0001] This application claims priority to U.S. Provisional Application No. 62/831,572, filed April 9, 2019, and EP Application No. EP19306311.2, filed October 8, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 183952032040SEQLIST.TXT, date recorded: April 6, 2020, size: 526 KB).

FIELD

[0003] The disclosure relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen binding sites that specifically bind one or more target proteins, wherein a first pair of polypeptides forming the binding protein possess dual variable domains having a cross-over orientation. The disclosure also relates to methods for making trispecific and/or trivalent binding proteins and uses of such binding proteins.

BACKGROUND

[0004] Monoclonal antibody based biotherapeutics have become an important avenue for new drug development. Monoclonal antibody technology offers specific targeting, precise signaling delivery and/or payload to specific cell population, and provides long lasting biological effect through its Fc functions. Efforts in antibody engineering have allowed developing bispecific antibodies combining 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 biotherapeutics to treat complexed diseases such as cancer, arthritis, and/or inflammatory disorders.

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

[0006] As part of the human adaptive immunity, T cell immunity plays crucial role in controlling viral infection and cancer, possibly eliminating infected cells and malignant cells which result in clearance of viral infection or cure of cancer. In chronic infectious diseases such as Herpes viral infection (HSV, CMV, EBV, etc.), HIV, and HBV, viruses establish their persistence in humans by various mechanisms including immune

suppression, T cell exhaustion, and latency establishment. Nevertheless, viral infection generally induces viral antigen specific immunity including antigen specific CD8 T cells that can readily recognize infected cells for controlling or killing through cytokine release or cytotoxic T cell (CTL) mediated killing processes.

[0007] Thus, viral antigen specific T cell activation and/or amplification in vivo and/or ex vivo may provide therapeutic strategies against chronic viral infections.

[0008] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot Accession numbers are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

[0009] To meet these and other needs, provided herein are trispecific binding proteins ( e.g ., antibodies) that form three antigen binding sites. These binding proteins can specifically bind one, two, or three antigen targets or target proteins, such as CD28, CD3, and a tumor target protein. 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 carcinomas. Optimal activation of T cells requires two factors: 1. Antigen recognition and 2. Co-stimulation. Using the trispecific

HER2/CD28xCD3 trispecific binding proteins described herein, Signal 1 is provided by an agonist anti-CD3 binding site, and Signal 2 is provided by an agonist anti-CD28 binding site. The trispecific binding protein recruits T cells to the tumor via HER2, CD38, or a binding site recognizing another tumor target protein and activates the engaged T cells via anti-CD3 and -CD28. The resulting activation induces the killing potential of the immune cells against the nearby tumor cells. In addition, anti-CD3 binding sites are described with high affinity binding to human CD3 polypeptides and potential manufacturing liabilities ( e.g ., deamidation sites) removed.

[0010] Further provided herein are anti-CD38/CD28xCD3 trispecific antibodies that were developed and evaluated for their potential in activating T cells, and subsequent proliferation and/or amplification of antigen specific T cells. These trispecific Abs can effectively expand CD4 and CD8 effector and memory populations, including antigen specific CD8 T central memory and effector memory cells in vitro. Specifically, in vitro expansion of CMV, EBV, HIV-1, Influenza specific CD8 central memory and effector memory cells were demonstrated. The anti-CD38/CD28xCD3 trispecific antibodies described herein exhibited novel properties by engaging CD3/CD28/CD38, providing signaling pathways to stimulate and expand T cells, which may offer an effective strategy treating chronic infectious diseases such as HSV, CMV, EBV, HIV-1, and HBV infections.

[0011] To meet these and other needs, provided herein are binding proteins that bind CD38 polypeptides (e.g., human and cynomolgus monkey CD38 polypeptides), including monospecific, bispecific, or trispecific binding proteins with at least one antigen binding site that binds a CD38 polypeptide. Advantageously, these binding proteins have the ability to recruit T cells to the proximity of cancer cells, subsequently to activate T cells and promote the activated T cells killing of adjacent cancer cells through a Granzyme/Perforin mechanism, providing a different mode of action for anti -tumor activity from anti-CD38 antibodies such as DARZALEX® (daratumumab). Moreover, the ability to bind both human and cynomolgus monkey CD38 polypeptides allows binding proteins to be readily tested in preclinical toxicological studies, e.g., to evaluate their safety profiles for later clinical use.

[0012] In some embodiments, provided herein are binding proteins comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-Cm-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and Cm domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site;

wherein Vm and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of

QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site.

[0013] In some embodiments, the first binding site binds a CD28 polypeptide. In some embodiments, the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the VL1 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNIYVW (SEQ ID NO:52), a CDR-L2 sequence comprising the amino acid sequence of KAS (SEQ ID NO:53), and a CDR-L3 sequence comprising the amino acid sequence of QQGQTYPY

(SEQ ID NO: 54). In some embodiments, the Vm domain comprises the amino acid sequence of

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGN VNTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDV WGKGTTVTVSS (SEQ ID NO:91), and/or the VL1 domain comprises the amino acid sequence of

DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHT GVPSRF SGSGSGTDFTLTIS SLQPEDIAT YYCQQGQTYP YTF GQGTKLEIK (SEQ ID NO: 92).

[0014] In some embodiments, the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of QSLVHQNAQTY (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and QSLVHENLRTY (SEQ ID NO:62). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHQNAQTY (SEQ ID NO:59), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO: 64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of QSLVHENLQTY (SEQ ID NO:60), a CDR-L2 sequence comprising the amino acid sequence of KVS (SEQ ID NO:64), and a CDR-L3 sequence comprising the amino acid sequence of GQGTQYPFT (SEQ ID NO:65). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-

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

RGVYYALSPFDY (SEQ ID NO:57); and/or an antibody light chain variable (VL) domain comprising a CDR-L1 sequence comprising the amino acid sequence of 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 VH2 domain comprises the amino acid sequence of

QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD K SN S Y AT Y Y AD S VKGRFTI SRDD SKNTL YLQMN SLR AEDT A V Y Y CRGV Y Y AL SPF DYWGQGTLVTVSS (SEQ ID NO:93), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK

(SEQ ID NO: 95),

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S W YLQKPGQ SPQ SLI YK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK

(SEQ ID NO: 96),

DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVS NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK

(SEQ ID NO: 97), and

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK

(SEQ ID NO: 98). In some embodiments, the VH2 domain comprises the amino acid sequence of

QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD K SN S Y AT Y Y AD S VKGRFTI SRDD SKNTL YLQMN SLRAEDT A V Y Y CRGV Y Y AL SPF DYWGQGTLVTVSS (SEQ ID NO:93) or

QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKD KSNSYATYYASSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPF DYWGQGTLVTVSS (SEQ ID NO:302), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQ YPFTFGSGTKVEIK

(SEQ ID NO: 95),

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK

(SEQ ID NO: 96),

DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVS NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK

(SEQ ID NO: 97), and

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V S NRF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK

(SEQ ID NO:98). In some embodiments, a binding protein of the present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) 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 present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) 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 present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VET) 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 present disclosure comprises an antigen binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, a binding protein of the present disclosure comprises an antigen

binding site comprising: an antibody heavy chain variable (VH) domain comprising the amino acid sequence of SEQ ID NO:93, and/or an antibody light chain variable (VL) domain comprising the amino acid sequence of SEQ ID NO:98.

[0015] In some embodiments, the third antigen binding site binds a tumor target protein. In some embodiments, the tumor target protein is a CD38 polypeptide ( e.g ., a human CD38 polypeptide). In some embodiments, the tumor target protein is a HER2 polypeptide (e.g., a human HER2 polypeptide). In some embodiments, a tumor target protein of the present disclosure includes, without limitation, 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-la), CCL4 (also known as MIP-lb), 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-ld), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL2 0 (also known as MIP-3a), CCL2 1 (also known as MIP-2),

CCL24 (also known as MPIF-2/eotaxin-2), CCL2 5 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD 19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L,

CD 152 (also known as CTLA4), CD 154 (also known as CD40L), CD 160, CD272, CD273 (also known as PDL2 ), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), 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, IFNa, IgE, IGF1R, IL2 Rbeta, IL1, ILIA, IL1B, IL1F10, IL2 , IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhILlO, IL12, IL13, IL13Ral, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL2 5), IL18, IL22, IL23, IL2 5, IL2 7, IL33, IL35, ITGB4 (also known as b4 integrin), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, MUC-1, NCR3LG1, NKG2D, NTPDase-1, 0X40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREMl, TSLP (also known as a co-receptor for IL7Ra),

TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as

GPR5/CCXCR1). In some embodiments, one or more of the above antigen targets are human antigen targets.

[0016] In some embodiments, the third antigen binding site binds a human CD38 polypeptide. In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVSSYGQGF (SEQ ID NO: 16), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO: 17), and a CDR-L3 sequence comprising the amino acid sequence of QQNKEDPWT (SEQ ID NO: 18). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSVISRF (SEQ ID NO:22), a CDR-L2 sequence comprising the amino acid sequence of GAS (SEQ ID NO:23), and a CDR-L3 sequence comprising the amino acid sequence of QQDSNLPIT (SEQ ID NO:24). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QNVGTA (SEQ ID NO:28), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO:29), and a CDR-L3 sequence comprising the amino acid sequence of QQYSTYPFT (SEQ ID NO:30). In some embodiments, the VH3 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

ARRFEGF YY SMD Y (SEQ ID NO:33), and the VL3 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, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL3 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). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL3 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). In some embodiments, the VH3 domain comprises the amino acid sequence of

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPG QGGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTY WGQGTLVTVSS (SEQ ID NO:79), and/or the VL3 domain comprises the amino acid sequence of

DIVLTQ SP ATLSL SPGERATISCRASQ S V S S YGQGFMHW YQQKPGQPPRLLI Y GAS S RAT GIP ARF S GS GS GTDFTLTI SPLEPEDF A V Y Y C QQNKEDPWTF GGGTKLEIK

(SEQ ID NO: 80). In some embodiments, the VH3 domain comprises the amino acid sequence of

Q VQL VQSGAEVKKPGAS VKVSCKVSGYTLTEF SIHWVRQ APGQGLEWMGGFDPE DGETIY AQKF QGRVIMTEDT STDT AYMEMN SLRSEDT AIYYCTT GRFFDWF W GQG TLVTVSS (SEQ ID NO:81), and/or the VL3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIP VRF SGSGSGTDF SLTIS SLQPEDC AVYYCQQDSNLPITF GQGTRLEIK (SEQ ID NO:82). In some embodiments, the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPG SGSTNY AQKF QGRVTMT VDRS STT AYMEL SRLRSDDT AVYY CARY AY GYW GQG TLVTVSS (SEQ ID NO:83), and/or the VL3 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQNVGTAVAWYQQKPGKSPKQLIYSASNRYT

NO:84). In some embodiments, the VH3 domain comprises the amino acid sequence of Q VQLVESGGGVVQPGRSLRLSC AASGFTF S S Y GMYWVRQAPGKGLEWVAVIWYD GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYHCARDPGLRYFDGG MDVWGQGTTVTVSS (SEQ ID NO:87), and/or the VL3 domain comprises the amino acid sequence of

NO:88). In some embodiments, the VH3 domain comprises the amino acid sequence of

and/or the VL3 domain comprises the amino acid sequence of

(SEQ ID NO:85), and/or the VL3 domain comprises the amino acid sequence of

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

[0019] In some embodiments, the third antigen binding site binds a human HER2 polypeptide. In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) 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), SRWGGEGFY AMDY (SEQ ID NO:7), or SRWGGSGFYAMDY (SEQ ID NO: 8), and the VLI domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9) or QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino

acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), 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

SRW GGDGF YAMD Y (SEQ ID NO:6), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO: 9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID

NO: 7), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVNTA (SEQ ID NO:9), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), 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

SRW GGDGF YAMD Y (SEQ ID NO:6), and the VL3 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QDVQTA (SEQ ID NO: 10), a CDR-L2 sequence comprising the amino acid sequence of SAS (SEQ ID NO: 11), and a CDR-L3 sequence comprising the amino acid sequence of QQHYTTP (SEQ ID NO: 12). In some embodiments, the VH3 domain comprises the amino acid sequence of

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF YAMD Y WGQGTLVTVSS (SEQ ID NO: 72),

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:73),

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO: 74),

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO: 75), or

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or

DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF Y AMD Y WGQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:73), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO:75), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQ GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGSGF YAMD Y WGQGTLVTVSS (SEQ ID NO:74), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTN AYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD Y WGQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL Y S GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID

NO:77). In some embodiments, the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN GYTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGDGF Y AMD Y WGQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (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: l 16; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 117; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 121; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 125; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 129; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 133; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 135. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 137; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 146; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 147. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 148; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 150; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:286; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:287 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:287; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:288 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:288; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:289 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:289. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:290 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:290; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:291 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:291; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:292 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:292; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:293 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:293. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:294 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:294; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:295 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:295; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:296 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:296; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:297 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:297. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:298 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:298; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:299 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:299; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:300 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:300; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:301 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:301.

[0021] In some embodiments that may be combined with any other embodiments described herein, at least one of Li, L2, L3 or L4 is independently 0 amino acids in length.

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

embodiments, L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and

GGSGSSGSGG (SEQ ID NO:71). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO:68), L3 comprises the sequence S, and L4 comprises the sequence RT. 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).

[0022] In some embodiments that may be combined with any other embodiments described herein, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236. In some embodiments, the hinge-CH2-Cro domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-Cro domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S. In some embodiments, the hinge-CH2-Cro domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[0023] In some embodiments, provided herein are isolated nucleic acid molecules comprising a nucleotide sequence encoding the binding protein of any one of the above embodiments. In some embodiments, provided herein are expression vectors comprising the nucleic acid molecule of any one of the above embodiments. In some embodiments, provided herein are isolated host cells 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 are pharmaceutical compositions comprising the binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0025] In some embodiments, provided herein are methods of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein or pharmaceutical composition of any one of the above embodiments. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in a method of preventing and/or treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of the binding protein or pharmaceutical composition. In some embodiments, provided herein is a binding protein or pharmaceutical composition according to any one of the above embodiments for use in manufacturing a medicament for preventing and/or treating cancer in a patient.

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

[0027] In some embodiments, the third antigen binding site binds a human CD38 polypeptide, and wherein 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 a B cell lymphoma. In some embodiments, prior to administration of the binding protein, the patient has been treated with daratumumab without a wash-out period.

[0028] In some embodiments, the third antigen binding site binds a human HER2 polypeptide, and wherein 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, provided herein is a method for expanding virus-specific memory T cells, comprising contacting a virus-specific memory T cell with a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH3 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid

sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide

[0030] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of

GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for use in expanding virus-specific memory T cells.

[0031] In some embodiments, the virus-specific memory T cell is contacted with the binding protein in vitro or ex vivo. In some embodiments, contacting the virus-specific memory T cell with the binding protein causes activation and/or proliferation of virus-specific memory T cells.

[0032] In some embodiments, provided herein is a method for expanding T cells, comprising contacting a T cell with a binding protein in vitro or ex vivo , wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

[0033] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

Vm is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for use in a method for expanding T cells.

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

[0035] In some embodiments, provided herein is a method for treating chronic viral infection, comprising administering to an individual or patient in need thereof an effective amount of a binding protein, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO:55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

[0036] In some embodiments, provided herein is a binding protein that comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide, wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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), and wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide for use in a method for treating chronic viral infection, wherein said method comprises administering to an individual or patient in need thereof an effective amount of the binding protein.

[0037] In some embodiments, the individual or patient is a human. In some

embodiments, the binding protein is administered to the individual or patient in

pharmaceutical formulation comprising the binding protein and a pharmaceutically

acceptable carrier. In some embodiments, administration of the binding protein results in activation and/or proliferation of virus-specific memory T cells in the individual or patient.

[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 (TCM) or effector memory T cells (TEM).

[0039] In some embodiments that may be combined with any other embodiments described herein, the virus is a 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 1 (HSV-2).

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

[0041] In some embodiments, provided herein is a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a 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, provided herein are kits comprising one, two, three, or four polypeptide chains of a binding protein according to any one of the above

embodiments. In some embodiments, the kits further comprise instructions for using the polypeptide chain or binding protein according to any of the methods or uses described herein, e.g. , supra.

[0043] In some embodiments, provided herein are kits comprising one, two, three, or four polynucleotides according to any one of the above embodiments. In some

embodiments, provided herein are kits of polynucleotides comprising one, two, three, or four polynucleotides of a kit of polynucleotides comprising: (a) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 189, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 190, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 191, and a fourth polynucleotide

comprising the polynucleotide sequence of SEQ ID NO: 192; (b) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 193, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 194, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 195, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 196; (c) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 197, a second polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 198, a third polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 199, and a fourth polynucleotide comprising the polynucleotide sequence of SEQ ID NO:200; (d) a first polynucleotide comprising the polynucleotide sequence of SEQ ID NO: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; (1) 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.

[0044] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and HER2. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes HER2. The trispecific binding protein shown in FIG. 1A uses a constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.

[0046] FIG. IB provides the fold change (vs. parental) in binding affinities of anti-CD28/CD3/HER2 trispecific antibody variants using the indicated anti-HER2, anti-CD3, and anti-CD28 binding domains. Mutations 3233QQ to QEQ (top to bottom) refer to mutations introduced into residues 32-35 of the VL domain of the anti-CD3 binding site (indicated by *); the remaining mutations were introduced into the VH or VL domain of the trastuzumab anti-HER2 binding site (indicated by #; numbering according to Kabat). For the mutations in the anti-HER2 binding site, mutation 30Q was introduced into the VL domain, and the remaining mutations were introduced into the VH domain. The binding affinities were measured by ELISA, and the values provided are relative to parental trispecific antibody.

[0047] FIG. 1C provides binding curves for the indicated trispecific antibodies binding to human HER2, human CD28, and CD3, as determined by ELISA.

[0048] FIG. ID provides a proposed mechanism of action for HER2/CD28xCD3 trispecific antibody-mediated T cell activation and HER2+ cancer cell killing.

[0049] FIG. 2A provides a schematic representation of a trispecific binding protein comprising four polypeptide chains that form three antigen binding sites that binds three target proteins: CD28, CD3, and CD38. A first pair of polypeptides possess dual variable domains having a cross-over orientation (VH1-VH2 and VL2-VL1) forming two antigen binding sites that recognize CD3 and CD28, and a second pair of polypeptides possess a single variable domain (VH3 and VL3) forming a single antigen binding site that recognizes CD38. The trispecific binding protein shown in FIG. 2A uses an IgG4 constant region with a“knobs-into-holes” mutation, where the knob is on the second pair of polypeptides with a single variable domain.

[0050] FIGS. 2B-2E show the binding affinities, as measured by ELISA, of

CD38/CD28sup x CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated anti-CD38 binding domains for the target antigens human CD38 (FIG. 2B), cynomolgus monkey CD38 (FIG. 2C), human CD3 (FIG. 2D), and human CD28 (FIG. 2E).

[0051] FIG. 3 shows SPR competition assays for binding to CD38 by Daratumumab and anti-CD38 monospecific antibodies with the indicated anti-CD38 binding domains. If an antibody recognized an epitope on CD38 which was different from that of

Daratumumab, injection of the antibody resulted in an increased SPR signal. If an antibody recognized an overlapping epitope as Daratumumab, injection of the antibody did not increase SPR signal.

[0052] FIGS. 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+). The assays were carried out in the presence of 5 nM isotype control antibody (FIG. 4A) or Daratumumab (FIG. 4B). In the presence of daratumumab, the trispecific antibodies continued to exhibit cell killing activity.

[0053] FIGS. 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-Lyl9 cells (CD38+/CD28-). The assays were carried out in the presence of 5 nM isotype control antibody (FIG. 5A) or

Daratumumab (FIG. 5B). Daratumumab caused a decrease in the cell killing activity of anti-CD38/CD28xCD3 trispecific antibodies.

[0054] FIGS. 6A-6J show the characterization of in vitro T cell subset expansion in

PBMCs collected from CMV-infected Donor D in response to CD38/CD28sup x

CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated alternative anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control (DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA). T cell populations were measured at 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 CMV-specific CD8+ T cells (FIGS. 6A-6B), CMV-specific Tcm CD8+ cells (FIGS. 6C-6D), and CMV-specific Tem CD8+ cells (FIGS. 6E-6F). In addition, the percentages of CMV-specific Tcm (FIGS. 6G-6H) and Tem (FIGS. 6I-6J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in a dose-responsive manner.

[0055] FIGS. 7A-7J show the 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 antibodies with the indicated anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA).

Antibodies shown in legend from top to bottom are shown in the graphs from left to right.

T cell populations were measured at 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 quantify CMV-specific CD8+ T cells

(FIGS. 7A-7B), CMV-specific TCm CD8+ cells (FIGS. 7C-7D), and CMV-specific Tem CD8+ cells (FIGS. 7E-7F). In addition, the percentages of CMV-specific Tcm (FIGS. 7G-7H) and Tem (FIGS. 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 with different potency and kinetics in dose response manner.

[0056] FIGS. 8A-8J show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor C in response to CD38/CD28sup x

CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated alternative anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VH1/(DCD28sup x (DCD3mid IgG4 FALA). T cell populations were measured at 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 (FIGS. 8A-8B), CMV-specific Tcm CD8+ cells (FIGS. 8C-8D), and CMV-specific Tem CD8+ cells (FIGS. 8E-8F). In addition, the percentages of EBV-specific Tcm (FIGS. 8G-8H) and Tem (FIGS. 8I-8J) CD8+ cells were quantified at the indicated time points. All tested trispecific antibodies promoted the proliferation of CMV-specific memory CD8+ T cells with different potency and kinetics in dose response manner.

[0057] FIGS. 9A-12 show the characterization of in vitro T cell subset expansion in PBMCs collected from EBV-infected Donor D in response to CD38/CD28sup x

CD3mid_ENLQ DKTHT IgG4 FALA trispecific antibodies with the indicated alternative anti-CD38 binding domains. A trispecific antibody lacking the CD38VH1 anti-CD38 binding domain was used as a negative control ((DCD38VHl/(DCD28sup x (DCD3mid IgG4 FALA). T cell populations were measured at 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 (FIGS. 9A-9B), EBV-specific Tcm CD8+ cells (FIGS. 9C-9D), and EBV-specific Tem CD8+ cells (FIGS. 9E-9F). In addition, the percentages of EBV-specific Tcm (FIGS. 9G-10) and Tem (FIGS. 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 with different potency and kinetics in dose response manner.

[0058] FIGS. 13A-13D show the change over time (days) in tumor volume (FIG. 13A) and body weight (FIG. 13B) in ZR-75-1 tumor bearing NSG mice engrafted with in vitro expanded human CD3+ T cells. Groups of 10 mice were either treated with vehicle or

Her2/CD28 x CD3 trispecific antibody at the indicated dosages. Arrow heads indicate days of administration. Tumor volume is depicted as mean ± SEM, mm3. Body weight change is depicted as % change, mean ± SEM. X-axis shows days after implantation with ZR-75-1 cells. Tumor volume (mm3) over time for individual mice in each treatment group are shown in FIG. 13C. Tumor weight (mg) for each treatment group is shown in FIG. 13D.

** = p<0.001; *** = p<0.0003 (two-way ANOVA, control vs. 100 & lOug/kg).

[0059] FIGS. 14A-14C show the effect of Her2/CD28 x CD3 trispecific antibody treatment on T cells from whole blood. FIG. 14A shows the analysis of hCD45+, CD8+, CD4+, and mCD45+ cells by flow cytometry. FIG. 14B shows the effect of control or Her2/CD28 x CD3 trispecific antibody treatment (at the indicated doses) on hCD45+, CD8+, CD4+, and mCD45+ cell counts. FIG. 14C shows the effect of control or

Her2/CD28 x CD3 trispecific antibody treatment (at the indicated doses) on human cell ratios (CD4+/CD45+ and CD8+/CD45+). For each x-axis parameter shown in FIGS. 14B & 14C, conditions are (left to right): control, lOOug/kg trispecific antibody, lOug/kg trispecific antibody, lug/kg trispecific antibody, and 0. lug/kg trispecific antibody.

Percentages shown in FIGS. 14B & 14C are based on control sample vs. lOOug/kg.

[0060] FIGS. 15A-15C show the effect of Her2/CD28 x CD3 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.

Tipper images are at IX magnification; lower images are at 20X magnification. In both sets of images, staining for human CD45, human CD4, and human CD8 are shown from left to right. Shown are tumors from mice treated with vehicle control (FIG. 15A), lOOug/kg trispecific antibody (FIG. 15B), or 0. lug/kg trispecific antibody (FIG. 15C).

[0061] FIGS. 16A-16C show quantitation of the effect of Her2/CD28 x CD3 trispecific antibody treatment on TILs as measured by IHC. Each dot represents one tumor from an individual mouse; rectangles represent group means; and error bars indicate standard deviation. * = p<0.05 compared to vehicle control group (ANOVA). Numbers of CD45+ (FIG. 16 A), CD4+ (FIG. 16B), or CD8+ (FIG. 16C) cells are shown. In FIG. 16C, a $ area quantitation approach was used for CD8+ cells instead of cell counting algorithm due to excessive non-specific signal in the CD8 IHC slide.

[0062] FIGS. 17A-17F show in vitro cell lysis of HER2+ breast cancer target cells in the presence of human CD8+ T cells by Her2/CD28 x CD3 trispecific antibody with wild-type trastuzumab antigen binding domain and an anti-CD3 antigen binding domain without without 32/35 QQ mutations in the VL domain (“ctl”) as compared to a Her2/CD28 x CD3 trispecific antibodies having mutations in the anti-HER2 arm and the VL domain of the anti-CD3 arm (numbering as shown in Table 1). Cell killing activities against cell lines with varying expression of HER2 are depicted: HCC1954 for high HER2 expression (FIG. 17A), BT20 for intermediate HER2 expression (FIG. 17C), and MDA-MD-231 for low HER2 expression (FIG. 17E). Graphs depicting cell killing as a function of antibody concentration against target cells HCC1954 (FIG. 17B), BT20 (FIG. 17D), and MDA-MD-231 (FIG. 17F) are shown, comparing binding protein #2 vs. ctl or binding protein #1 and #5 vs. ctl.

[0063] FIGS. 18A & 18B summarize the mean EC50 (pM) of in vitro cell killing by experimental or control Her2/CD28 x CD3 trispecific antibodies against the indicated breast cancer (FIG. 18A) or gastric cancer cell lines (FIG. 18B). Amino acid sequences of the indicated trispecific antibodies are provided in Table 1.

DETAILED DESCRIPTION

[0064] The 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 possess dual variable domains having a cross-over orientation.

General Definitions

[0065] 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 shall include pluralities and plural terms shall include the singular.

[0066] It is understood that aspects and embodiments of the disclosure described herein include“comprising,”“consisting,” and“consisting essentially of’ aspects and

embodiments.

[0067] The term "polynucleotide" as used herein refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or

deoxyribonucleotides or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as

phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,

phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term

"polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

What is claimed is:

1. A binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of

IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site.

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

3. The binding protein of claim 2, wherein the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the VL1 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).

4. The binding protein of claim 3, wherein the Vm domain comprises the amino acid sequence of

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNV NTNYAQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWG KGTTVTVSS (SEQ ID NO:91), and/or the VL1 domain comprises the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTG VPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK (SEQ ID NO: 92).

5. The binding protein of any one of claims 1-4, wherein the CDR-L1 sequence of the VL2 domain comprises an amino acid sequence selected from the group consisting of Q SLVHQNAQT Y (SEQ ID NO:59), QSLVHENLQTY (SEQ ID NO:60), QSLVHENLFTY (SEQ ID NO:61), and Q SL VHENLRT Y (SEQ ID NO:62).

6. The binding protein of claim 5, wherein the VH2 domain comprises the amino acid sequence of

QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AD S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD Y WGQGTLVTVSS (SEQ ID NO:93) or

QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKS N S YAT YY AS S VKGRFTISRDD SKNTLYLQMN SLRAEDT AVYY CRGVYY AL SPFD YW GQGTLVTVSS (SEQ ID NO:302), and/or the VL2 domain comprises an amino acid sequence selected from the group consisting of

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHQNAQT YL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:95),

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLQTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 96),

DIVMTQTPLSLSVTPGQPASISCKSSQSLVHENLFTYLSWYLQKPGQSPQSLIYKVSNR F SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO: 97), and

DIVMTQTPLSL S VTPGQP ASISCKS SQ SLVHENLRTYL S WYLQKPGQ SPQ SLIYK V SN RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98).

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

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

9. The binding protein of claim 8, wherein:

(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL3 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. The binding protein of claim 9, wherein:

(a) the VH3 domain comprises the amino acid sequence of

(b) the VH3 domain comprises the amino acid sequence of

(c) the VH3 domain comprises the amino acid sequence of

(d) the VH3 domain comprises the amino acid sequence of

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYAVHWVRQAPGQGLEWMGVISPY Y GDTT Y AQKF QGRVTMTVDRS S ST AYMEL SRLRSDDT AVYY C ARRFEGF YY SMD Y WGQGTL VTVSS (SEQ ID NO:85), and/or the VL3 domain comprises the amino acid sequence of

(e) the VH3 domain comprises the amino acid sequence of

sequence of

or

(f) the VH3 domain comprises the amino acid sequence of

1 1. The binding protein of claim 8, wherein:

(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; 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;

(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;

(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; 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;

(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; 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;

(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; 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;

(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; 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;

(g) 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; 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.

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

13. The binding protein of claim 12, wherein:

(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l) 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 SRW GGDGF YAMD Y (SEQ ID NO: 6), SRW GGEGF YAMD Y (SEQ ID NO: 7), or SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIKDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTQGYT (SEQ ID NO:4), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGSGFYAMDY (SEQ ID NO:8), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO:2), a CDR-H2 sequence comprising the amino acid sequence of IYPTNAYT (SEQ ID NO:5), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGEGFYAMDY (SEQ ID NO:7), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFNIRDTY (SEQ ID NO: l), a CDR-H2 sequence comprising the amino acid sequence of IYPTNGYT (SEQ ID NO:3), and a CDR-H3 sequence comprising the amino acid sequence of SRWGGDGFYAMDY (SEQ ID NO:6), and the VL3 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).

14. The binding protein of claim 13, wherein:

(a) the VH3 domain comprises the amino acid sequence of

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO: 72),

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:73),

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG

YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW

GQGTLVTVSS (SEQ ID NO: 74),

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO: 75), or

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 77) or

DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:78);

(b) the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);

(c) the VH3 domain comprises the amino acid sequence of

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AVYY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:73), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGK APKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);

(d) the VH3 domain comprises the amino acid sequence of

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA

YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:75), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGKAPKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);

(e) the VH3 domain comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTQG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGSGFYAMDYW GQGTLVTVSS (SEQ ID NO:74), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGKAPKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77);

(f) the VH3 domain comprises the amino acid sequence of

EVQLVESGGGLVQPGGSLRLSCAASGFNIRDTYIHWVRQAPGKGLEWVARIYPTNA YTRY AD S VKGRFTIS ADTSKNT AYLQMN SLRAEDT AV YY C SRW GGEGF YAMD YW GQGTLVTVSS (SEQ ID NO:76), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQ SP S SLS AS VGDRVTIT CRASQD VNT AVAW Y QQKPGKAPKLLI Y S ASFL YSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO: 77); or

(g) the VH3 domain comprises the amino acid sequence of

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:72), and/or the VL3 domain comprises the amino acid sequence of

DIQMTQSPSSLSASVGDRVTITCRASQDVQTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRF SGSRSGTDFTLTIS SLQPEDF AT YY CQQHYTTPPTF GQGTKVEIK (SEQ ID NO:78).

15. The binding protein of claim 12, wherein:

(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; 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;

(b) 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;

(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; 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;

(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; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 123;

(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; 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;

(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; 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;

(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; 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;

(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; 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;

(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; 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;

(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; 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;

(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; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151;

(m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 152; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 153; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 154; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 155;

(n) 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;

(o) 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;

(p) 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; or

(q) 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.

16. The binding protein of any one of claims 1-15, wherein at least one of L1, L2, L3 or L4 is independently 0 amino acids in length.

17. The binding protein of any one of claims 1-15, wherein (a) L1, L2, L3 and L4 each independently are zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO: 70), S, RT, TKGPS (SEQ ID NO: 68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO: 71); or (b) L1, L2, L3 and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO:69), GGGGSGGGGSGGGGS (SEQ ID NO:70), S, RT, TKGPS (SEQ ID NO:68), GQPKAAP (SEQ ID NO: 67), and GGSGSSGSGG (SEQ ID NO:71).

18. The binding protein of any one of claims 1-15, wherein L1 comprises the sequence GQPKAAP (SEQ ID NO: 67), L2 comprises the sequence TKGPS (SEQ ID NO:68), L3 comprises the sequence S, and L4 comprises the sequence RT.

19. The binding protein of any one of claims 1-15, wherein at least one of L1, L2, L3 or L4 comprises the sequence DKTHT (SEQ ID NO:66).

20. The binding protein of claim 19, wherein L1, L2, L3 and L4 comprise the sequence DKTHT (SEQ ID NO:66).

21. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to EU Index, wherein the amino acid substitutions are F234A and L235A.

22. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 233-236 of human IgG4 according to EU Index, wherein the amino acid substitutions are E233P, F234V, L235A, and a deletion at 236.

23. The binding protein of any one of claims 1-22, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgG4 hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to EU Index, wherein the amino acid substitutions are S228P and R409K.

24. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234, 235, and 329 of human IgGl according to EU Index, wherein the amino acid substitutions are L234A, L235A, and P329A.

25. The binding protein of any one of claims 1-20, wherein the hinge-CH2-CH3 domains of the second and the third polypeptide chains are human IgGl hinge-CH2-CH3 domains, and wherein the hinge-CH2-CH3 domains each comprise amino acid substitutions at positions corresponding to positions 298, 299, and 300 of human IgGl according to EU Index, wherein the amino acid substitutions are S298N, T299A, and Y300S.

26. The binding protein of any one of claims 1-25, wherein the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W.

27. The binding protein of any one of claims 1-25, wherein the hinge-CH2-CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are S354C and T366W; and wherein the hinge-CH2-CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgGl or IgG4 according to EU Index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

42. The binding protein of any one of claims 7-27 or the pharmaceutical composition of claim 32 for use in preventing and/or treating cancer in a patient.

43. The binding protein for use or the composition for use of claim 42, wherein the at least one binding protein is to be co-administered with a chemotherapeutic agent.

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

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

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

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

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

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

50. The binding protein for use or the 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 for expanding T cells, comprising contacting a T cell with a binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

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. 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 contacting a virus-specific memory T cell with a binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

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

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

57. A method for treating chronic viral infection, comprising administering to a patient in need thereof an effective amount of a binding protein comprising four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and

L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

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

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

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

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

62. The method of any one of claims 53-56, 60, and 61, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).

63. The method of any one of claims 54-62, wherein the virus is a 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 1 (HSV-2).

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

65. A binding protein for use in expanding T cells, wherein the binding protein is to be contacted with a T cell, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

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

68. A binding protein for use in expanding virus-specific memory T cells, wherein the binding protein is to be contacted with a virus-specific memory T cell, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

Vm is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein Vm and VL1 form a first antigen binding site that binds a CD28 polypeptide;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

69. The binding protein for use of claim 68, wherein the virus-specific memory T cell is to be contacted with the binding protein in vitro or ex vivo.

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

71. A binding protein for use in treating chronic viral infection, wherein the binding protein is to be administered to a patient in need thereof, wherein the binding protein comprises four polypeptide chains that form the three antigen binding sites, wherein a first polypeptide chain comprises a structure represented by the formula:

VL2-L1-VL1-L2-CL [I]

and a second polypeptide chain comprises a structure represented by the formula:

VHi-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

and a third polypeptide chain comprises a structure represented by the formula:

VH3-CH1-hinge-CH2-CH3 [III]

and a fourth polypeptide chain comprises a structure represented by the formula:

VL3-CL [IV]

wherein:

VL1 is a first immunoglobulin light chain variable domain;

VL2 is a second immunoglobulin light chain variable domain;

VL3 is a third immunoglobulin light chain variable domain;

VH1 is a first immunoglobulin heavy chain variable domain;

VH2 is a second immunoglobulin heavy chain variable domain;

VH3 is a third immunoglobulin heavy chain variable domain;

CL is an immunoglobulin light chain constant domain;

CH1 is an immunoglobulin CH1 heavy chain constant domain;

CH2 is an immunoglobulin CH2 heavy chain constant domain;

CH3 is an immunoglobulin CH3 heavy chain constant domain;

hinge is an immunoglobulin hinge region connecting the CH1 and CH2 domains; and L1, L2, L3 and L4 are amino acid linkers;

wherein the polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair; and

wherein VH1 and VL1 form a first antigen binding site that binds a CD28 polypeptide;

wherein VH2 and VL2 form a second antigen binding site that binds a CD3 polypeptide, wherein the VH2 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFTKAW (SEQ ID NO: 55), a CDR-H2 sequence comprising the amino acid sequence of IKDKSNSYAT (SEQ ID NO:56), and a CDR-H3 sequence comprising the amino acid sequence of RGVYYALSPFDY (SEQ ID NO:57), and the VL2 domain comprises a CDR-L1 sequence comprising the amino acid sequence of QSLVHX1NX2X3TY, wherein X1 is E or Q, X2 is A or L, and X3 is Q, R, or F (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); and

wherein VH3 and VL3 form a third antigen binding site that binds a CD38 polypeptide.

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

73. The binding protein for use of claim 71 or claim 72, wherein the binding protein is to be administered to the patient in pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable carrier.

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

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

76. The binding protein for use of any one of claims 67-70, 74, and 75, wherein the memory T cells are central memory T cells (TCM) or effector memory T cells (TEM).

77. The binding protein for use of any one of claims 68-76, wherein the virus is a 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 1 (HSV-2).

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

79. The method or binding protein for use of any one of claims 51-78, wherein the Vm domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYY (SEQ ID NO:49), a CDR-H2 sequence comprising the amino acid sequence of IYPGNVNT (SEQ ID NO:50), and a CDR-H3 sequence comprising the amino acid sequence of TRSHYGLDWNFDV (SEQ ID NO:51), and the VL1 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. The method or binding protein for use of claim 79, wherein the Vm domain comprises the amino acid sequence of

81. The method or binding protein for use of any one of claims 51-80, wherein the CDR- L1 sequence of the VL2 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 Q SL VHENLRT Y (SEQ ID NO:62).

82. The method or binding protein for use of claim 81, wherein the VH2 domain comprises the amino acid sequence of

RF SGVPDRF SGSGSGTDFTLKISRVEAED VGVYYCGQGTQYPFTFGSGTKVEIK (SEQ ID NO:98).

83. The method or binding protein for use of any one of claims 51-82, wherein:

(a) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTFTSYA (SEQ ID NO: 13), a CDR-H2 sequence comprising the amino acid sequence of IYPGQGGT (SEQ ID NO: 14), and a CDR-H3 sequence comprising the amino acid sequence of ARTGGLRRAYFTY (SEQ ID NO: 15), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYTLTEFS (SEQ ID NO: 19), a CDR-H2 sequence comprising the amino acid sequence of FDPEDGET (SEQ ID NO:20), and a CDR-H3 sequence comprising the amino acid sequence of TTGRFFDWF (SEQ ID NO:21), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYAFTTYL (SEQ ID NO:25), a CDR-H2 sequence comprising the amino acid sequence of INPGSGST (SEQ ID NO:26), and a CDR-H3 sequence comprising the amino acid sequence of ARYAYGY (SEQ ID NO:27), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GYSFTNYA (SEQ ID NO:31), a CDR-H2 sequence comprising the amino acid sequence of ISPYYGDT (SEQ ID NO:32), and a CDR-H3 sequence comprising the amino acid sequence of ARRFEGFYYSMDY (SEQ ID NO:33), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:37), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:38), and a CDR-H3 sequence comprising the amino acid sequence of ARDPGLRYFDGGMDV (SEQ ID NO:39), and the VL3 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) the VH3 domain comprises a CDR-H1 sequence comprising the amino acid sequence of GFTFSSYG (SEQ ID NO:43), a CDR-H2 sequence comprising the amino acid sequence of IWYDGSNK (SEQ ID NO:44), and a CDR-H3 sequence comprising the amino acid sequence of ARMFRGAFDY (SEQ ID NO:45), and the VL3 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. The method or binding protein for use of claim 83, wherein:

(a) the VH3 domain comprises the amino acid sequence of QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYAMHWVKEAPGQRLEWIGYIYPGQ GGTNYNQKFQGRATLTADTSASTAYMELSSLRSEDTAVYFCARTGGLRRAYFTYWG QGTLVTVSS (SEQ ID NO:79), and/or the VL3 domain comprises the amino acid sequence of DIVLTQ SP ATLSL SPGERATISCRASQ S V S S YGQGFMHW YQQKPGQPPRLLI Y GAS SR ATGIP ARE SGSGSGTDFTLTISPLEPEDF AVYYCQQNKEDPWTF GGGTKLEIK (SEQ ID NO:80);

(b) the VH3 domain comprises the amino acid sequence of

Q VQL VQSGAEVKKPGAS VKVSCKVSGYTLTEF SIHWVRQ APGQGLEWMGGFDPED GETIY AQKFQGRVIMTEDT STDT AYMEMN SLRSEDT AIYYCTT GRFFDWF W GQGTL VTVSS (SEQ ID NO:81), and/or the VL3 domain comprises the amino acid sequence of EIILTQSPAILSLSPGERATLSCRASQSVISRFLSWYQVKPGLAPRLLIYGASTRATGIPV RF SGSGSGTDF SLTIS SLQPEDC AVYYCQQDSNLPITF GQGTRLEIK (SEQ ID NO:82);

(c) the VH3 domain comprises the amino acid sequence of

QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYLVEWIRQRPGQGLEWMGVINPGS GSTNY AQKF QGRVTMTVDRS STT AYMEL SRLRSDDT AVYY CARY AY GYW GQGTL

(d) the VH3 domain comprises the amino acid sequence of

sequence of

S

(e) the VH3 domain comprises the amino acid sequence of

sequence of

or

(f) the VH3 domain comprises the amino acid sequence of

85. The method or binding protein for use of any one of claims 51-82, wherein:

(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; 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;

(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;

(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; 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;

(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; 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;

(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; 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;

(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; 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;

(g) 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; 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 a first, second, third, and fourth polypeptide chain of a binding protein of any one of claims 1-27.

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

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.

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