IgG4 according to the EU index. In some embodiments, the amino acid substitution is 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 positions corresponding to positions 228, 233-236, and / or 409 of human IgG4 according to the EU index. In some embodiments, the amino acid mutations are S228P; E233P, F234V, L235A, and a deletion at 236; and / or R409K.

[0211] In some embodiments, the Fc region comprises an Fc receptor binding and / or Fc receptor subunit of the Fc region. The antibody or antibody may comprise one or more mutations that reduce or eliminate vector function (e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC)).

[0212] 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 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.

[0213] In some embodiments, the binding proteins of the disclosure comprise one or more mutations to improve stability, e.g., stability of the hinge region and / or dimer interface of 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, the amino acid substitutions being S228P and R409K. In some embodiments, the binding proteins comprise one or more mutations to improve stability, e.g., stability of the hinge region and / or dimer interface of 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, the amino acid substitutions being S228P and R409K. H1 a second polypeptide chain further comprising a first Fc region linked to said first polypeptide chain, said first Fc region comprising an immunoglobulin hinge region and a C H2 and C H3 a second polypeptide chain comprising an immunoglobulin heavy chain constant domain, and H1 a third polypeptide chain further comprising a second Fc region linked to said third polypeptide chain, said second Fc region being linked to a hinge region and a C region of an immunoglobulin; H2 and C H3and a third polypeptide chain comprising an immunoglobulin heavy chain constant domain; the first and second Fc regions are human IgG4 Fc regions; the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, respectively, the amino acid substitutions being S228P and R409K. In some embodiments, the binding protein of the present disclosure comprises 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.

[0214] 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 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.

[0215] nucleic acid Another aspect of the present disclosure relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the binding proteins described herein. Exemplary and non-limiting nucleic acid sequences are provided in Table 5.

[0216] Another aspect of the disclosure relates to a kit of polynucleotides, wherein the polynucleotides encode one or more polypeptides of a binding protein, for example, as described herein. In some embodiments, the kit of polynucleotides of the disclosure includes: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189; (b) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:193, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:194, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:195, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:196; (c) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:197, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:198, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:199, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:200; (d) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:201, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:202, and a polynucleotide of SEQ ID NO:203. (e) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:205, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:206, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:207, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:208; (f) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:209, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:210, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:211, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:212; (g) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:213, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:214, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:215, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID 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, (l) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:233, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:234, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:235, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:236; (m) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:237, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:238, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:239, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:240; (n) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:241, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:242, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:243, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:244;(o) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO:245, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:246, and 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 a polynucleotide sequence of SEQ ID NO:261, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:262, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:263, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:264; (t) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:265, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:266, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:267, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:268; (u) a first polynucleotide comprising a polynucleotide sequence of SEQ ID NO:269, a second polynucleotide comprising a polynucleotide sequence of SEQ ID NO:270, a third polynucleotide comprising a polynucleotide sequence of SEQ ID NO:271, and a fourth polynucleotide comprising a polynucleotide sequence of SEQ ID NO:272;or (v) one, two, three, or four of the following kit of polynucleotides: 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;

[0217] Another aspect of the present disclosure relates to a vector system comprising one or more vectors encoding the first, second, third, and fourth polypeptide chains of any of the binding proteins 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, for example as shown in the polynucleotides of 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. One or more vectors of the vector system may be any vector described herein. In some embodiments, 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, for example, 1, 2, 3 or 4 expression vectors.

[0218] Standard recombinant DNA methodology is used to construct polynucleotides encoding polypeptides that form binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Unless specific definitions are provided, the nomenclature utilized in the context of analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical manufacturing, formulation, delivery, and treatment of patients.

[0219] In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to direct the transcription of the binding protein-encoding nucleic acid sequence. A promoter can refer to a nucleic acid control sequence that directs the transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a binding protein-encoding sequence when the promoter affects the transcription or expression of the coding sequence. Examples of promoters include, but are not limited to, promoters derived from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40)), heterologous eukaryotic promoters (such as actin promoters, immunoglobulin promoters, and heat shock promoters), CAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), phosphoglycerate kinase (PGK)-promoter, tetracycline-inducible promoter (Masui et al., Nucleic Acid Res. 33:e43, 2005), the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of phage lambda, the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, and the promoter of yeast alpha mating factor. A polynucleotide encoding a 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 readily recognized by one of skill in the art.

[0220] 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 linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers include, but are not limited to, enhancer sequences from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), as well as enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer on the late side of the replication origin (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, adenovirus enhancer, etc.). Examples of suitable vectors include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus, vaccinia virus, Sindbis virus, measles, herpes virus, lentivirus, retrovirus, adeno-associated virus vectors, etc.). Expression vectors can 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.

[0221] host cell Other aspects of the disclosure relate to a host cell (e.g., an isolated host cell) comprising one or more isolated polynucleotides, vectors, and / or vector systems described herein. In some embodiments, the isolated host cell of the disclosure is cultured in vitro. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., a S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells can include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Exemplary mammalian host cells include, for example, human embryonic kidney cells (e.g., 293 cells or 293 cells subcloned for growth in suspension culture), Expi293™ cells, 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 carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (eg, Hep G2), and myeloma cells (eg, NS0 and Sp2 / 0 cells).

[0222] Another aspect of the present disclosure relates to a method for producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and / or vector system (e.g., any of the 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 for culturing a host cell under conditions for protein expression are well known to those skilled in the art. Methods for isolating a protein from a cultured host cell are well known to those skilled in the art, and include, for example, affinity chromatography (e.g., two-step affinity chromatography including Protein A affinity chromatography followed by size exclusion chromatography).

[0223] Pharmaceutical Compositions Therapeutic or pharmaceutical compositions comprising the binding proteins are within the scope of this disclosure. Such therapeutic or pharmaceutical compositions can include a therapeutically effective amount of the binding protein, or binding protein-drug conjugate, in combination with a pharma- ceutical or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0224] Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.

[0225] Pharmaceutical compositions can contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition.Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, ammonium chloride, sodium lauryl sulfate ... Examples of suitable pharmaceutical agents include, but are not limited to, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, 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 tyloxapol), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides, e.g., sodium or potassium chloride, or mannitol, sorbitol), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants (e.g., see REMINGTON'S TECHNOLOGY ROADMAP FOR SEMICONDUCTORS, 2003, incorporated herein by reference for any purpose). PHARMACEUTICAL SCIENCES (18th Ed., edited by A. R. Gennaro, Mack Publishing Company 1990), and subsequent editions thereof).

[0226] Optimal pharmaceutical compositions can be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

[0227] The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, suitable vehicles or carriers for injection can be water, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, the binding protein composition can be prepared for storage by mixing the selected composition having the desired purity with optional formulating agents in the form of a lyophilized cake or aqueous solution. Additionally, the binding protein can be formulated as a lyophilizate using appropriate excipients, such as sucrose.

[0228] The pharmaceutical composition of the present disclosure can be selected for parenteral or subcutaneous delivery.Alternatively, the composition can be selected for inhalation or for delivery through the digestive tract, for example, orally.The manufacture of such pharmacologic acceptable compositions is within the skill of the art.

[0229] Formulation components are present in concentrations that are acceptable for the site of administration, for example, buffers are used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0230] When parenteral administration is intended, the therapeutic composition for use can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution containing the desired binding protein in a pharma- ceutically acceptable medium.A particularly suitable medium for parenteral injection is sterile distilled water, in which the binding protein is formulated as a sterile, isotonic solution that is appropriately preserved.Yet another product can involve the formulation of the desired molecule with an agent that provides controlled or sustained release of the product that can be delivered via depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes.Hyaluronic acid can also be used, which can have the effect of promoting sustained duration in the circulation.Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.

[0231] 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 nebulized.

[0232] It is also contemplated that certain formulations can be administered orally.In one embodiment of the present disclosure, the binding protein administered in this manner can be formulated with or without carriers that are conventionally used in the preparation of solid dosage forms, such as tablets and capsules.For example, capsules can be designed to release the active portion of the formulation at the location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to promote the absorption of binding protein.Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0233] Another pharmaceutical composition can involve an effective amount of binding protein in a mixture with non-toxic excipients suitable for tablet manufacture.By dissolving tablets in sterile water or other suitable medium, the solution can be manufactured into unit dose form.Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0234] Additional pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including formulations with binding proteins in sustained or controlled delivery formulations. Techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma 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 produced by any of several methods known in the art.

[0235] Pharmaceutical compositions to be used for in vivo administration must typically be sterile. This can be accomplished by filtration through sterile filtration membranes. If sterilization using this method can be performed either prior to or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Additionally, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0236] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution prior to administration (e.g., lyophilized).

[0237] The present disclosure also includes kits for producing single-dose administration units.Each kit can contain both a first container with dry protein and a second container with aqueous formulation.Kits containing single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of the present disclosure.

[0238] The effective amount of the binding protein pharmaceutical composition used therapeutically depends, for example, on the therapeutic context and purpose.Those skilled in the art will understand that the appropriate dosage level for treatment will vary depending in part on the molecule to be...

Claims

1. A binding protein comprising four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site; V Ｈ２ and V Ｌ２ forms a second antigen-binding site that binds to the CD3 polypeptide, and V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site, The binding protein.

2. The binding protein of claim 1 , wherein the first binding site binds to a CD28 polypeptide.

3. V 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), Ｌ１ The domain is C 3. The binding protein of claim 2, comprising a DR-L1 sequence, 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).

4. V Ｈ１ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); Ｌ１ The domain is DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92), The binding protein of claim 3.

5. V Ｌ２ 5. The binding protein of any one of claims 1 to 4, wherein 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).

6. V Ｈ２ The domain is QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESG and / or comprising the amino acid sequence of V Ｌ２ The domain is DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLS VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97); and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98), The binding protein of claim 5.

7. The binding protein of any one of claims 1 to 6, wherein the third antigen-binding site binds to a tumor targeting protein.

8. The binding protein of any one of claims 1 to 6, wherein the third antigen-binding site binds to a human CD38 polypeptide.

9. (a) V 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), Ｌ３ The 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); (b) 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); Ｌ３ The 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); (c) 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); Ｌ３ The 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); (d) V Ｈ３ The 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), 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); (e) V Ｈ３ The 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), Ｌ３ The 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); or (f) V Ｈ３ The 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), Ｌ３ 9. The binding protein of claim 8, wherein the domain comprises 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).

10. (a) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79); Ｌ３ The domain is DIVLTQSPATTLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:80); (b) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSDTAYMEMNSLRSETAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81); Ｌ３ The domain is EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO:82); (c) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83); Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO:84); (d) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); Ｌ３ The domain is DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86); (e) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87); Ｌ３ The domain is or comprising the amino acid sequence of: DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO:88); or (f) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89); Ｌ３ The domain is AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90), The binding protein of claim 9.

11. (a) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:156 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:156; and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:157 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

157. a third polypeptide chain comprises an amino acid sequence of SEQ ID NO:158 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:158; a fourth polypeptide chain comprises an amino acid sequence of SEQ ID NO:159 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:159; (b) 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; 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; (c) 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; 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; (d) 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; 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; (e) 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; 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; (f) 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; 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; (g) the first polypeptide chain comprises an 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 an 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 an 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; the fourth polypeptide chain comprises an 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; or (h) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO:185 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:185; and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO:186 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

186. the third polypeptide chain comprises an 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 an 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.

12. The binding protein of any one of claims 1 to 7, wherein the third antigen-binding site binds to a human HER2 polypeptide.

13. (a) V Ｈ３ The 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), Ｌ３ The 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); (b) 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), 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); (c) 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), 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); (d) 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 SRWGGSGGFYAMDY (SEQ ID NO: 8), 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); (e) 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 SRWGGSGGFYAMDY (SEQ ID NO: 8), 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); (f) 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), 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); or (g) V Ｈ３ The domain is 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), 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); The binding protein of claim 12.

14. (a) V Ｈ３ The domain is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 72), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWV RQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 73), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTS KNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQGTLVTVSS (SEQ ID NO: 74), EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYWGQ GTLVTVSS (SEQ ID NO: 75), or EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS (SEQ ID NO: 76), and / or Ｌ３ The domain is comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78); (b) V Ｈ３ The domain is and / or comprising the amino acid sequence of V Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77); (c) V Ｈ３ The domain is and / or comprising the amino acid sequence of V Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77); (d) V Ｈ３ The domain is and / or comprising the amino acid sequence of V Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77); (e) V Ｈ３ The domain is and / or comprising the amino acid sequence of V Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77); (f) V Ｈ３ The domain is and / or comprising the amino acid sequence of V Ｌ３ The domain is or comprising the amino acid sequence of: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77); or (g) V Ｈ３ The domain is and / or comprising the amino acid sequence of V Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 78), The binding protein of claim 13.

15. (a) 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; 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; (b) the first polypeptide chain comprises an 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 an 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 an 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 an 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. comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 107; (c) 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; (d) 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; 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; (e) 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; 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; (f) 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; 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; (g) 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; 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; (h) 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; 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; (i) 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; 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; (j) 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; 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; (k) 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; 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; (l) 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; 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; (m) the first polypeptide chain comprises an 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 an 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 an 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; the fourth polypeptide chain comprises an 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; (n) the first polypeptide chain comprises an 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 an 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 an 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; the fourth polypeptide chain comprises an 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; (o) the first polypeptide chain comprises an 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 an 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 an 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; the fourth polypeptide chain comprises an 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; (p) the first polypeptide chain comprises an 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 an 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 an 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; the fourth polypeptide chain comprises an 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; or (q) the first polypeptide chain comprises an 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 an 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 an 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 an 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.

16. L １ , L ２ , L ３ Or L ４ 16. The binding protein of any one of claims 1 to 15, wherein at least one of is independently 0 amino acids in length.

17. (a) L １ , L ２ , L ３ and L ４ are each independently 0 amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S,RT,TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71); or (b) L １ , L ２ , L ３ and L ４ each independently comprises a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGGS (SEQ ID NO:70), S,RT,TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO:67), and GGSGSSGSGG (SEQ ID NO:71).

18. L １ comprises the sequence GQPKAAP (SEQ ID NO:67), ２ comprises the sequence TKGPS (SEQ ID NO:68), ３ contains a sequence S, and L ４ The binding protein of any one of claims 1 to 15, wherein:

19. L １ , L ２ , L ３ Or L ４ The binding protein of any one of claims 1 to 15, wherein at least one of said amino acids comprises the sequence DKTHT (SEQ ID NO: 66).

20. L １ , L ２ , L ３ and L ４ The binding protein of claim 19, comprising the sequence DKTHT (SEQ ID NO: 66).

21. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is human IgG4 hinge-C Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 21. The binding protein of any one of claims 1 to 20, wherein each of the domains comprises amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, the amino acid substitutions being F234A and L235A.

22. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is human IgG4 hinge-C Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 21. The binding protein of any one of claims 1 to 20, wherein each of the domains comprises an amino acid substitution at a position corresponding to 233-236 of human IgG4 according to the EU index, the amino acid substitutions being E233P, F234V, L235A, and a deletion at 236.

23. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is human IgG4 hinge-C Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 23. The binding protein of any one of claims 1 to 22, wherein each of the domains comprises amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, the amino acid substitutions being S228P and R409K.

24. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is human IgG1 hinge-C Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 21. The binding protein of any one of claims 1 to 20, wherein the domains comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgG1 according to the EU index, respectively, and the amino acid substitutions are L234A, L235A, and P329A.

25. Hinge-C of the second and third polypeptide chains Ｈ２ -C Ｈ３ The domain is human IgG1 hinge-C Ｈ２ -C Ｈ３ domain, hinge-C Ｈ２ -C Ｈ３ 21. The binding protein of any one of claims 1 to 20, wherein the domains comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgG1 according to the EU index, respectively, and the amino acid substitutions are S298N, T299A, and Y300S.

26. Hinge-C of the second polypeptide chain Ｈ２ -C Ｈ３ The domain comprises amino acid substitutions at positions corresponding to 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being Y349C, T366S, L368A, and Y407V; Ｈ２ -C Ｈ３ 26. The binding protein of any one of claims 1 to 25, wherein the domain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, the amino acid substitutions being S354C and T366W.

27. Hinge-C of the second polypeptide chain 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, the amino acid substitutions being S354C and T366W; Ｈ２ -C Ｈ３ 26. The binding protein of any one of claims 1 to 25, wherein 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, the amino acid substitutions being Y349C, T366S, L368A, and Y407V.

28. 28. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding protein according to any one of claims 1 to 27.

29. 29. An expression vector comprising the nucleic acid molecule of claim 28.

30. 30. An isolated host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29.

31. 31. The isolated host cell of claim 30, which is a mammalian or insect cell.

32. A pharmaceutical composition comprising the binding protein of any one of claims 1 to 27 and a pharma- ceutically acceptable carrier.

33. 32. A method for preventing and / or treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of at least one binding protein according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 32.

34. 34. The method of claim 33, wherein the at least one binding protein is administered in combination with a chemotherapeutic agent.

35. 35. The method of claim 33 or claim 34, wherein the patient is a human.

36. The method of any one of claims 33 to 35, wherein the third antigen-binding site binds to a human CD38 polypeptide and the cancer cells from the patient express CD38.

37. 37. The method of claim 36, wherein the cancer is multiple myeloma.

38. 37. The method of claim 36, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma.

39. 39. The method of any one of claims 36 to 38, wherein prior to administration of the binding protein, the patient has been treated with daratumumab without a washout period.

40. The method of any one of claims 33 to 35, wherein the third antigen-binding site binds to a human HER2 polypeptide and the cancer cells from the patient express HER2.

41. 41. The method of claim 40, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

42. A binding protein according to any one of claims 7 to 27 or a pharmaceutical composition according to claim 32 for use in the prevention and / or treatment of cancer in a patient.

43. 43. The binding protein for use or composition for use of claim 42, wherein at least one binding protein is administered in combination with a chemotherapeutic agent.

44. 44. The binding protein for use or composition for use according to claim 42 or claim 43, wherein the patient is a human.

45. 45. The binding protein for use or composition for use according to any one of claims 42 to 44, wherein the third antigen-binding site binds to a human CD38 polypeptide and the cancer cells from the patient express CD38.

46. 46. ​​The binding protein for use or composition for use of claim 45, wherein the cancer is multiple myeloma.

47. 46. ​​The binding protein for use or composition for use of claim 45, wherein the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or B-cell lymphoma.

48. 48. The binding protein for use or composition for use according to any one of claims 45 to 47, wherein prior to administration of the binding protein the patient has been treated with daratumumab without a washout period.

49. 45. The binding protein for use or composition for use according to any one of claims 42 to 44, wherein the third antigen-binding site binds to a human HER2 polypeptide and the cancer cells from the patient express HER2.

50. 50. The binding protein for use or composition for use of claim 49, wherein the cancer is breast cancer, colorectal cancer, gastric cancer, or non-small cell lung cancer (NSCLC).

51. A method of expanding T cells, comprising contacting T cells with a binding protein comprising four polypeptide chains forming three antigen-binding sites, The do chain has the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site which binds to the CD28 polypeptide; V Ｈ２ and V Ｌ２ forms a second antigen-binding site that binds to the CD3 polypeptide, and V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site which binds to the CD38 polypeptide; The method.

52. 52. The method of claim 51 , wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.

53. 53. The method of claim 51 or claim 52, wherein the T cell is a memory T cell or an effector T cell.

54. A method for expanding virus-specific memory T cells, comprising: binding the virus-specific memory T cells to a binding protein comprising four polypeptide chains forming three antigen-binding sites. and contacting the first polypeptide chain with a protein, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site which binds to the CD28 polypeptide; V Ｈ２ and V Ｌ２ forms a second antigen-binding site that binds to the CD3 polypeptide, and V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site which binds to the CD38 polypeptide; The method.

55. 55. The method of claim 54, wherein the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo.

56. 56. The method of claim 54 or claim 55, wherein contacting the virus-specific memory T cells with the binding protein causes activation and / or proliferation of the virus-specific memory T cells.

57. A method for treating a chronic viral infection comprising administering to a patient in need thereof an effective amount of The method includes administering a binding protein comprising four polypeptide chains forming one antigen-binding site, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site which binds to the CD28 polypeptide; V Ｈ２ and V Ｌ２ forms a second antigen-binding site that binds to the CD3 polypeptide, and V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site which binds to the CD38 polypeptide; The method.

58. 58. The method of claim 57, wherein the patient is a human.

59. 59. The method of claim 57 or claim 58, wherein the binding protein is administered to the patient in a pharmaceutical formulation comprising the binding protein and a pharma- ceutically acceptable carrier.

60. 60. The method of any one of claims 57 to 59, wherein administration of the binding protein results in activation and / or expansion of virus-specific memory T cells in the patient.

61. The method of any one of claims 53 to 56 and 60, wherein the memory T cells are CD8+ or CD4+ memory T cells.

62. Memory T cells are central memory T cells (T ＣＭ ) or effector memory T cells (T ＥＭ 62. The method of any one of claims 53 to 56, 60, and 61, wherein

63. 63. The method of any one of claims 54 to 62, wherein 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).

64. The method of any one of claims 51 to 63, wherein the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide, and the CD38 polypeptide is a human CD38 polypeptide.

65. 1. A binding protein for use in the expansion of T cells, the binding protein contacting a T cell, the binding protein comprising four polypeptide chains forming three antigen binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site which binds to the CD28 polypeptide; V Ｈ２ and V Ｌ２ forms a second antigen-binding site which binds to the CD3 polypeptide, V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site which binds to the CD38 polypeptide; A binding protein for said use.

66. 66. The binding protein for use of claim 65, wherein the T cell expresses a chimeric antigen receptor (CAR) on its cell surface or comprises a polynucleotide encoding a CAR.

67. 67. The binding protein for use according to claim 65 or claim 66, wherein the T cell is a memory T cell or an effector T cell.

68. 1. A binding protein for use in the expansion of virus-specific memory T cells, the binding protein contacting a virus-specific memory T cell, the binding protein comprising four polypeptide chains forming three antigen-binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site which binds to the CD28 polypeptide; V Ｈ２ and V Ｌ２ forms a second antigen-binding site that binds to the CD3 polypeptide, and V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site which binds to the CD38 polypeptide; A binding protein for said use.

69. 69. The binding protein for use according to claim 68, wherein the virus-specific memory T cells are contacted with the binding protein in vitro or ex vivo.

70. 70. The binding protein for use according to claim 68 or claim 69, wherein contacting virus-specific memory T cells with the binding protein causes activation and / or proliferation of the virus-specific memory T cells.

71. 1. A binding protein for use in the treatment of a chronic viral infection, the binding protein being administered to a patient in need thereof, the binding protein comprising four polypeptide chains which form three antigen binding sites, the first polypeptide chain having the formula: V Ｌ２ -L １ -V Ｌ１ -L ２ -C Ｌ [I] The structure includes: The second polypeptide chain has the formula: V Ｈ１ -L ３ -V Ｈ２ -L ４ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [II] The structure includes: The third polypeptide chain has the formula: V Ｈ３ -C Ｈ１ - Hinge - C Ｈ２ -C Ｈ３ [III] The structure includes: The fourth polypeptide chain has the formula: V Ｌ３ -C Ｌ [IV] The structure includes: Where: V Ｌ１ is a first immunoglobulin light chain variable domain; V Ｌ２ is a second immunoglobulin light chain variable domain; V Ｌ３ is a third immunoglobulin light chain variable domain; V Ｈ１ is a first immunoglobulin heavy chain variable domain; V Ｈ２ is a second immunoglobulin heavy chain variable domain; V Ｈ３ is a third immunoglobulin heavy chain variable domain; C Ｌ is an immunoglobulin light chain constant domain; C Ｈ１ is immunoglobulin C Ｈ１ a heavy chain constant domain; C Ｈ２ is immunoglobulin C Ｈ２ a heavy chain constant domain; C Ｈ３ is immunoglobulin C Ｈ３ a heavy chain constant domain; The hinge is C Ｈ１ and C Ｈ２ an immunoglobulin hinge region connecting the domains; L １ , L ２ , L ３ and L ４ is an amino acid linker; a polypeptide of formula I and a polypeptide of formula II form a crossover light chain-heavy chain pair; V Ｈ１ and V Ｌ１ forms a first antigen-binding site that binds to the CD28 polypeptide. ； V Ｈ２ and V Ｌ２ forms a second antigen-binding site that binds to the CD3 polypeptide, and V Ｈ２ The 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), Ｌ２ The domain is QSLVHX １ NX ２ X ３ TY, where X １ is E or Q, and X ２ is A or L, and X ３ is Q, R, or F; １ NX ２ X ３ a CDR-L1 sequence comprising the amino acid sequence of TY (SEQ ID NO: 180), 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); V Ｈ３ and V Ｌ３ forms a third antigen-binding site which binds to the CD38 polypeptide; A binding protein for said use.

72. 72. The binding protein for use according to claim 71, wherein the patient is a human.

73. 73. The binding protein for use according to claim 71 or claim 72, wherein the binding protein is administered to a patient in a pharmaceutical formulation comprising the binding protein and a pharma- ceutically acceptable carrier.

74. 74. The binding protein for use according to any one of claims 71 to 73, wherein administration of the binding protein results in the activation and / or expansion of virus-specific memory T cells in the patient.

75. 75. The binding protein for use according to any one of claims 67 to 70 and 74, wherein the memory T cells are CD8+ or CD4+ memory T cells.

76. Memory T cells are central memory T cells (T ＣＭ ) or effector memory T cells (T ＥＭ 76. The binding protein for use according to any one of claims 67 to 70, 74 and 75, wherein

77. 77. The binding protein for use according to any one of claims 68 to 76, wherein 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).

78. 78. The binding protein for use according to any one of claims 65 to 77, wherein the CD28 polypeptide is a human CD28 polypeptide, the CD3 polypeptide is a human CD3 polypeptide and the CD38 polypeptide is a human CD38 polypeptide.

79. V 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), Ｌ１ 79. A binding protein for the method or use of any one of claims 51 to 78, wherein 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).

80. V Ｈ１ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS (SEQ ID NO: 91); Ｌ１ The domain is DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO:92), 80. A binding protein for the method or use of claim 79.

81. V Ｌ２ 81. The binding protein for the method or use of any one of claims 51 to 80, wherein 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).

82. V Ｈ２ The domain is QVQLVESGGGVVQPGRSLRLSCAASGFFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS (SEQ ID NO: 93) or QVQLVESG and / or comprising the amino acid sequence of V Ｌ２ The domain is DIVMTQTPLSLSVTPGQPASISCKSSQSLVHQNAQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 95), DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLQTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96), DIVMTQTPLSLS VTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:97); and DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLRTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98), 82. A binding protein for the method or use according to claim 81.

83. (a) V 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), Ｌ３ The 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); (b) 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). Columns, V Ｌ３ The 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); (c) 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); Ｌ３ The 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); (d) V Ｈ３ The 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), 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); (e) V Ｈ３ The 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), Ｌ３ The 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); or (f) V Ｈ３ The 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), Ｌ３ 83. A binding protein for the method or use of any one of claims 51 to 82, wherein the domain comprises 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).

84. (a) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWGQGTLVTVSS (SEQ ID NO: 79); Ｌ３ The domain is DIVLTQSPATTLSLSPGERATISCRASQSVSSYGQGFMHWYQQKPGQPPRLLIYGASSRATGIPARFSGSGSGTDFTLTISPLEPEDFAVYYCQQNKEDPWTFGGGTKLEIK (SEQ ID NO:80); (b) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKVSGYTLTEFSIHWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVIMTEDTSDTAYMEMNSLRSETAIYYCTTGRFFDWFWGQGTLVTVSS (SEQ ID NO: 81); Ｌ３ The domain is EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPVRFSGSGSGTDFSLTISSLQPEDCAVYYCQQDSNLPITFGQGTRLEIK (SEQ ID NO:82); (c) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGSGSTNYAQKFQGRVTMTVDRSSTTAYMELSRLRSDDTAVYYCARYAYGYWGQGTLVTVSS (SEQ ID NO: 83); Ｌ３ The domain is DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYSTYPFTFGQGTKLEIK (SEQ ID NO:84); (d) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPYYGDTTYAQKFQGRVTMTVDKSSSTAYMELSRLRSDDTAVYYCARRFEFYYSMDYWGQGTLVTVSS (SEQ ID NO: 85); Ｌ３ The domain is DVVMTQSPLSLPVTLGQPASISCRPSQSLVHSNGNTYLNWYQQRPGQSPKLLIYKVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGTKVEIK (SEQ ID NO:86); (e) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGGMDVWGQGTTVTVSS (SEQ ID NO: 87); Ｌ３ The domain is or comprising the amino acid sequence of: DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIFAASTLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSFPYTFGQGTKLEIK (SEQ ID NO:88); or (f) V Ｈ３ The domain is and / or comprising the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCARMFRGAFDYWGQGTLVTVSS (SEQ ID NO: 89); Ｌ３ The domain is AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDSATYYCLQDYIYYPTFGQGTKVEIK (SEQ ID NO: 90), 84. A binding protein for the method or use of claim 83.

85. (a) 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; 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; (b) 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. comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 163; (c) 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; 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; (d) 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; 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; (e) 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; 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; (f) 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; 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; (g) the first polypeptide chain comprises an 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 an 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 an 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; the fourth polypeptide chain comprises an 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; or (h) 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.

86. A vector system comprising one or more vectors encoding the first, second, third and fourth polypeptide chains of the binding protein of any one of claims 1 to 27.

87. 87. The vector system of claim 86, comprising a first vector encoding a first polypeptide chain of a binding protein, a second vector encoding a second polypeptide chain of the binding protein, a third vector encoding a third polypeptide chain of the binding protein, and a fourth vector encoding a fourth polypeptide chain of the binding protein.

88. 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:201, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO:202, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO:203, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:204; (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: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:232; (l) 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; (m) 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; (n) 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; (o) 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; (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; (t) 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; (u) 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 (v) 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. The kit comprising:

89. 89. The kit of claim 88, wherein the first, second, third, and fourth polynucleotides are present on one or more expression vectors.

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