Multispecific constructs and uses thereof

Abstract

Bispecific and multispecific antibodies, including agonistic anti-4-1BB single domain antibodies, capable of effector function-mediated tumor inhibition are provided. Furthermore, these agonistic antibodies bind to 4-1BB but cannot activate 4-1BB signaling in the absence of tumor antigen-expressing cells. However, in the presence of tumor antigen-expressing cells, these antibodies can induce tumor antigen-dependent 4-1BB signaling, resulting in a strong immune response against tumor antigen-expressing tumor cells.

Description

[Technical Field]

[0001] Field of the Disclosure The present application relates to multispecific molecules, such as anti-tumor antigen / anti-4-1BB bispecific antibodies, and uses thereof, including treating diseases or conditions. [Background technology]

[0002] background 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF-receptor superfamily (TNFRSF) and a costimulatory molecule expressed after activation of immune cells, including both innate and adaptive immune cells. 4-1BB plays an important role in modulating immune cell activity. 4-1BB agonists can enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Many studies have shown that activation of 4-1BB enhances immune responses to eliminate tumors in mice, indicating 4-1BB as a promising target molecule in cancer immunology.

[0003] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. Summary of the Invention [Means for solving the problem]

[0004] overview Prior to the present disclosure, conventional wisdom was that agonistic anti-4-1BB antibodies should not be ADCC capable, at least because ADCC-inducing FcγR binding would deplete 4-1BB+ cells, thereby undermining the agonistic effect of the antibody and potentially causing toxicity (e.g., liver toxicity) due to FcγR engagement. For example, the superior anti-4-1BB antibody drug candidate urelumab contains an IgG4 Fc with no or limited effector function.

[0005] In an unexpected discovery, the inventors demonstrated that when a proprietary anti-4-1BB single-domain antibody (represented by SEQ ID NO: 27) was used in a bispecific format with a second antibody moiety targeting a tumor-associated antigen (TAA), the use of WT IgG1 actually enhanced its tumor-inhibitory activity. More specifically, in Experimental Example 4, two different versions of an anti-claudin 6 (CLDN6-1) / anti-4-1BB bispecific antibody were compared. One of them contained wild-type (WT) IgG1 Fc, and the other had a silencing mutation (N297A or NA). As shown in Figure 7B, both the WT (with effector function) and NA (without effector function) versions exhibited good anti-tumor activity, but the WT version achieved significantly higher and more sustained tumor inhibition.

[0006] Without being bound by any particular theory, such unique effector function-enhancing properties of bispecific antibodies may be attributed to the activity or format (eg, single domain) of the anti-4-1BB antibodies of the present invention.

[0007] Thus, according to one embodiment of the present disclosure, there is provided a multispecific construct, e.g., a multispecific antibody, comprising: (1) a first antibody portion that specifically binds to a tumor antigen; and (2) a second antibody portion that specifically binds to 4-1BB. In some embodiments, the 4-1BB-activating activity of the second antibody portion depends on the binding of the first antibody portion to the corresponding tumor antigen.

[0008] In some embodiments, the multispecific construct further comprises an Fc domain with maintained or improved effector function.

[0009] In some embodiments, the first antibody portion is selected from the group consisting of a full length antibody, a half antibody, a single chain half antibody, a Fab, a Fab', a F(ab')2, and a scFv.

[0010] In some embodiments, the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the second antibody portion is selected from the group consisting of a full-length antibody, a half antibody, a single-chain half antibody, Fab, Fab', F(ab')2, scFv, and an sdAb. In some embodiments, the second antibody portion is an sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, CDR1, CDR2, and CDR3 follow the Kabat numbering scheme. In some embodiments, the sdAb comprises (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO:24; (2) sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25; and (3) sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the second antibody portion comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27.

[0011] In some embodiments, it is a bispecific antibody or bispecific binding fragment.

[0012] In some embodiments, the multispecific construct further comprises an Fc domain. In some embodiments, the Fc domain is derived from any one selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc domain is derived from IgG1. In some embodiments, the Fc domain comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 46-56, 285-286, and 288-289.

[0013] In some embodiments, activation of 4-1BB by the second antibody moiety is enhanced by at least 10-fold after binding of the first antibody moiety to a tumor antigen. In some embodiments, activation of 4-1BB by the second antibody moiety results in an increase in IFNγ levels, IL-2 levels, or NFκB signaling. In some embodiments, the first antibody moiety enhances activation of 4-1BB by at least 10-fold.-7 M~about 10 -13 It has a binding affinity of M.

[0014] In some embodiments, the first antibody portion is fused to the C-terminus of the second antibody portion. In some embodiments, the first antibody portion is fused to the N-terminus of the second antibody portion. In some embodiments, the first antibody portion and the second antibody portion are fused to each other via a linker.

[0015] In some embodiments, the first antibody moiety is a Fab' fused to the N-terminus of an IgG Fc domain and the second antibody moiety is an sdAb fused to the C-terminus of the IgG Fc domain, hi some embodiments, the second antibody moiety is fused to the IgG Fc domain via a linker.

[0016] In some embodiments, the multispecific construct further comprises a third antibody portion that specifically binds to a second tumor antigen, which in some embodiments has the same epitope as the tumor antigen but a different epitope.

[0017] In some embodiments, the third antibody portion is selected from the group consisting of a full length antibody, a half antibody, a single chain half antibody, a Fab, a Fab', a F(ab')2, and a scFv.

[0018] In some embodiments, the first antibody moiety is a Fab' fused to the N-terminus of an IgG Fc domain, the second antibody moiety is an sdAb fused to the C-terminus of the IgG Fc domain, and the third antibody moiety is an scFv fused to the N-terminus of the first antibody moiety. In some embodiments, the second antibody moiety is fused to the IgG Fc domain via a linker, and the third antibody moiety is fused to the first antibody moiety via a linker.

[0019] In some embodiments, the first antibody moiety is a Fab' fused to the N-terminus of the IgG Fc domain, the second antibody moiety is an sdAb fused to the C-terminus of the IgG Fc domain, the third antibody moiety is an scFv fused to the N-terminus of the paired IgG Fc domain, and the second antibody moiety is an sdAb fused to the C-terminus of the paired IgG Fc domain, wherein the paired IgG Fc domain forms a heterodimer with the IgG Fc domain. In some embodiments, the second antibody moiety is fused to the IgG Fc domain via a linker, and the second antibody moiety is fused to the paired IgG Fc via a linker.

[0020] In some embodiments, the third antibody moiety is fused to the N-terminus of the paired IgG Fc domain via a tether that comprises, from N- to C-terminus, the short linker "AA" and / or the hinge variant of "EPKSSDKTHT" (SEQ ID NO: 291).

[0021] In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 285 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any greater than 99%) sequence identity to the sequence set forth in SEQ ID NO: 285, and the paired Fc domain comprises the amino acid sequence of SEQ ID NO: 288 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any greater than 99%) sequence identity to the sequence set forth in SEQ ID NO: 288.

[0022] In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO:286 or a variant thereof having at least about 80% (e.g., including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any of higher than 99%) sequence identity to the sequence set forth in SEQ ID NO:286, and the paired Fc domain comprises the amino acid sequence of SEQ ID NO:289 or a variant thereof having at least about 80% (e.g., including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any of higher than 99%) sequence identity to the sequence set forth in SEQ ID NO:289.

[0023] In another aspect, provided herein is a pharmaceutical composition comprising a multispecific construct described herein and a pharmaceutically acceptable carrier.

[0024] In another aspect, provided herein are nucleic acids encoding the multispecific constructs described herein.

[0025] In another aspect, provided herein is a vector comprising a nucleic acid described herein.

[0026] In another aspect, provided herein is a host cell comprising a nucleic acid described herein or a vector described herein.

[0027] In another aspect, provided herein is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a multispecific construct described herein or a pharmaceutical composition described herein. In some embodiments, the disease or condition is cancer.

[0028] In another aspect, provided herein is the use of a multispecific construct as described herein in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

[0029] The present disclosure also provides an antibody or antigen-binding fragment thereof having specificity for human mucin 16 (MUC16) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are: (a) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: DSRKYYYDSSGPALWGFDAFDI (SEQ ID NO: 59), LCDR1: RASQSISSYLN (SEQ ID NO: 60), LCDR2: AASSLQS (SEQ ID NO: 61), and LCDR3: QQSYSTLST (SEQ ID NO: 62), (b) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: EPPLSNYGDYATEQYYYGMDV (SEQ ID NO: 67), LCDR1: RASQSISSYLN (SEQ ID NO: 60), LCDR2: AASSLQS (SEQ ID NO: 61), and LCDR3: QQSYSTPLT (SEQ ID NO: 70); (c) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: APMVRGVPPTPYYYYYGMDV (SEQ ID NO: 75), LCDR1: RASQSVSNYLA (SEQ ID NO: 76), LCDR2: DASNRAT (SEQ ID NO: 77), and LCDR3: QQRSNWPS (SEQ ID NO: 78), (d) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: TPELLWFGELGGAYYFDY (SEQ ID NO: 83), LCDR1: RASESISSWLA (SEQ ID NO: 84), LCDR2: KASTLEN (SEQ ID NO: 85), and LCDR3: QQYRSHWSST (SEQ ID NO: 86); (e) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: ANFNIYYYYYGMDV (SEQ ID NO: 91), LCDR1: RSSQSLLHSNGYNYLD (SEQ ID NO: 92), LCDR2: LGSNRAS (SEQ ID NO: 93), and LCDR3: MQGTHWPRT (SEQ ID NO: 94), (f) HCDR1: SYEMN (SEQ ID NO: 97), HCDR2: RIKSKTDGGTTDYAAPV (SEQ ID NO: 98), HCDR3: DLAAVAGLFDY (SEQ ID NO: 99), LCDR1: QASQDISNYLN (SEQ ID NO: 100), LCDR2: DASNLET (SEQ ID NO: 101), and LCDR3: QQSYSTPWK (SEQ ID NO: 102), (g) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: RIIPIFGIANYAQKFQG (SEQ ID NO: 106), HCDR3: TGDYDILTGSYYYGMDV (SEQ ID NO: 107), LCDR1: RASQGIRNDLG (SEQ ID NO: 108), LCDR2: AASSLQS (SEQ ID NO: 61), and LCDR3: LQDYNYPFT (SEQ ID NO: 120), (h) HCDR1: DYYLS (SEQ ID NO: 123), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: GGPHYDFWSGYTPGQHGGAFDI (SEQ ID NO: 125), LCDR1: RASQSVSSSYLA (SEQ ID NO: 126), LCDR2: GASSRAT (SEQ ID NO: 127), and LCDR3: QQRSNWRNT (SEQ ID NO: 128), (i) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: DSGSSITMVRGGDYYYMDV (SEQ ID NO: 133), LCDR1: RASQSVSSYLA (SEQ ID NO: 134), LCDR2: DASNRAT (SEQ ID NO: 77), and LCDR3: QQRSNWPPT (SEQ ID NO: 136), (j) HCDR1: YHAIS (SEQ ID NO: 139), HCDR2: GIIPILGTANYAQKFQG (SEQ ID NO: 140), HCDR3: GTTAARYYYYYYYMDV (SEQ ID NO: 141), LCDR1: QASQDISNYLN (SEQ ID NO: 100), LCDR2: DASNLET (SEQ ID NO: 101), and LCDR3: QQYDNLPLT (SEQ ID NO: 144), (k) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: SITDYYDSSGYYFRPHFNTGYYYGMDV (SEQ ID NO: 149), LCDR1: RASQGINNYLA (SEQ ID NO: 150), LCDR2: AASTLQS (SEQ ID NO: 151), and LCDR3: QQYDTFSET (SEQ ID NO: 152), (l) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: GGPHYDFWSGYTPGQHGGAFDI (SEQ ID NO: 125), LCDR1: RASQSISGWLA (SEQ ID NO: 158), LCDR2: RTSYLES (SEQ ID NO: 159), and LCDR3: QHYDTFSRA (SEQ ID NO: 160), or (m) HCDR1: YHAIS (SEQ ID NO: 139), HCDR2: SISSGGNTYYPDTVKGR (SEQ ID NO: 38), HCDR3: EGPDYGDYSWSMDYYYGMDV (SEQ ID NO: 165), LCDR1: RASQSVNSRYLA (SEQ ID NO: 166), LCDR2: GASTRAT (SEQ ID NO: 167), and LCDR3: QQYGTFSIT (SEQ ID NO: 168) is selected from the group consisting of:

[0030] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161, and 169, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161, and 169.

[0031] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162, and 170, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162, and 170.

[0032] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80; (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88; (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96; (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 103 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 104; (g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 121 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 122; (h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 129 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130; (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 138; (j) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 146; (k) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 153 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154; (l) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 161 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 162; or (m) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 169 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 170 Includes.

[0033] In some embodiments, the antibodies provided herein are chimeric or humanized antibodies.

[0034] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0035] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are bifunctional molecules comprising a first antibody portion that has specificity for human MUC16 protein and a second antibody portion that has specificity for a second protein, wherein the first antigen-binding portion comprises an antibody or antigen-binding fragment thereof provided herein.

[0036] In some embodiments, the second protein is 4-1BB. In some embodiments, the first antibody portion is a full-length antibody. In some embodiments, the second antibody portion is an sdAb. In some embodiments, the second antibody portion is fused to the C-terminus of the first antibody portion. In some embodiments, the first and second antibody portions are fused to each other via a linker. In some embodiments, the second antibody portion comprises an HCDR1 of SNCMG (SEQ ID NO: 24), an HCDR2 of VICTGGGSPSYADSVKG (SEQ ID NO: 25), and an HCDR3 of DLLRAGTPLSSYEFNY (SEQ ID NO: 26). In some embodiments, the second antibody portion comprises the amino acid sequence of SEQ ID NO: 27 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0037] In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof provided herein, or a bifunctional molecule provided herein, and a pharmaceutically acceptable carrier.

[0038] In one aspect, the present disclosure provides an isolated cell comprising one or more polynucleotides encoding an antibody or antigen-binding fragment thereof provided herein, or a bifunctional molecule provided herein.

[0039] In one aspect, the disclosure provides polynucleotides encoding one or more chains of an antibody or antigen-binding fragment thereof provided herein, or a bifunctional molecule provided herein.

[0040] In one aspect, the present disclosure provides a method of treating cancer in a patient in need thereof, the method comprising administering to the patient an antibody or antigen-binding fragment thereof provided herein, or a bifunctional molecule provided herein.

[0041] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer.

[0042] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described in the following detailed description. [Brief explanation of the drawings]

[0043] [Figure 1-1] 1A to 1C show the binding affinity of the CLDN6x4-1BB bispecific antibody (BsAb) disclosed herein to CLDN6. [Figure 1-2] Same as above.

[0044] [Figure 2-1] 2A to 2C show the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to CLDN6-expressing cells. [Figure 2-2] Same as above.

[0045] [Figure 3-1] 3A-3D show the binding affinity of the CLDN6x4-1BB BsAb disclosed herein to 4-1BB. [Figure 3-2] Same as above.

[0046] [Figure 4] 4A-4B show the binding of the CLDN6x4-1BB BsAb disclosed herein to soluble 4-1BB and cells expressing 4-1BB.

[0047] [Figure 5-1]5A-5D show CLDN6-dependent 4-1BB activation of the CLDN6x4-1BB BsAb disclosed herein. [Figure 5-2] Same as above.

[0048] [Figure 6-1] 6A-6H show activation of PBMCs by the CLDN6x4-1BB BsAb disclosed herein. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0049] [Figure 7] 7A-7B show tumor inhibition in hu4-1BB mice after treatment with the CLDN6x4-1BB BsAb disclosed herein.

[0050] [Figure 8] 8A-8B show liver function after treatment with the CLDN6x4-1BB BsAb of the present application.

[0051] [Figure 9-1] 9A-9C show the binding activity of 13 selected monoclonal antibodies to human MUC16 protein as measured by ELISA. [Figure 9-2] Same as above.

[0052] [Figure 10] FIG. 10 shows the binding activity of 13 selected monoclonal antibodies to cynomolgus monkey MUC16 protein as measured by ELISA.

[0053] [Figure 11-1] 11A-11C show cell-based binding of 13 selected monoclonal antibodies to human MUC16 positive and negative cells. [Figure 11-2] Same as above.

[0054] [Figure 12-1] 12A-12C show the cell-based binding activity of 13 selected monoclonal antibodies to human MUC16 in OVCAR3, SNU216, and MUC16-overexpressing HEK293 cells, respectively. [Figure 12-2] Same as above.

[0055] [Figure 13-1] 13A-13D show the cell-based binding activity of five selected monoclonal antibodies to human MUC16 in the OVCAR3 cell line in the presence or absence of CA125. [Figure 13-2] Same as above.

[0056] [Figure 14-1] Figures 14A-14B show the kinetics of binding activity of three selected monoclonal antibodies to human MUC16. [Figure 14-2] Same as above.

[0057] [Figure 15-1] 15A-15E show MUC16-dependent 4-1BB activation in a 4-1BB NFκB reporter assay for cell lines with different MUC16 expression levels. [Figure 15-2] Same as above. [Figure 15-3] Same as above.

[0058] [Figure 16-1] Figures 16A-16F show MUC16-dependent 4-1BB activation for cell lines with different MUC16 expression levels, which induces T cell costimulatory activity in PBMCs, leading to the release of human IFNγ and IL-2 cytokines. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0059] [Figure 17-1] Figures 17A-17F show MUC16-dependent 4-1BB activation for cell lines with different MUC16 expression levels, which induces T cell costimulatory activity in CD8+ T cells, leading to the release of human IFNγ and IL-2 cytokines. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 17-4] Same as above.

[0060] [Figure 18-1] 18A-18B show the cell binding activity of bispecific anti-ROR1 / anti-4-1BB antibodies as measured by FACS. [Figure 18-2] Same as above.

[0061] [Figure 19-1] 19A-19C show ROR1-dependent 4-1BB activation of bispecific anti-ROR1 / anti-4-1BB antibodies in a reporter gene assay. [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0062] [Figure 20-1] 20A-20C show epitope binning of anti-ROR1 antibodies. A. Epitope binning workflow; B-C. Epitope binning results for 3C5 and 8F5, respectively. [Figure 20-2] Same as above.

[0063] [Figure 21-1] 21A-21C show ROR1-dependent 4-1BB activation by biparatopic anti-ROR1 / anti-4-1BB antibodies in a reporter gene assay. [Figure 21-2] Same as above.

[0064] [Figure 22-1]Figures 22A-22D show (A-B) the cell binding activity of anti-ROR1 mAb to ROR1 as measured by FACS before and after humanization, and (C-D) the ROR1-dependent 4-1BB activation of bispecific anti-ROR1 / anti-4-1BB antibody in a reporter gene assay before and after humanization. [Figure 22-2] Same as above. [Figure 22-3] Same as above.

[0065] [Figure 23-1] Figures 23A-23F show (A-C) cell binding activity of bispecific and biparatopic anti-ROR1 / anti-4-1BB antibodies as measured by FACS, and (D-F) ROR1-dependent 4-1BB activation of bispecific and biparatopic anti-ROR1 / anti-4-1BB antibodies in reporter gene assays using ROR1-positive and -negative tumor cell lines. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above.

[0066] [Figure 24-1] 24A-24C show SPR results for humanized bispecific and biparatopic anti-ROR1 / anti-4-1BB antibodies. [Figure 24-2] Same as above.

[0067] [Figure 25-1] 25A-25B show in vivo efficacy studies of humanized bispecific and biparatopic anti-ROR1 / anti-4-1BB antibodies. (A) Study design; (B) In vivo anti-tumor efficacy in 4-1BB knock-in mice. [Figure 25-2] Same as above.

[0068] [Figure 26-1] 26A-26E show ROR1-dependent 4-1BB activation of affinity-matured bispecific anti-ROR1 / anti-4-1BB antibodies in a reporter gene assay. [Figure 26-2] Same as above. [Figure 26-3] Same as above.

[0069] [Figure 27-1] 27A-27C show ROR1-dependent 4-1BB activation of affinity-matured biparatopic anti-ROR1 / anti-4-1BB antibodies in a reporter gene assay. [Figure 27-2] Same as above.

[0070] [Figure 28-1] 28A-28B show ROR1-dependent 4-1BB-induced cytokine release of affinity-matured bispecific and biparatopic anti-ROR1 / anti-4-1BB antibodies in reporter gene assays. [Figure 28-2] Same as above.

[0071] [Figure 29-1] 29A-29C show in vivo efficacy studies of affinity-matured biparatopic anti-ROR1 / anti-4-1BB antibodies. (A) Study design; (B) In vivo anti-tumor efficacy in 4-1BB knock-in C57 mice inoculated with hROR1-expressing MC38 cells; (C) Summary tumor growth inhibition (TGI) for each treatment group. [Figure 29-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0072] Detailed Description of the Application definition The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0073] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains can be referred to as "VH" and "VL," respectively. The variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs, including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein can be defined or identified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant regions of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0074] The term "half antibody" as used herein refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain. Those skilled in the art will readily understand that half antibodies can encompass fragments thereof and can also have an antigen-binding domain consisting of a single variable domain, for example, of camelid origin. In certain embodiments, half antibodies comprise Fab' and Fc domains. Fab' is fused to the N-terminus of the Fc domain.

[0075] The term "single-chain half antibody," as used herein, refers to a single-chain polypeptide comprising a VL domain, optionally a CL domain, a tether, a VH domain, optionally a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, which domains are arranged relative to one another in the N-terminal to C-terminal direction as follows: VL-tether-VH-hinge-CH2-CH3, VL-tether-VH-partial hinge-CH2-CH3, VL-tether-VH-hinge variant-CH2-CH3, or VL-CL-tether-VH-CH1-hinge-CH2-CH3.

[0076] The expression "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) is capable of conferring antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived recombinantly from human and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trends Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO2005 / 035572; WO03 / 035694; Febs Lett (1994) 339:285-290; WO00 / 29004; WO02 / 051870). If the sdAb contains only one heavy chain, it may be used interchangeably as "VHH" or "single heavy chain variable domain antibody" or "nanobody".

[0077] The term "antigen-binding fragment" as used herein refers to antibody fragments, including, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv), bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain Fvs (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of antibodies comprising one or more CDRs, single-domain antibodies (e.g., camelized single-domain antibodies), nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen as the parent antibody or parent antibody fragment (e.g., the parent scFv). In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies.

[0078] "Fab," with respect to an antibody, refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain. Fab can be obtained by papain digestion of an antibody at residues proximal to the N-terminus of the inter-heavy chain disulfide bond in the hinge region.

[0079] "Fab'" refers to a Fab fragment containing a portion of the hinge region that can be obtained by pepsin digestion of an antibody at residues proximal to the C-terminus of the inter-heavy chain disulfide bond in the hinge region and therefore differs from Fab by a small number of residues in the hinge region (including one or more cysteines).

[0080] "F(ab)2" refers to a dimer of Fab' containing two light chains and part of two heavy chains.

[0081] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plueckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0082] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are Kabat et al., J. Biol. Chem. 252:6609-6616 (1977);Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991);Chothia et al., J. Mol. Biol. 196:901-917 (1987);Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997);MacCallum et al., J. Mol. Biol. 262:732-745 (1996);Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008);Lefranc MP et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and these definitions include overlapping amino acid residues or subsets of amino acid residues when compared to each other. Nevertheless, application of either definition to refer to the CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined by each of the above-cited references are shown in Table 1 below for comparison.CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are hereby incorporated by reference in their entirety for use in this application and for possible inclusion in one or more claims herein. [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al., supra. 2 Residue numbering follows the nomenclature of Chothia et al., supra 3 Residue numbering follows the nomenclature of MacCallum et al., supra 4 Residue numbering follows the nomenclature of Lefranc et al., supra 5 Residue numbering follows the nomenclature of Honegger and Plückthun, supra

[0083] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of an antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or hypervariable region (HVR) of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a "standard" Kabat-numbered sequence in the region of homology.

[0084] Unless otherwise indicated herein, the numbering of residues in immunoglobulin heavy chains is the EU index numbering as in Kabat et al., supra, with minor modifications. Briefly, we added five additional residues in the hypervariable loop before the heavy chain CDR1. "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody.

[0085] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as herein defined.

[0086] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a recipient hypervariable region (HVR) are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs correspond to those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0087] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human and / or is produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). For the preparation of human monoclonal antibodies, the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouses, whose endogenous loci have been disabled but which have been engineered to produce such antibodies in response to antigen challenge (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). For example, regarding human antibodies generated via human B cell hybridoma technology, see also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0088] "Percent (%) amino acid sequence identity" or "homology" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, with respect to the polypeptide and antibody sequences identified herein, after aligning the sequences and considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0089] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparison is performed when the two sequences are aligned to maximize homology.

[0090] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2 and C H 3 domains (collectively, C H ) and light chain CHL (or C L ) domain.

[0091] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0092] The "CH1 domain" (also called "C1" for "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0093] The "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S—S bonds in the same positions.

[0094] The "CH2 domain" (also called the "C2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it does not closely pair with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in an intact native IgG molecule. It has been speculated that the carbohydrates may provide a surrogate for domain-domain pairing and may help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0095] The "CH3 domain" (also referred to as the "C3" domain) comprises the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residue 446 or 447 of IgG).

[0096] The terms "Fc region," "Fc domain," or "fragment crystallizable region" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, intact antibody compositions can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0097] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Additionally, a preferred FcR is an FcR (gamma receptor) that binds to IgG antibodies, including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. Daeoron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein.

[0098] The term "epitope," as used herein, refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding for that antigen.

[0099] As used herein, a first antibody or fragment thereof "competes" with a second antibody or fragment thereof for binding to a target antigen if the first antibody or fragment thereof inhibits target antigen binding of the second antibody or fragment thereof by at least about 50% (e.g., at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the first antibody or fragment. A high-throughput process for "binning" antibodies based on their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0100] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody or antibody portion, that determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than its binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤ 10 -12 Dissociation constant of M (K D) In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE™-test, and peptide scan.

[0101] An "isolated" antibody (or construct) is an antibody (or construct) that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from association with all other components from its production environment.

[0102] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free from all components associated with the production environment. An isolated nucleic acid molecule encoding a polypeptide or antibody described herein is in a form other than the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from the nucleic acid encoding the polypeptide or antibody described herein that naturally exists in a cell. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0103] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0104] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that have integrated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0105] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0106] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its progeny, regardless of the number of transfers. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened and selected for in the originally transformed cell are included herein.

[0107] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. Reduction of pathological consequences of cancer (e.g., tumor volume, etc.) is also encompassed by "treatment." The methods of the present application contemplate any one or more of these aspects of treatment.

[0108] With respect to cancer, the term "treating" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0109] The term "inhibition" or "inhibiting" refers to a decrease or cessation of any phenotypic characteristic, or a decrease or cessation in the incidence, degree, or likelihood of that characteristic. "Reducing" or "inhibiting" refers to a decrease, reduction, or cessation of an activity, function, and / or amount compared to that of a reference. In certain embodiments, "reducing" or "inhibiting" refers to the ability to cause a 20% or greater overall reduction. In another embodiment, "reducing" or "inhibiting" refers to the ability to cause a 50% or greater overall reduction. In yet other embodiments, "reducing" or "inhibiting" refers to the ability to cause a 75%, 85%, 90%, 95% or greater overall reduction.

[0110] The term "agonizing" or "agonize" refers to an increase or enhancement of any phenotypic characteristic, or the incidence, degree, or likelihood of that characteristic. "Increasing" or "enhancing" refers to a decrease, reduction, or cessation of an activity, function, and / or amount compared to that of a reference. In certain embodiments, "increasing" or "enhancing" refers to the ability to cause an overall increase in activity, function, and / or amount of at least about one-fold or greater, e.g., an overall increase in activity, function, and / or amount. In another embodiment, "increasing" or "enhancing" refers to the ability to cause an overall increase in activity, function, and / or amount of at least about five-fold or greater, e.g., an overall increase in activity, function, and / or amount, compared to a reference. In yet another embodiment, "increase" or "enhance" refers to the ability to cause an overall increase in activity, function and / or amount, e.g., of at least about any one of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold or 1000-fold (including any ranges therebetween), or greater than about 100-fold, as compared to a reference.

[0111] " Reference " as used herein refers to any sample, standard or level used for comparison purposes. Reference can be obtained from healthy and / or non-diseased sample. In some cases, reference can be obtained from untreated sample. In some cases, reference is obtained from non-diseased or non-treated sample of individual. In some cases, reference is obtained from one or more healthy individuals who are not individuals or patients.

[0112] As used herein, "delaying the onset of disease" means to postpone, prevent, slow down, delay, stabilize, inhibit, and / or postpone the onset of a disease (e.g., cancer). This delay can be of varying duration, depending on the history of the disease and / or individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention in individuals who do not develop the disease. For example, the onset of late-stage cancer, e.g., metastasis, can be delayed.

[0113] "Preventing," as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual who may be predisposed to the disease but has not yet been diagnosed with the disease.

[0114] As used herein, to "inhibit" a function or activity is to reduce the function or activity when compared to conditions that are otherwise the same except for the state or parameter of interest, or alternatively, when compared to another condition. For example, an antibody that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the antibody.

[0115] As used herein, "based on" includes assessing, determining, or measuring an individual's characteristics as described herein (and preferably selecting an individual suitable to receive treatment). When the status of claudin-18 abnormalities is "used as a basis" for selecting, evaluating, measuring, or determining a method of treatment as described herein, the CLDN6 abnormality determined before and / or during treatment, and the resulting status (including the presence, absence, expression level, activity level, and / or phosphorylation level of CLDN6) are used by the clinician in assessing any of the following: (a) probable or likely suitability of the individual to initially receive the treatment(s); (b) probable or likely incompetence of the individual to initially receive the treatment(s); (c) responsiveness to treatment; (d) probable or likely suitability of the individual to continue receiving the treatment(s); (e) probable or likely incompetence of the individual to continue receiving the treatment(s); (f) dosage adjustment; or (g) prediction of the likelihood of clinical benefit.

[0116] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0117] Embodiments of the present application described herein are understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0118] Reference herein to "about" a value or parameter includes (and describes) variations on that value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0119] As used herein, a reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0120] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0121] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. Antibody binding affinity

[0122] The binding specificity of the antibody moieties of the multispecific constructs described herein can be determined experimentally by methods known in the art, including, but not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™ test and peptide scan.

[0123] In some embodiments, the binding affinity is determined by the dissociation constant K D The dissociation constant can be determined via any analytical technique known in the art, including biochemical or biophysical techniques, such as fluorescence-activated cell sorting (FACS), flow cytometry, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), BioLayer interferometry (see, e.g., the Octet System by ForteBio), meso scale discover assays (see, e.g., MSD-SET), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analysis; or cell binding assays.

[0124] In some embodiments, the K of binding between the antibody moiety and 4-1BB Dis about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10 -8 M~about 10 -10 M, or about 10 -7 M~about 10 -9 In some embodiments, the K of binding between the antibody moiety and 4-1BB is D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 or 10 -13 M. In some embodiments, 4-1BB is a human antigen.

[0125] In some embodiments, the K of binding between the antibody moiety and 4-1BB on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 3 M -1 s-1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and 4-1BB is on is about 10 3 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 or about 10 5 M -1 s -1 ~about 10 8 M -1 s-1 In some embodiments, the K of binding between the antibody moiety and 4-1BB is on is about 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 In some embodiments, 4-1BB is a human antigen.

[0126] In some embodiments, the K of binding between the antibody moiety and 4-1BB off is about 1 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -2 s -1 , about 10 -2 s -1 ~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10-6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 or about 10 -3 s -1 ~about 10 -5 s -1 In some embodiments, the K of binding between the antibody moiety and 4-1BB is off is at least about 1 s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 In some embodiments, 4-1BB is a human antigen.

[0127] In some embodiments, the K of binding between the antibody moiety and the tumor antigen D is about 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -7 M~about 10 -10 M, about 10-8 M~about 10 -10 M, or about 10 -7 M~about 10 -9 In some embodiments, the K of binding between the antibody moiety and the tumor antigen is D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M. In some embodiments, the tumor antigen is a human antigen.

[0128] In some embodiments, the K of binding between the antibody moiety and the tumor antigen on is about 10 3 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 3 M -1 s -1 ~about 10 4 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and the tumor antigen is on is about 10 3 M -1 s -1 ~about 10 5 M -1s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 or about 10 5 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and the tumor antigen is on is about 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 In some embodiments, the tumor antigen is a human antigen.

[0129] In some embodiments, the K of binding between the antibody moiety and the tumor antigen off is about 1 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -2 s -1 , about 10-2 s -1 ~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 , about 1 s -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 or about 10 -3 s -1 ~about 10 -5 s -1 In some embodiments, the K of binding between the antibody moiety and the tumor antigen is off is at least about 1 s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 In some embodiments, the tumor antigen is a human antigen. Chimeric or humanized antibodies

[0130] In some embodiments, one or more of the antibody moieties of the multispecific construct of the present application are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0131] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0132] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0133] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996). Human antibodies

[0134] In some embodiments, one or more of the antibody moieties of the multispecific constructs of the present application are human antibodies (known as human domain antibodies or human DAbs). Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single-domain antibodies (or DAbs) are known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0135] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0136] Human antibodies (e.g., human DAbs) can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0137] Human antibodies (e.g., human DAbs) can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below. Anti-MUC16 antibody

[0138] MUC16 is a tumor-associated antigen polypeptide expressed by human ocular surface epithelium, in the mucosa of the bronchi, fallopian tubes, and uterus. One proposed function of MUC16 is to provide a protective lubricating barrier against particles and infectious agents at mucosal surfaces. Highly polymorphic MUC16 consists of three domains: a Ser- / Thr-rich N-terminal domain, a partially conserved tandem repeat domain of between 11 and more than 60 amino acids, each averaging 156 amino acids, and a C-terminal non-repetitive domain containing a transmembrane sequence and a short cytoplasmic tail. MUC16 is heavily O- and N-glycosylated. It has been reported that MUC16 is strongly overexpressed in certain types of human cancerous ovarian, breast, and pancreatic tumors compared with corresponding normal human ovarian, breast, and pancreatic tissues, respectively. Due to its overexpression in certain human tumors, the MUC16 polypeptide and the nucleic acid encoding it are targets for quantitative and qualitative comparison between various mammalian tissue samples. The unique expression profile of the MUC16 polypeptide and the nucleic acid encoding that polypeptide can be exploited for the diagnosis and therapeutic treatment of certain types of cancerous tumors in mammals.

[0139] The present disclosure provides antibodies, including antibodies or antigen-binding fragments thereof, that have binding specificity for human MUC16 protein. As demonstrated in experimental examples, 13 anti-human MUC16 antibodies with high binding affinity to human MUC16 protein were obtained. Antibody clones D57, B218, C25, and D100 were selected for further multispecific antibody construction. The human antibodies bound to human MUC16 with high affinity, and the bispecific antibodies efficiently induced MUC16-dependent 4-1BB activation in T cells.

[0140] According to one embodiment of the present disclosure, there is provided an antibody or antigen-binding fragment thereof comprising heavy and light chain variable domains having the CDR regions of the antibody prepared in the Examples. The CDRs and variable regions are summarized in Table 4 of the Examples (Kabat numbering).

[0141] In some embodiments, the VH CDR1, CDR2 and CDR3 are selected from any set of VH CDR1, CDR2 and CDR3 shown in Table 1, and the VL CDR1, CDR2 and CDR3 are selected from any set of VL CDR1, CDR2 and CDR3 shown in Table 1. In some embodiments, the VH CDR1, CDR2 and CDR3 and the VL CDR1, CDR2 and CDR3 are selected from those derived from the same antibodies as in the Examples.

[0142] In some embodiments, at least one or two or three or four or five or six of the VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 are modified by one, two or three amino acid additions, deletions, substitutions, or a combination thereof.

[0143] The CDRs, heavy and light chain variable regions of the present disclosure may be further modified, in some embodiments, the modified heavy or light chain variable region retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity and is still capable of binding to MUC16.

[0144] In some embodiments, the modification is a substitution at one or less hotspot positions from each CDR. In some embodiments, the modification is a substitution at one, two or three of these hotspot positions. In one embodiment, the modification is a substitution at one of the hotspot positions. In some embodiments, such substitution is a conservative substitution.

[0145] It will also be understood by those skilled in the art that the antibodies disclosed herein may be modified such that their amino acid sequences vary from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0146] In certain embodiments, the antibodies provided herein further comprise a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0147] In certain embodiments, the present disclosure provides bifunctional molecules comprising a first antigen-binding portion that has specificity for human MUC16 protein and a second portion that has specificity for a second protein, wherein the first antigen-binding portion comprises an anti-MUC16 antibody or fragment thereof provided herein.

[0148] In certain embodiments, the second part is an antibody or an antigen-binding fragment thereof. In certain embodiments, the second part has specificity for an immune checkpoint. In certain embodiments, the second part has specificity for another tumor antigen.

[0149] In certain embodiments, the second moiety is a 4-1BB antigen-binding moiety.

[0150] In certain embodiments, a first antigen-binding moiety provided herein has a full-length antibody / IgG format, and a second antigen-binding moiety provided herein has a single-domain antibody (sdAb) format.

[0151] In certain embodiments, the sdAb is fused to the N-terminus or C-terminus of an IgG. In certain embodiments, the sdAb is fused to the N-terminus of an IgG heavy chain variable region. In certain embodiments, the sdAb is fused to the C-terminus of an IgG heavy chain constant region (IgG(CH)) comprising an Fc domain.

[0152] In certain embodiments, the multispecific constructs provided herein comprise a first polypeptide comprising, from N- to C-terminus: VH(MUC16)-IgG(CH)-VHH(4-1BB), and a second polypeptide comprising, from N- to C-terminus: VL(MUC16)-IgG light chain constant region (IgG(CL)). The first and second polypeptides are paired via VH(MUC16)-VL(MUC16) pairing.

[0153] In certain embodiments, the multispecific constructs provided herein comprise two first polypeptides comprising, from N- to C-terminus: VH(MUC16)-IgG(CH)-VHH(4-1BB), and two second polypeptides comprising, from N- to C-terminus: VL(MUC16)-IgG light chain constant region (IgG(CL)). The first and second polypeptides are paired via VH(MUC16)-VL(MUC16) pairing. The two first polypeptides may be paired via IgG(CH) pairing.

[0154] The first antigen-binding moiety and the second antigen-binding moiety are fused via a linker. Substitution, insertion, and deletion variants

[0155] In some embodiments, antibody variants containing one or more amino acid substitutions are included in the multispecific constructs or antibodies described herein. Sites of interest for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading of "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 2]

[0156] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0157] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0158] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study have an alteration (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0159] Alterations (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such alterations can be made in HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction or reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0160] In some embodiments, substitutions, insertions, or deletions may be present within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside of HVR "hot spots" or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0161] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0162] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Glycosylation variants

[0163] In some embodiments, one or more antibody portions of the multispecific constructs or antibodies of the present application are altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0164] If the antibody portion comprises an Fc region, the carbohydrate attached thereto can be varied. Native antibodies produced by mammalian cells typically contain the C of the Fc region. H The biantennary oligosaccharides comprise branched, biantennary oligosaccharides, typically linked by an N-linkage to Asn297 in the 2 domain. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody moiety may be performed to create antibody variants with certain improved properties.

[0165] In some embodiments, an antibody portion has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 compared to the sum of all glycostructures (e.g., complex hybrid high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. US2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0 132140;US2004 / 0110704;US2004 / 0110282;US2004 / 0109865;WO2003 / 085119;WO200 3 / 084570;WO2005 / 035586;WO2005 / 035778;WO2005 / 053742;WO2002 / 031140;Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0166] In some embodiments, the antibody portion has bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). Fc domain

[0167] In some embodiments, the first antibody portion or the second antibody portion comprises an Fc region (also referred to herein as an "Fc fragment"). In some embodiments, the Fc region is an Fc domain, i.e., an Fc region that retains some or all effector functions, including, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In some embodiments, the Fc domain is derived from IgG1 or IgG3.

[0168] In some embodiments, the Fc region has maintained or improved effector function, eg, ADCC and / or CDC.

[0169] In some embodiments, one or more amino acid modifications may be introduced into the Fc domain, thereby generating an Fc domain variant. The Fc domain variant may comprise a human Fc domain sequence (e.g., derived from a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc domain variant alters one or more functional and / or pharmacokinetic properties of the antibody.

[0170] In some embodiments, the Fc domain possesses some, but not all, effector functions, making it a desirable candidate for applications in which the in vivo half-life of the antibody moiety is important, but certain effector functions (e.g., CDC and ADCC) are unnecessary or deleterious.

[0171] In vitro and / or in vivo cytotoxicity assays can be performed to analyze the CDC and / or ADCC activity of the Fc region. For example, Fc receptor (FcR) binding assays can be performed to determine whether an antibody possesses FcγR binding (and thus potentially ADCC activity) and / or FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assay can also be carried out to confirm that antibody cannot bind to C1q and therefore lacks CDC activity.For example, see the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402.To evaluate complement activation, CDC assay can be carried out (for example, see Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0172] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581), which has substitutions of residues 265 and 297 to alanine. In some embodiments, the Fc region of a multispecific construct does not comprise a mutation that reduces its effector function, e.g., one or more of the mutations described herein. In some embodiments, the Fc region of a multispecific construct comprises one or more of these mutations.

[0173] Certain antibody variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). In some embodiments, the Fc region of a multispecific construct does not comprise a variant with improved or reduced binding to FcR. In some embodiments, the Fc region of a multispecific construct comprises a variant with improved binding to FcγRI. In some embodiments, the Fc region of a multispecific construct comprises a variant with improved binding to FcγRII. In some embodiments, the Fc region of a multispecific construct comprises a variant with improved binding to FcγRIII. In some embodiments, the Fc region of a multispecific construct comprises a variant with reduced binding to FcγRI. In some embodiments, the Fc region of a multispecific construct comprises a variant with reduced binding to FcγRII. In some embodiments, the Fc region of the multispecific construct comprises a variant with reduced binding to FcγRIII.

[0174] In some embodiments, the Fc domain is derived from human IgG1. In some embodiments, the Fc domain derived from human IgG1 does not comprise a L234A mutation and / or a L235A mutation. In some embodiments, the Fc domain derived from human IgG1 comprises a L234A mutation and / or a L235A mutation. In some embodiments, the Fc domain is derived from human IgG3. In some embodiments, the Fc domain is derived from human IgG2 or IgG4. In some embodiments, the Fc domain is derived from human IgG4. In some embodiments, the human IgG4-derived Fc domain comprises a S228P, F234A, and / or L235A mutation. In some embodiments, the human IgG4-derived Fc domain does not comprise a S228P, F234A, and / or L235A mutation.

[0175] In some embodiments, the multispecific construct comprises an Fc domain with one or more amino acid substitutions that improve ADCC, hi some embodiments, the one or more substitutions are at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

[0176] In some embodiments, alterations are made in the Fc domain that result in altered (i.e., either improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0177] In some embodiments, the multispecific construct comprises a variant Fc domain containing one or more amino acid substitutions that alter half-life and / or binding to the neonatal Fc receptor (FcRn). Antibodies with improved binding to the neonatal Fc receptor (FcRn), responsible for increased half-life and transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that alter binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the Fc region residues, for example, a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0178] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0179] In some embodiments, the multispecific construct comprises an Fc domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one selected from the group consisting of SEQ ID NOs: 46 to 56. In some embodiments, the multispecific construct comprises an Fc domain comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 46 to 56. Cysteine ​​Engineered Antibody Variants and Heterodimers

[0180] In some embodiments, it may be desirable to create cysteine-engineered antibody moieties, e.g., "thioMAbs," in which one or more residues in one or more antibody moieties in the multispecific constructs herein are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues can be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibody moieties can be generated, for example, as described in U.S. Pat. No. 7,521,541. heterodimer

[0181] Typically, two identical Fc regions form homodimers. However, two different Fc regions can form heterodimers, for example, through knobs-into-holes (KIH), disulfide bonds (-SS-), or with mutations to one or two individual chains, such as hydrophobic, electrostatic, or hydrophilic interactions, or increased flexibility.

[0182] The term "knobs-into-holes" or "KIH" technology, as used herein, refers to a technique that directs pairing of two polypeptides in vitro or in vivo by introducing a convex portion (knob) into one polypeptide and a concave portion (hole) into the other polypeptide at their interaction interface. For example, KIH has been introduced into the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of an antibody (see, for example, US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, Zhu et al., 1997, Protein Science 6:781-788, and WO2012 / 106587). In some embodiments, KIH facilitates pairing of two different heavy chains during the production of multispecific antibodies. For example, multispecific antibodies with KIH in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise different heavy chain variable domains paired with similar or different light chain variable domains. KIH technology can also be used to pair two different receptor extracellular domains together, or any other polypeptide sequence containing different target recognition sequences (including, for example, affibodies, peptibodies, and other Fc fusions).

[0183] The term "knob mutation," as used herein, refers to a mutation that introduces a protrusion (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0184] The term "hole mutation," as used herein, refers to a mutation that introduces a recess (hole) in a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0185] In some embodiments, the knob mutation in the IgG1 constant region is T366W (EU numbering). In some embodiments, the hole mutation in the IgG1 constant region comprises one or more mutations selected from T366S, L368A, and Y407V (EU numbering).

[0186] In some embodiments, the knob mutations in the IgG1 constant region are S354C and T366W (EU numbering). In some embodiments, the hole mutations in the IgG1 constant region comprise one or more mutations selected from Y349C, T366S, L368A and Y407V (EU numbering).

[0187] Multispecific antibodies can be made by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al, J. Immunol, 148(5): 1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al, J. Immunol, 152:5368 (1994)); as well as by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0188] In some embodiments, an Fc domain provided herein comprises a knob mutation and the paired Fc domain comprises hole mutation(s), or vice versa.

[0189] In some embodiments, the multispecific construct comprises an Fc domain comprising the amino acid sequence of any one of SEQ ID NOs: 285-286 and 288-289.

[0190] In some embodiments, the multispecific construct comprises an Fc domain comprising the amino acid sequence of SEQ ID NO:285 or a variant thereof having at least about 80% (including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any higher than 99%) sequence identity to the sequence set forth in SEQ ID NO:285, and a paired Fc domain comprising the amino acid sequence of SEQ ID NO:288 or a variant thereof having at least about 80% (including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any higher than 99%) sequence identity to the sequence set forth in SEQ ID NO:288.

[0191] In some embodiments, the multispecific construct comprises an Fc domain comprising the amino acid sequence of SEQ ID NO: 286 or a variant thereof having at least about 80% (including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any higher than 99%) sequence identity to the sequence set forth in SEQ ID NO: 286, and a paired Fc domain comprising the amino acid sequence of SEQ ID NO: 289 or a variant thereof having at least about 80% (including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any higher than 99%) sequence identity to the sequence set forth in SEQ ID NO: 289.

[0192] In some embodiments, the Fc domain and / or paired Fc domain provided herein is an IgG Fc domain. In some embodiments, the IgG is an IgG1, IgG2, IgG3, or IgG4. Anti-4-1BB antibody part

[0193] The anti-4-1BB antibody portion of the multispecific construct described in this application includes any antibody portion that specifically binds to 4-1BB. In some embodiments, 4-1BB is human 4-1BB ("h4-1BB"). h4-1BB is a type I transmembrane receptor with four extracellular cysteine-rich domains ("CRDs", i.e., CRD1, CDR2, CRD3, and CRD4), followed by a short transmembrane domain and a C-terminal cytoplasmic region. CRD2 and CRD3 of h4-1BB interact with the ligand 4-1BBL (Bitra et al. (2018) J Biol Chem. 293(26): 9958-9969). In contrast to other TNFRs, h4-1BB exists as a disulfide-linked dimer, and dimerization occurs via an unpaired cysteine ​​(Cys) found within CRD4 of h4-1BB. 121 ) In some embodiments, h4-1BB comprises the sequence set forth in SEQ ID NO: 41 or a variant thereof (e.g., a post-translationally modified variant and / or a conformational variant). In some embodiments, the anti-4-1BB antibody portion binds to the CRD3 / CRD4 region of 4-1BB.

[0194] The anti-4-1BB antibody moiety may be in any suitable format known in the art. In some embodiments, the anti-4-1BB antibody moiety is selected from the group consisting of a full-length antibody, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the anti-4-1BB antibody moiety comprises a single domain antibody that binds to 4-1BB. Exemplary Anti-4-1BB Antibody Moieties

[0195] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% (e.g., including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or any of greater than 99%) sequence identity to the sequence set forth in SEQ ID NO:27.

[0196] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence set forth in SEQ ID NO: 27, wherein CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme. In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb), which comprise sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, which comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence set forth in SEQ ID NO: 27, wherein CDR1, CDR2, and CDR3 are according to the IMGT numbering scheme. In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence set forth in SEQ ID NO: 27, wherein CDR1, CDR2 and CDR3 are according to the Kabat numbering scheme.

[0197] In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27.

[0198] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising: a) an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; b) an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and c) an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3.

[0199] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising: a) an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26.

[0200] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any of greater than 99%) sequence identity to the sequence set forth in SEQ ID NO: 27. In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. tumor antigens

[0201] The tumor antigen to which the first moiety (e.g., the first antibody moiety) specifically binds can be any suitable tumor antigen known in the art. In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6.

[0202] In some embodiments, the tumor antigen is HER2, Nectin-4, 5T4, GPC3, MSLN, FAP, CLDN18.2, PD-L1, PD-L2, ILT-4, B7-H3, CS1, CD19, CD2, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD81, CD123, CD133, CD151, CD171, CD276, CLL1, BCMA, VEGFR-2, GPC3, PMSA, CEACAM6, c-Met, ErbB3, HER3, ErbB4 / HER -4, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PSCA, HVEM, PSMA, RANK, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, PTCH1, WT-1, Robo1, Frizzled, Notch-1 to 4, APRIL, MAGE3, folate receptor alpha, folate receptor beta, GPC2, CD70, BAFF-R, and TROP-2.

[0203] In some embodiments, the tumor antigen is mucin 16 (MUC16). MUC16 is a member of the mucin family of glycoproteins and is also known as mucin CA125. MUC16 has been shown to play a role in tumorigenesis and tumor growth. MUC16 is thought to be involved in cell-cell interactions that enable metastasis through the binding of mesothelin. MUC16 may also play a role in promoting cell motility and invasion through its C-terminal domain. An exemplary protein sequence of MUC16 can be found, for example, in UniProtKB Q8WX17.

[0204] In some embodiments, the first antibody portion binds to MUC16. Any anti-MUC16 antibody whose binding to MUC16 triggers the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-MUC16 antibodies include Bast et al. (J. Clin. Invest. 1981;68(5):1331-1337), Nustad et al. (Tumour Biol, 1996;17(4):196-219), Lloyd et al. (Lloyd et al., Int. J. Cancer, 1997;71(5):842-850), Marcos-Silava et al. (Glycobiology, 2015;25(11:1172-1182)), Chen et al. (Cancer Res., 2007;67(10):4924-4932), Aithal et al. (PLoS One, 2018;13(4):e01293907), Gipson et al. (Glycobiology, 2017;27(1):920-926), Davies et al. (nt. J. Biochem. Cell Biolo., 2007; 39(1):1943-1954), WO2002 / 092836, WO2020 / 102555, WO2020 / 227538, WO2016 / 149368, U.S. Patent Application Publication No. 2021 / 0309758, U.S. Patent Application Publication No. 2020 / 0317810, U.S. Patent No. 10,941,208, WO2007 / 001851, U.S. Patent No. 7,078,188, WO2007 / 001851, WO2019 / 213747, and WO2008 / 141044, which are hereby incorporated by reference in their entireties. In some embodiments, the anti-MUC16 antibody is Mab-AR-9.6 (Quest PharmaTech), Oregovamab (Quest PharmaTech), RG-7458 (Genentech), RG-7882 (Genentech), EDO-772P (Mundipharma EDO GmbH), Abagovomab, NAV-005 (Navrogen).In some embodiments, the anti-MUC16 antibody binds to the N-terminal tandem repeat region of MUC16. In some embodiments, the anti-MUC16 antibody binds to the carboxy-terminal region of MUC16. In some embodiments, the anti-MUC16 antibody binds to the juxtamembrane domain, the cytoplasmic tail, or the region C-terminal to the mucin repeat domain.

[0205] In some embodiments, the tumor antigen is ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3). ENPP3 is also known as NPP3, PDNP3, CD203c, and PD-IBETA. ENPP3 is an ectoenzyme, a class of transmembrane proteins involved in the hydrolysis of extracellular nucleotides, and has been found to be expressed in several cancers and cancerous cells, including neoplastic mast cells, acute basophilic leukemia, colon cancer, renal cell carcinoma, hepatocellular carcinoma, and neoplastic cholangiocytes. An exemplary protein sequence of ENPP3 can be found, for example, in UniProtKB O14638.

[0206] In some embodiments, the first antibody portion binds to ENPP3. Any anti-ENPP3 antibody whose binding to ENPP3 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-ENPP3 antibodies include, but are not limited to, those described in Donate et al. (Clin Cancer Res (2016) 22 (8): 1989-1999), U.S. Patent No. 7,427,399, U.S. Patent Application Publication No. 2010 / 0099111, U.S. Patent No. 8,562,989, U.S. Patent Application Publication No. 2016 / 0176977, and U.S. Patent Application Publication No. 2019 / 0092874, the entire contents of which are hereby incorporated by reference herein.

[0207] In some embodiments, the tumor antigen is tyrosine-protein kinase transmembrane receptor 1 (ROR1). ROR1 is also known as NTRKR1. ROR1 is believed to play a role in tumor cell survival, proliferation, migration and chemotaxis, and is highly expressed in several cancer types, including chronic lymphocytic leukemia, mantle cell lymphoma, ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma and colorectal cancer. The exemplary protein sequence of ROR2 can be found, for example, in UniProtKB Q01973.

[0208] In some embodiments, the first antibody portion binds to ROR1. Any anti-ROR1 antibody whose binding to ROR1 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-ROR1 antibodies include, but are not limited to, those described in U.S. Patent No. 9,758,591, U.S. Patent No. 10,618,959, WO2010 / 124188, WO2016 / 187220, WO2012 / 045085, WO2017 / 072361, WO2019 / 008377, WO2019 / 005636, WO2014 / 031174, and WO2017 / 127664, which are hereby incorporated by reference in their entireties. In some embodiments, the first antibody portion is cirmtuzumab.

[0209] In some embodiments, the tumor antigen is SLC7A11. SLC7A11 is also known as CCBR1, xCT and solute carrier family 7 member 11, and is a cysteine / glutamate transporter. Overexpression of SLC7A11 is thought to promote tumor growth, in part through the suppression of ferroptosis. The exemplary protein sequence of SLC7A11 can be found, for example, in UniProtKB Q9UPY5.

[0210] In some embodiments, the first antibody portion binds to SLC7A11. Any anti-SLC7A11 antibody whose binding to SLC7A11 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-SLC7A11 antibodies include but are not limited to those described in WO2018 / 204278 and WO2020 / 227640, which are hereby incorporated by reference in their entirety.

[0211] In some embodiments, the tumor antigen is Delta-like 3 (DLL3). DLL3 is a member of the Delta protein ligand family that functions as a Notch ligand. High DLL3 expression has been observed in some cancer types, particularly neuroendocrine-related tumors, and has been investigated as a potential target for some cancer types, including small cell lung cancer, non-small cell lung cancer, and large cell neuroendocrine carcinoma. High DLL3 expression is part of the DLL3 protein sequence. An exemplary DLL3 protein sequence can be found, for example, in UniProtKB Q9NYJ7.

[0212] In some embodiments, the first antibody portion binds to DLL3. Any anti-DLL3 antibody whose binding to DLL3 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-DLL3 antibodies include, but are not limited to, those described in Sauders et al. (Sci Transl Med., 2015; 7(302): 302ra136), WO2017 / 031458, WO2019 / 217145, WO2013 / 126746, WO2015 / 127407, WO2011 / 093097, and WO2021 / 007371, which are hereby incorporated by reference in their entirety. In some embodiments, the anti-DLL3 antibody is rovalpituzumab.

[0213] In some embodiments, the tumor antigen is B7H4, also known as V-set domain-containing T-cell activation inhibitor 1 (VTCN1) and B7x. B7H4 belongs to the immunoglobulin superfamily and is involved in regulating T cell proliferation and expansion. B7H4 is highly expressed in several cancers, including ovarian cancer, renal cell carcinoma, pancreatic cancer, hepatocellular carcinoma, gastric cancer, lung cancer, glioma, breast cancer, prostate cancer, urothelial cancer, cervical cancer, and melanoma, and this increased expression is thought to help tumors evade the immune system. An exemplary protein sequence of B7H4 can be found, for example, in UniProtKB Q7Z7D3.

[0214] In some embodiments, the first antibody portion binds to B7H4. Any anti-B7H4 antibody whose binding to B7H4 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-B7H4 antibodies include, but are not limited to, those described in WO2019 / 040780, U.S. Patent No. 9,562,099, WO2012 / 145568, WO2013 / 067492 and WO2014 / 100483, which are hereby incorporated by reference in their entirety. In some embodiments, the antibody is Alsevalimab (FP150).

[0215] In some embodiments, the tumor antigen is EPH receptor A2 (EPHA2). EPHA2 is also known as ECK, CPTA, ARCC2, CTPP1, and CTRCT6. EPHA2 is a member of the ephrin receptor subfamily of the protein tyrosine kinase family. EPHA2 is thought to play a role in tumor growth, invasion, metastatic progression, and drug resistance, and has been reported to be overexpressed in several cancer types, including prostate cancer, lung cancer, esophageal cancer, colorectal cancer, cervical cancer, ovarian cancer, breast cancer, and skin cancer. An exemplary protein sequence of EPHA2 can be found, for example, in UniProtKB P29317.

[0216] In some embodiments, the first antibody portion binds to EPHA2. Any anti-EPHA2 antibody whose binding to EPHA2 triggers the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-EPHA2 antibodies include Kinch et al. (Cancer Res. 2002;62(10):2840-2847), Coffman et al. (Cancer Res. 2003;63(22):7907-7912), Goldgur et al. (Growth Factors. 2014;32(6):214-222), Sakamoto et al. (AntiCancer Res. 2018;38(6):3273-3282), Bruckheimer et al. (Neoplasia. 2009, 11(6):509-517), Hasegawa et al. (Cancer Biol. Ther., 2016;17(11): 1158-1167), U.S. Patent No. 7,101,976, U.S. Patent No. 7,776,327, WO2004 / 014292, U.S. Patent Application Publication No. 2007 / 0086943, U.S. Patent Application Publication No. 2010 / 0298545, WO2016 / 081601, U.S. Patent No. 7,659,374, WO2006 / 023403, U.S. Patent No. 10,406,225, and U.S. Patent Application Publication No. 2016 / 0031987, which are hereby incorporated by reference in their entirety.

[0217] In some embodiments, the tumor antigen is CD318. CD318 is also known as CUB domain-containing protein 1 (CDCP1), SIMA135, and TRASK. CD318 is a transmembrane glycoprotein with an extracellular domain containing two CUB domains. Phosphorylation of CD318 has been observed in many cancer types, including pre-invasive cancer, invasive cancer, and tumor metastasis. An exemplary protein sequence of CD318 can be found, for example, in UniProtKB Q9H5V8.

[0218] In some embodiments, the first antibody portion binds to CD318. Any anti-CD318 antibody whose binding to CD318 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-CD318 antibodies include, but are not limited to, those described in WO2011 / 023389, WO2018 / 112334, WO2019 / 084319, WO2021 / 132427, and WO2011 / 023390, which are hereby incorporated by reference in their entirety.

[0219] In some embodiments, the tumor antigen is claudin 6 (CLDN6). Claudin-6 (CLDN6) is a member of the claudin family and functions as a tight junction molecule that plays an important role in cell-to-cell adhesion in epithelial or endothelial cell sheets. It encodes a tetraspan membrane protein with a size of 220 amino acids and a molecular weight of 23,292 Da. CLDN6 has been identified as the origin of cell adhesion signaling, involved in regulating nuclear receptor activity via targeting molecules of the nuclear receptor superfamily and controlling their gene expression (Sugimoto et al. (2019). "Cell adhesion signals regulate the nuclear receptor activity." Proc. Natl. Acad. Sci. USA 116, 24600-24609). CLDN6 appears to be significantly upregulated in 20 types of human cancer (Zhang et al. (2021) Front. Cell. Dev. Biol. 9: 726656). In some embodiments, the CLDN6 is human CLDN6 ("hCLDN6"). In some embodiments, the hCLDN6 comprises the amino acid sequence set forth in SEQ ID NO: 40 or a variant thereof (e.g., a post-translationally modified variant and / or a conformational variant).

[0220] In some embodiments, the first antibody portion binds to CLDN6. Any anti-CLDN6 antibody whose binding to CLDN6 induces the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-CLDN6 antibodies include, but are not limited to, any one of the anti-CLDN6 antibodies listed herein, U.S. Patent No. 9,274,119, WO2012 / 156018, WO2019 / 056023, and U.S. Patent No. 10,053,511, which are hereby incorporated by reference in their entirety.

[0221] In some embodiments, the tumor antigen is programmed death-ligand 1 (PD-L1), also known as CD274 and B7-H1. PD-L1 is a transmembrane protein that plays a role in suppressing the adaptive immune system. PD-L1 binds to PD-1, which is expressed on T cells, B cells, and myeloid cells. Overexpression of PD-L1 on tumor cells is thought to help cancer evade the immune system. An exemplary protein sequence of PD-L1 can be found, for example, in UniProtKB Q9NZ17.

[0222] In some embodiments, the first antibody portion binds to PD-L1. Any anti-PD-L1 antibody whose binding to PD-L1 triggers the second antibody portion to activate 4-1BB can be used in the present invention. Suitable anti-PD-L1 antibodies include, but are not limited to, atezolizumab, avelumab, durvalumab, atezolizumab (e.g., Tecentriq®), avelumab (e.g., Bavencio®), and durvalumab (e.g., IMFINZI™). Multispecific construct

[0223] In one aspect, provided herein is a multispecific construct comprising a first portion (e.g., a first antibody portion) that specifically binds to a tumor antigen and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion to the tumor antigen triggers the second antibody portion to activate 4-1BB. In some embodiments, the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB.

[0224] In some embodiments, provided herein are multispecific constructs comprising a first antibody portion that specifically binds to a tumor antigen and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion to the tumor antigen induces the second antibody portion to activate 4-1BB. In some embodiments, activation of 4-1BB by the second antibody portion is enhanced by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold (including any range therebetween) after binding of the first antibody portion to the tumor antigen. In some embodiments, the multispecific construct activates 4-1BB signaling in the absence of binding to the tumor antigen. In some embodiments, the second portion does not activate 4-1BB signaling in the absence of binding to the tumor antigen.

[0225] In some embodiments of the multispecific construct of the present application, the second antibody moiety is an sdAb. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, each comprising the amino acid sequences of CDR1, CDR2, and CDR3, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein CDR1, CDR2, and CDR3 are numbered according to the Kabat numbering scheme.

[0226] In some embodiments, the sdAb comprises an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3.

[0227] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (e.g., including at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any of greater than 99%) sequence identity to the sequence set forth in SEQ ID NO: 27. In some embodiments, the affinity of such an sdAb for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an sdAb comprising SEQ ID NO: 27.

[0228] In some embodiments, the tumor antigen to which the first antibody moiety specifically binds can be any suitable tumor antigen known in the art, hi some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6.

[0229] In some embodiments, the present application provides multispecific constructs that bind to both MUC16 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies that include an anti-MUC16 antibody portion and an anti-4-1BB antibody portion. The anti-MUC16 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0230] In some embodiments, the present application provides a multispecific construct that binds to both ENPP3 and 4-1BB. In some embodiments, the multispecific construct described herein is a bispecific antibody comprising an anti-ENPP3 antibody portion and an anti-4-1BB antibody portion. The anti-ENPP3 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0231] In some embodiments, the present application provides multispecific constructs that bind to both ROR1 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies comprising an anti-ROR1 antibody portion and an anti-4-1BB antibody portion. The anti-ROR1 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0232] In some embodiments, the present application provides a multispecific construct that binds to both SLC7A11 and 4-1BB. In some embodiments, the multispecific construct described herein is a bispecific antibody comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion. The anti-SLC7A11 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0233] In some embodiments, the present application provides a multispecific construct that binds to both DLL3 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies that include an anti-DLL3 antibody portion and an anti-4-1BB antibody portion. The anti-DLL3 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0234] In some embodiments, the present application provides a multispecific construct that binds to both B7H4 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies that include an anti-B7H4 antibody portion and an anti-4-1BB antibody portion. The anti-B7H4 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0235] In some embodiments, the present application provides a multispecific construct that binds to both EPHA2 and 4-1BB. In some embodiments, the multispecific construct described herein is a bispecific antibody comprising an anti-EPHA2 antibody portion and an anti-4-1BB antibody portion. The anti-EPHA2 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0236] In some embodiments, the present application provides a multispecific construct that binds to both CD318 and 4-1BB. In some embodiments, the multispecific construct described herein is a bispecific antibody comprising an anti-CD318 antibody portion and an anti-4-1BB antibody portion. The anti-CD318 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0237] In some embodiments, the present application provides a multispecific construct that binds to both CLDN6 and 4-1BB. In some embodiments, the multispecific construct described herein is a bispecific antibody comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion. The anti-CLDN6 antibody portion and the anti-4-1BB antibody portion can be any of those described herein.

[0238] In some embodiments, the multispecific construct is biparatopic and comprises a third antibody portion that specifically binds to the same tumor antigen as the first antibody portion, but with a different epitope.

[0239] In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb comprising the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values).

[0240] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a third antibody portion that specifically binds to the same tumor antigen as the first antibody portion but with a different epitope. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. The format of the third antibody portion can be the same or different from that of the first antibody portion. For example, both the first antibody portion and the third antibody portion can be in the form of a full-length antibody. As another example, the first antibody portion is a full-length antibody and the third antibody portion is an scFv, or vice versa. As yet another example, the first antibody portion is a half antibody and the third antibody portion is a single-chain half antibody, or vice versa.

[0241] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The multispecific construct binds to a tumor antigen with an affinity of M. In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to the tumor antigen induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0242] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-MUC16 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-MUC16 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a second anti-MUC16 portion that specifically binds to the same tumor antigen as the first anti-MUC16 portion but with a different epitope. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. The format of the second anti-MUC16 moiety can be the same as or different from the first anti-MUC16 moiety. For example, both the first anti-MUC16 moiety and the third anti-MUC16 moiety can be full-length antibody formats. As another example, the first anti-MUC16 moiety can be a full-length antibody and the second anti-MUC16 moiety can be an scFv, or vice versa. As yet another example, the first anti-MUC16 moiety can be a half antibody and the second anti-MUC16 moiety can be a single-chain half antibody, or vice versa.

[0243] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to MUC16 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0244] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-ENPP3 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-ENPP3 antigen antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a second anti-ENPP3 portion that specifically binds to the same tumor antigen as the first anti-ENPP3 portion but with a different epitope. The format of the second anti-ENPP3 portion can be the same or different from the first anti-ENPP3 portion. For example, both the first anti-ENPP3 portion and the third anti-ENPP3 portion can be full-length antibody formats. As another example, the first anti-ENPP3 moiety is a full-length antibody and the second anti-ENPP3 moiety is an scFv, or vice versa. As yet another example, the first anti-ENPP3 moiety is a half antibody and the second anti-ENPP3 moiety is a single-chain half antibody, or vice versa.

[0245] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7M~about 10 -13 The first antibody moiety (and / or third antibody moiety) binds to ENPP3 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to ENPP3 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0246] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-ROR1 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-ROR1 antigen antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific constructs are biparatopic and thus comprise a second anti-ROR1 portion that specifically binds to the same tumor antigen as the first anti-ROR1 portion but with a different epitope. The format of the second anti-ROR1 portion can be the same or different from the first anti-ROR1 portion. For example, both the first anti-ROR1 portion and the third anti-ROR1 portion can be full-length antibody formats. As another example, the first anti-ROR1 moiety is a full-length antibody and the second anti-ROR1 moiety is an scFv, or vice versa. As yet another example, the first anti-ROR1 moiety is a half antibody and the second anti-ROR1 moiety is a single-chain half antibody, or vice versa.

[0247] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13 and binds to ROR1 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to ROR1 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0248] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-SLC7A11 antigen-antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the multispecific constructs are biparatopic and thus comprise a second anti-SLC7A11 portion that specifically binds to the same tumor antigen as the first anti-SLC7A11 portion but with a different epitope. The format of the second anti-SLC7A11 portion can be the same or different from that of the first anti-SLC7A11 portion. For example, both the first anti-SLC7A11 moiety and the third anti-SLC7A11 moiety can be in the form of a full-length antibody. As another example, the first anti-SLC7A11 moiety can be a full-length antibody and the second anti-SLC7A11 moiety can be an scFv, or vice versa. As yet another example, the first anti-SLC7A11 moiety can be a half antibody and the second anti-SLC7A11 moiety can be a single-chain half antibody, or vice versa.

[0249] In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or third antibody moiety) binds to SLC7A11 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to SLC7A11 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0250] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-DLL3 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-DLL3 antigen antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific constructs are biparatopic and thus comprise a second anti-DLL3 portion that specifically binds to the same tumor antigen as the first anti-DLL3 portion but with a different epitope. The format of the second anti-DLL3 portion can be the same or different from the first anti-DLL3 portion. For example, both the first and third anti-DLL3 portions can be full-length antibody formats. As another example, the first anti-DLL3 moiety is a full-length antibody and the second anti-DLL3 moiety is an scFv, or vice versa. As yet another example, the first anti-DLL3 moiety is a half antibody and the second anti-DLL3 moiety is a single-chain half antibody, or vice versa.

[0251] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13 The first antibody moiety (and / or third antibody moiety) binds to DLL3 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to DLL3 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0252] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-B7H4 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-B7H4 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a second anti-B7H4 portion that specifically binds to the same tumor antigen as the first anti-B7H4 portion but with a different epitope. The format of the second anti-B7H4 portion can be the same or different from the first anti-B7H4 portion. For example, both the first and third anti-B7H4 portions can be full-length antibody formats. As another example, the first anti-B7H4 moiety is a full-length antibody and the second anti-B7H4 moiety is an scFv, or vice versa. As yet another example, the first anti-B7H4 moiety is a half antibody and the second anti-B7H4 moiety is a single-chain half antibody, or vice versa.

[0253] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or third antibody moiety) binds to B7H4 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to B7H4 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0254] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-EPHA2 antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-EPHA2 antigen antibody portion and an anti-4-1BB antibody portion, where the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a second anti-EPHA2 portion that specifically binds to the same tumor antigen as the first anti-EPHA2 portion but with a different epitope. The format of the second anti-EPHA2 portion can be the same or different from the first anti-EPHA2 portion. For example, both the first anti-EPHA2 portion and the third anti-EPHA2 portion can be full-length antibody formats. As another example, the first anti-EPHA2 moiety is a full-length antibody and the second anti-EPHA2 moiety is an scFv, or vice versa. As yet another example, the first anti-EPHA2 moiety is a half antibody and the second anti-EPHA2 moiety is a single-chain half antibody, or vice versa.

[0255] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13 The first antibody moiety (and / or third antibody moiety) binds to EPHA2 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to EPHA2 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0256] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-CD318 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-CD318 antigen-antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific constructs are biparatopic and thus comprise a second anti-CD318 portion that specifically binds to a tumor antigen with the same epitope as the first anti-CD318 portion but a different epitope. The format of the second anti-CD318 portion can be the same or different from the first anti-CD318 portion. For example, both the first anti-CD318 portion and the third anti-CD318 portion can be full-length antibody formats. As another example, the first anti-CD318 moiety is a full-length antibody and the second anti-CD318 moiety is an scFv, or vice versa. As yet another example, the first anti-CD318 moiety is a half antibody and the second anti-CD318 moiety is a single-chain half antibody, or vice versa.

[0257] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or third antibody moiety) binds to CD318 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to CD318 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0258] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific, biparatopic, or trispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion specifically binds to the CRD3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific antibodies) comprising an anti-CLDN6 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a second anti-CLDN6 portion that specifically binds to a tumor antigen having the same epitope as the first anti-CLDN6 portion but a different epitope. The format of the second anti-CLDN6 portion can be the same as or different from the first anti-CLDN6 portion. For example, both the first anti-CLDN6 portion and the third anti-CLDN6 portion can be full-length antibody formats. As another example, the first anti-CLDN6 moiety is a full-length antibody and the second anti-CLDN6 moiety is an scFv, or vice versa. As yet another example, the first anti-CLDN6 moiety is a half antibody and the second anti-CLDN6 moiety is a single-chain half antibody, or vice versa.

[0259] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13 The first antibody moiety (and / or third antibody moiety) binds to CLDN6 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to CLDN6 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0260] In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-CLDN6 moiety that specifically binds to the same tumor antigen as the first anti-CLDN6 moiety but with a different epitope. The format of the second anti-CLDN6 moiety can be the same as or different from that of the first anti-CLDN6 moiety. For example, both the first and third anti-CLDN6 moieties can be in the format of a full-length antibody. As another example, the first anti-CLDN6 moiety can be a full-length antibody and the second anti-CLDN6 moiety can be an scFv, or vice versa. As yet another example, the first anti-CLDN6 moiety can be a half antibody and the second anti-CLDN6 moiety can be a single-chain half antibody, or vice versa.

[0261] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-tumor antigen portion that specifically binds to a tumor antigen having the same epitope as the first anti-tumor antigen portion but a different epitope. The format of the second anti-tumor antigen portion can be the same or different from that of the first anti-tumor antigen portion. For example, both the first and third anti-tumor antigen portions can be in the format of a full-length antibody. As another example, the first anti-tumor antigen portion is a full-length antibody and the second anti-tumor antigen portion is an scFv, or vice versa. As yet another example, the first anti-tumor antigen portion is a half antibody and the second anti-tumor antigen portion is a single-chain half antibody, or vice versa.

[0262] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13and binds to the tumor antigen with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first binding moiety (and / or third antibody moiety) to the tumor antigen triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0263] In some embodiments, a multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-MUC16 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-MUC16 portion that specifically binds to MUC16 with the same epitope as the first anti-MUC16 portion, but a different epitope. The format of the second anti-MUC16 portion can be the same or different from the first anti-MUC16 portion. For example, both the first and third anti-MUC16 portions can be full-length antibody formats. As another example, the first anti-MUC16 portion is a full-length antibody and the second anti-MUC16 portion is an scFv, or vice versa. As yet another example, a first anti-MUC16 moiety is a half antibody and a second anti-MUC16 moiety is a single chain half antibody, or vice versa.

[0264] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or third antibody portion) binds to MUC16 with an affinity of M. In some embodiments, the first antibody portion (and / or third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or third antibody portion) to MUC16 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0265] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-ENPP3 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-ENPP3 portion that specifically binds to ENPP3 with the same epitope as the first anti-ENPP3 portion but a different epitope. The format of the second anti-ENPP3 portion can be the same or different from that of the first anti-ENPP3 portion. For example, both the first and third anti-ENPP3 portions can be in the format of a full-length antibody. As another example, the first anti-ENPP3 portion is a full-length antibody and the second anti-ENPP3 portion is an scFv, or vice versa. As yet another example, the first anti-ENPP3 portion is a half antibody and the second anti-ENPP3 portion is a single-chain half antibody, or vice versa.

[0266] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or third antibody moiety) binds to ENPP3 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety (and / or third antibody moiety) to ENPP3 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0267] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-ROR1 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-ROR1 portion that specifically binds to ROR1 with the same epitope as the first anti-ROR1 portion, but a different epitope. The format of the second anti-ROR1 portion can be the same or different from that of the first anti-ROR1 portion. For example, both the first and third anti-ROR1 portions can be full-length antibody formats. As another example, the first anti-ROR1 portion is a full-length antibody and the second anti-ROR1 portion is an scFv, or vice versa. As yet another example, the first anti-ROR1 portion is a half antibody and the second anti-ROR1 portion is a single-chain half antibody, or vice versa.

[0268] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13and binds to ROR1 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to ROR1 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0269] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-SLC7A11 portion that specifically binds to SLC7A11 with the same epitope as the first anti-SLC7A11 portion but a different epitope. The format of the second anti-SLC7A11 portion can be the same or different from that of the first anti-SLC7A11 portion. For example, both the first anti-SLC7A11 portion and the third anti-SLC7A11 portion can be in the format of a full-length antibody. As another example, the first anti-SLC7A11 portion is a full-length antibody and the second anti-SLC7A11 portion is an scFv, or vice versa. As yet another example, the first anti-SLC7A11 moiety is a half antibody and the second anti-SLC7A11 moiety is a single chain half antibody, or vice versa.

[0270] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or third antibody moiety) binds to SLC7A11 with an affinity of M. In some embodiments, the first antibody moiety (and / or third antibody moiety) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety (and / or third antibody moiety) to SLC7A11 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0271] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-DLL3 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-DLL3 portion that specifically binds to the same DLL3 as the first anti-DLL3 portion but with a different epitope. The format of the second anti-DLL3 portion can be the same or different from that of the first anti-DLL3 portion. For example, both the first and third anti-DLL3 portions can be in the format of a full-length antibody. As another example, the first anti-DLL3 portion is a full-length antibody and the second anti-DLL3 portion is an scFv, or vice versa. As yet another example, the first anti-DLL3 portion is a half antibody and the second anti-DLL3 portion is a single-chain half antibody, or vice versa.

[0272] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13and binds to DLL3 with an affinity of M. In some embodiments, the first antibody portion (and / or third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or third antibody portion) to DLL3 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0273] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-B7H4 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-B7H4 portion that specifically binds to B7H4 with the same epitope as the first anti-B7H4 portion but a different epitope. The format of the second anti-B7H4 portion can be the same or different from that of the first anti-B7H4 portion. For example, both the first and third anti-B7H4 portions can be in the format of a full-length antibody. As another example, the first anti-B7H4 portion is a full-length antibody and the second anti-B7H4 portion is an scFv, or vice versa. As yet another example, a first anti-B7H4 moiety is a half antibody and a second anti-B7H4 moiety is a single chain half antibody, or vice versa.

[0274] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or third antibody portion) binds to B7H4 with an affinity of M. In some embodiments, the first antibody portion (and / or third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or third antibody portion) to B7H4 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0275] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-EPHA2 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-EPHA2 portion that specifically binds to the same EPHA2 as the first anti-EPHA2 portion but with a different epitope. The format of the second anti-EPHA2 portion can be the same or different from that of the first anti-EPHA2 portion. For example, both the first anti-EPHA2 portion and the third anti-EPHA2 portion can be in the format of a full-length antibody. As another example, the first anti-EPHA2 portion is a full-length antibody and the second anti-EPHA2 portion is an scFv, or vice versa. As yet another example, the first anti-EPHA2 portion is a half antibody and the second anti-EPHA2 portion is a single-chain half antibody, or vice versa.

[0276] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to EPHA2 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to EPHA2 induces the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0277] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-CD318 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus comprises a second anti-CD318 portion that specifically binds to CD318 with the same epitope as the first anti-CD318 portion but a different epitope. The format of the second anti-CD318 portion can be the same or different from that of the first anti-CD318 portion. For example, both the first anti-CD318 portion and the third anti-CD318 portion can be in the format of a full-length antibody. As another example, the first anti-CD318 portion is a full-length antibody and the second anti-CD318 portion is an scFv, or vice versa. As yet another example, a first anti-CD318 moiety is a half antibody and a second anti-CD318 moiety is a single chain half antibody, or vice versa.

[0278] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to CD318 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to CD318 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0279] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range between these values). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-CLDN6 portion that specifically binds to the same CLDN6 as the first anti-CLDN6 portion but with a different epitope. The format of the second anti-CLDN6 portion can be the same or different from that of the first anti-CLDN6 portion. For example, both the first anti-CLDN6 portion and the third anti-CLDN6 portion can be in the format of a full-length antibody. As another example, the first anti-CLDN6 portion is a full-length antibody and the second anti-CLDN6 portion is an scFv, or vice versa. As yet another example, the first anti-CLDN6 portion is a half antibody and the second anti-CLDN6 portion is a single-chain half antibody, or vice versa.

[0280] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to CLDN6 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, a single-chain half antibody, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to CLDN6 induces the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0281] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, and thus includes a second anti-tumor antigen moiety that specifically binds to the same tumor antigen as the first anti-tumor antigen moiety but with a different epitope. The second anti-tumor antigen moiety can be in the same or different format as the first anti-tumor antigen moiety. For example, both the first and third anti-tumor antigen moieties can be in the format of a full-length antibody. As another example, the first anti-tumor antigen moiety can be a full-length antibody and the second anti-tumor antigen moiety can be an scFv, or vice versa. As yet another example, the first anti-tumor antigen moiety can be a half antibody and the second anti-tumor antigen moiety can be a single-chain half antibody, or vice versa.

[0282] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13and binds to the tumor antigen with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, a half antibody, and a full-length antibody. In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to the tumor antigen triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0283] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-MUC16 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, thus including a second anti-MUC16 portion that specifically binds to the same MUC16 as the first anti-MUC16 portion but with a different epitope. The format of the second anti-MUC16 portion can be the same or different from that of the first anti-MUC16 portion. For example, both the first and third anti-MUC16 portions can be full-length antibody formats. As another example, the first anti-MUC16 portion is a full-length antibody and the second anti-MUC16 portion is an scFv, or vice versa. As yet another example, the first anti-MUC16 portion is a half antibody and the second anti-MUC16 portion is a single-chain half antibody, or vice versa.

[0284] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion binds to MUC16 with an affinity of M. In some embodiments, the first antibody portion is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to MUC16 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0285] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-ENPP3 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-ENPP3 portion that specifically binds to the same ENPP3 as the first anti-ENPP3 portion but with a different epitope. The format of the second anti-ENPP3 portion can be the same as or different from that of the first anti-ENPP3 portion. For example, both the first and third anti-ENPP3 portions can be in the format of a full-length antibody. As another example, the first anti-ENPP3 portion can be a full-length antibody and the second anti-ENPP3 portion can be an scFv, or vice versa. As yet another example, the first anti-ENPP3 portion can be a half antibody and the second anti-ENPP3 portion can be a single-chain half antibody, or vice versa.

[0286] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion binds to ENPP3 with an affinity of M. In some embodiments, the first antibody portion is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to ENPP3 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0287] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-ROR1 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-ROR1 portion that specifically binds to the same ROR1 as the first anti-ROR1 portion but with a different epitope. The format of the second anti-ROR1 portion can be the same or different from that of the first anti-ROR1 portion. For example, both the first and third anti-ROR1 portions can be full-length antibody formats. As another example, the first anti-ROR1 portion is a full-length antibody and the second anti-ROR1 portion is an scFv, or vice versa. As yet another example, the first anti-ROR1 portion is a half antibody and the second anti-ROR1 portion is a single-chain half antibody, or vice versa.

[0288] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion binds to ROR1 with an affinity of M. In some embodiments, the first antibody portion is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion (and / or the third antibody portion) to ROR1 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0289] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody moiety is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-SLC7A11 moiety that specifically binds to the same SLC7A11 moiety but with a different epitope as the first anti-SLC7A11 moiety. The format of the second anti-SLC7A11 moiety can be the same as or different from the first anti-SLC7A11 moiety. For example, both the first anti-SLC7A11 moiety and the third anti-SLC7A11 moiety can be in the format of a full-length antibody. As another example, the first anti-SLC7A11 moiety is a full-length antibody and the second anti-SLC7A11 moiety is an scFv, or vice versa. As yet another example, the first anti-SLC7A11 moiety is a half antibody and the second anti-SLC7A11 moiety is a single-chain half antibody, or vice versa.

[0290] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or the third antibody moiety) binds to SLC7A11 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to SLC7A11 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0291] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-DLL3 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-DLL3 moiety that specifically binds to the same DLL3 as the first anti-DLL3 moiety but with a different epitope. The format of the second anti-DLL3 moiety can be the same or different from that of the first anti-DLL3 moiety. For example, both the first and third anti-DLL3 moieties can be in the format of a full-length antibody. As another example, the first anti-DLL3 moiety can be a full-length antibody and the second anti-DLL3 moiety can be an scFv, or vice versa. As yet another example, the first anti-DLL3 moiety can be a half antibody and the second anti-DLL3 moiety can be a single-chain half antibody, or vice versa.

[0292] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to DLL3 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion to DLL3 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0293] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or biparatopic or trispecific antibodies) comprising an anti-B7H4 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-B7H4 portion that specifically binds to the same B7H4 as the first anti-B7H4 portion but with a different epitope. The format of the second anti-B7H4 portion can be the same or different from that of the first anti-B7H4 portion. For example, both the first and third anti-B7H4 portions can be in the format of a full-length antibody. As another example, the first anti-B7H4 portion can be a full-length antibody and the second anti-B7H4 portion can be an scFv, or vice versa. As yet another example, the first anti-B7H4 portion can be a half antibody and the second anti-B7H4 portion can be a single-chain half antibody, or vice versa.

[0294] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to B7H4 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to B7H4 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0295] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-EPHA2 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, and thus includes a second anti-EPHA2 portion that specifically binds to the same EPHA2 as the first anti-EPHA2 portion but with a different epitope. The format of the second anti-EPHA2 portion can be the same or different from that of the first anti-EPHA2 portion. For example, both the first anti-EPHA2 portion and the third anti-EPHA2 portion can be in the format of a full-length antibody. As another example, the first anti-EPHA2 portion is a full-length antibody and the second anti-EPHA2 portion is an scFv, or vice versa. As yet another example, the first anti-EPHA2 portion is a half antibody and the second anti-CD318 portion is a single-chain half antibody, or vice versa.

[0296] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to EPHA2 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to EPHA2 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0297] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-CD318 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-CD318 portion that specifically binds to the same CD318 as the first anti-CD318 portion but with a different epitope. The format of the second anti-CD318 portion can be the same or different from that of the first anti-CD318 portion. For example, both the first and third anti-CD318 portions can be full-length antibody formats. As another example, the first anti-CD318 portion is a full-length antibody and the second anti-CD318 portion is an scFv, or vice versa. As yet another example, the first anti-CD318 portion is a half antibody and the second anti-CD318 portion is a single-chain half antibody, or vice versa.

[0298] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to CD318 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to CD318 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0299] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or biparatopic or trispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises: an sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR1; an sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR2; and an sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof comprising up to about three (e.g., about one, two, or three) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-CLDN6 portion that specifically binds to the same CLDN6 as the first anti-CLDN6 portion but with a different epitope. The format of the second anti-CLDN6 portion can be the same as or different from that of the first anti-CLDN6 portion. For example, both the first and third anti-CLDN6 portions can be in the format of a full-length antibody. As another example, the first anti-CLDN6 portion can be a full-length antibody and the second anti-CLDN6 portion can be an scFv, or vice versa. As yet another example, the first anti-CLDN6 portion can be a half antibody and the second anti-CLDN6 portion can be a single-chain half antibody, or vice versa.

[0300] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to CLDN6 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first antibody portion to CLDN6 induces the second antibody portion to activate 4-1BB, for example, activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0301] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, and thus includes a second anti-tumor antigen portion that specifically binds to the same tumor antigen as the first anti-tumor antigen portion but with a different epitope. The format of the second anti-tumor antigen portion can be the same as or different from that of the first anti-tumor antigen portion. For example, both the first and third anti-tumor antigen portions can be in the format of a full-length antibody. As another example, the first anti-tumor antigen portion can be a full-length antibody and the second anti-tumor antigen portion can be an scFv, or vice versa. As yet another example, the first anti-tumor antigen portion can be a half antibody and the second anti-tumor antigen portion can be a single-chain half antibody, or vice versa.

[0302] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to the tumor antigen with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion to the tumor antigen induces the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0303] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-MUC16 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any ranges therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, thus comprising a second anti-MUC16 moiety that specifically binds to the same MUC16 moiety but with a different epitope as the first anti-MUC16 moiety. The format of the second anti-MUC16 moiety can be the same or different from that of the first anti-MUC16 moiety. For example, both the first and third anti-MUC16 moieties can be full-length antibody formats. As another example, the first anti-MUC16 moiety can be a full-length antibody and the second anti-MUC16 moiety can be an scFv, or vice versa. As yet another example, the first anti-MUC16 moiety can be a half antibody and the second anti-MUC16 moiety can be a single-chain half antibody, or vice versa.

[0304] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to MUC16 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion to MUC16 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0305] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-ENPP3 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-ENPP3 moiety that specifically binds to the same ENPP3 as the first anti-ENPP3 moiety but with a different epitope. The format of the second anti-ENPP3 moiety can be the same as or different from that of the first anti-ENPP3 moiety. For example, both the first and third anti-ENPP3 moieties can be in the format of a full-length antibody. As another example, the first anti-ENPP3 moiety can be a full-length antibody and the second anti-ENPP3 moiety can be an scFv, or vice versa. As yet another example, the first anti-ENPP3 moiety can be a half antibody and the second anti-ENPP3 moiety can be a single-chain half antibody, or vice versa.

[0306] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or the third antibody moiety) binds to ENPP3 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to ENPP3 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0307] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-ROR1 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-ROR1 moiety that specifically binds to the same ROR1 as the first anti-ROR1 moiety but with a different epitope. The format of the second anti-ROR1 moiety can be the same or different from that of the first anti-ROR1 moiety. For example, both the first and third anti-ROR1 moieties can be in full-length antibody format. As another example, the first anti-ROR1 moiety is a full-length antibody and the second anti-ROR1 moiety is an scFv, or vice versa. As yet another example, the first anti-ROR1 moiety is a half antibody and the second anti-ROR1 moiety is a single-chain half antibody, or vice versa.

[0308] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to ROR1 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to ROR1 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0309] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, thus including a second anti-SLC7A11 moiety that specifically binds to the same SLC7A11 moiety but with a different epitope as the first anti-SLC7A11 moiety. The format of the second anti-SLC7A11 moiety can be the same as or different from the first anti-SLC7A11 moiety. For example, both the first anti-SLC7A11 moiety and the third anti-SLC7A11 moiety can be in the format of a full-length antibody. As another example, the first anti-SLC7A11 moiety can be a full-length antibody and the second anti-SLC7A11 moiety can be an scFv, or vice versa. As yet another example, the first anti-SLC7A11 moiety can be a half antibody and the second anti-SLC7A11 moiety can be a single-chain half antibody, or vice versa.

[0310] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety (and / or the third antibody moiety) binds to SLC7A11 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to SLC7A11 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0311] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-DLL3 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-DLL3 moiety that specifically binds to the same DLL3 as the first anti-DLL3 moiety but with a different epitope. The format of the second anti-DLL3 moiety can be the same or different from that of the first anti-DLL3 moiety. For example, both the first and third anti-DLL3 moieties can be in the format of a full-length antibody. As another example, the first anti-DLL3 moiety can be a full-length antibody and the second anti-DLL3 moiety can be an scFv, or vice versa. As yet another example, the first anti-DLL3 moiety can be a half antibody and the second anti-DLL3 moiety can be a single-chain half antibody, or vice versa.

[0312] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to DLL3 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody portion to DLL3 triggers the second antibody portion to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0313] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-B7H4 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-B7H4 moiety that specifically binds to the same B7H4 moiety but a different epitope as the first anti-B7H4 moiety. The format of the second anti-B7H4 moiety can be the same or different from that of the first anti-B7H4 moiety. For example, both the first and third anti-B7H4 moieties can be full-length antibody formats. As another example, the first anti-B7H4 moiety can be a full-length antibody and the second anti-B7H4 moiety can be an scFv, or vice versa. As yet another example, the first anti-B7H4 moiety can be a half antibody and the second anti-B7H4 moiety can be a single-chain half antibody, or vice versa.

[0314] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to B7H4 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to B7H4 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0315] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-EPHA2 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, which comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic, and thus includes a second anti-EPHA2 moiety that specifically binds to the same EPHA2 as the first anti-EPHA2 moiety but with a different epitope. The format of the second anti-EPHA2 moiety can be the same or different from that of the first anti-EPHA2 moiety. For example, both the first anti-EPHA2 moiety and the third anti-EPHA2 moiety can be in the format of a full-length antibody. As another example, the first anti-EPHA2 moiety can be a full-length antibody and the second anti-EPHA2 moiety can be an scFv, or vice versa. As yet another example, the first anti-EPHA2 moiety can be a half antibody and the second anti-EPHA2 moiety can be a single-chain half antibody, or vice versa.

[0316] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to EPHA2 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to EPHA2 induces the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0317] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific, biparatopic, or trispecific antibody) comprising an anti-CD318 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO:27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-CD318 moiety that specifically binds to the same CD318 as the first anti-CD318 moiety but with a different epitope. The format of the second anti-CD318 moiety can be the same or different from that of the first anti-CD318 moiety. For example, both the first and third anti-CD318 moieties can be in the format of a full-length antibody. As another example, the first anti-CD318 moiety can be a full-length antibody and the second anti-CD318 moiety can be an scFv, or vice versa. As yet another example, the first anti-CD318 moiety can be a half antibody and the second anti-CD318 moiety can be a single-chain half antibody, or vice versa.

[0318] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody moiety binds to CD318 with an affinity of M. In some embodiments, the first antibody moiety (and / or the third antibody moiety) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody moiety (and / or the third antibody moiety) and the second antibody moiety are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first antibody moiety to CD318 triggers the second antibody moiety to activate 4-1BB, e.g., activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0319] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific antibody) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb, wherein the sdAb comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO: 27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% sequence identity, including any range therebetween). In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable to (e.g., the same as) that of an anti-4-1BB antibody portion comprising SEQ ID NO: 27. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is biparatopic and thus includes a second anti-CLDN6 moiety that specifically binds to the same CLDN6 as the first anti-CLDN6 moiety but with a different epitope. The format of the second anti-CLDN6 moiety can be the same as or different from that of the first anti-CLDN6 moiety. For example, both the first and third anti-CLDN6 moieties can be in the format of a full-length antibody. As another example, the first anti-CLDN6 moiety can be a full-length antibody and the second anti-CLDN6 moiety can be an scFv, or vice versa. As yet another example, the first anti-CLDN6 moiety can be a half antibody and the second anti-CLDN6 moiety can be a single-chain half antibody, or vice versa.

[0320] In some embodiments, the first antibody portion (and / or the third antibody portion) is about 10 -7 M~about 10 -13The first antibody portion (and / or the third antibody portion) binds to CLDN6 with an affinity of M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from the group consisting of an scFv, a Fab, and a full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are connected via an Fc domain (e.g., an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first antibody portion to CLDN6 induces the second antibody portion to activate 4-1BB, for example, activation that is enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0321] In some embodiments, multispecific constructs (e.g., bispecific antibodies) are provided that include an anti-tumor antigen antibody portion that includes an antibody (e.g., a full-length antibody) that specifically binds to a tumor antigen (e.g., a human tumor antigen) and an anti-4-1BB antibody portion that specifically binds to 4-1BB, where the anti-4-1BB antibody portion is fused to the N-terminus of one or both heavy chains of the anti-tumor antigen antibody (e.g., an scFv, Fab, or full-length anti-tumor antigen antibody). In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct includes, from N-terminus to C-terminus, a second antibody moiety (e.g., an sdAb), an optional first linker, an optional Fc domain, an optional second linker, and the heavy chain of the first antibody moiety. In some embodiments, the multispecific construct comprises, from N-terminus to C-terminus, a second antibody moiety (e.g., an sdAb), an optional first linker, and a heavy chain of a first antibody moiety (e.g., a full-length antibody, e.g., a full-length antibody comprising an Fc domain).

[0322] In some embodiments, a multispecific construct (e.g., bispecific antibody) is provided that includes an anti-tumor antigen antibody portion that includes an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen and an anti-4-1BB antibody portion that specifically binds to 4-1BB, wherein the anti-4-1BB antibody portion is fused to the C-terminus of one or both heavy chains of the anti-tumor antigen antibody (e.g., scFv, Fab, or full-length anti-tumor antigen antibody). In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct includes, from N-terminus to C-terminus, a heavy chain of a first antibody portion (e.g., sdAb, scFv, or Fab), an optional first linker, an optional Fc domain, an optional second linker, and a second antibody portion (e.g., sdAb). In some embodiments, the multispecific construct comprises, from N-terminus to C-terminus, the heavy chain of a first antibody moiety (e.g., a full-length antibody, e.g., a full-length antibody comprising an Fc domain), an optional first linker, and a second antibody moiety (e.g., an sdAb).

[0323] In some embodiments, multispecific constructs (e.g., bispecific antibodies) are provided that include an anti-tumor antigen antibody portion that includes an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen and an anti-4-1BB antibody portion that specifically binds to 4-1BB, where the anti-4-1BB antibody portion is fused to the N-terminus of one or both light chains of the anti-tumor antigen antibody (e.g., scFv, Fab, or full-length anti-tumor antigen antibody). In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct includes, from N-terminus to C-terminus, a second antibody portion (e.g., an sdAb), an optional first linker, an optional Fc domain, an optional second linker, and the light chain of the first antibody portion. In some embodiments, the multispecific construct comprises, from N-terminus to C-terminus, a second antibody moiety (e.g., an sdAb), an optional first linker, and the heavy chain of one antibody moiety (e.g., a full-length antibody, e.g., a full-length antibody comprising an Fc domain).

[0324] In some embodiments, a multispecific construct (e.g., bispecific antibody) is provided comprising an anti-tumor antigen antibody portion comprising an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen, and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion is fused to the C-terminus of one or both light chains of the anti-tumor antigen antibody (e.g., scFv, Fab, or full-length anti-tumor antigen antibody). In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises, from N-terminus to C-terminus, a light chain of a first antibody portion (e.g., sdAb, scFv, or Fab), an optional first linker, an optional Fc domain, an optional second linker, and a second antibody portion (e.g., sdAb). In some embodiments, the multispecific construct comprises, from N-terminus to C-terminus, the light chain of a first antibody moiety (e.g., a full-length antibody comprising an Fc domain), an optional first linker, and a second antibody moiety (e.g., an sdAb).

[0325] In some embodiments, a multispecific construct (e.g., bispecific antibody) is provided that includes an anti-tumor antigen antibody portion comprising an antibody (e.g., a full-length antibody) that specifically binds to a tumor antigen (e.g., a human tumor antigen) and an anti-4-1BB antibody portion comprising a single domain antibody that binds to 4-1BB (e.g., human 4-1BB), where the single domain antibody is fused to the N-terminus of one or both heavy chains of the anti-tumor antigen antibody (e.g., an scFv, Fab, or full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., bispecific antibody) is provided that includes an anti-tumor antigen antibody portion comprising an antibody (e.g., an scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen and an anti-4-1BB antibody portion comprising a single domain antibody that binds to 4-1BB, where the single domain antibody is fused to the C-terminus of one or both heavy chains of the anti-tumor antigen antibody (e.g., an scFv, Fab, or full-length anti-CLDN6 antibody).

[0326] In some embodiments, a multispecific construct (e.g., bispecific antibody) is provided that includes an anti-tumor antigen antibody portion that includes an antibody that specifically binds to a tumor antigen (e.g., an scFv, Fab, or full-length antibody) and an anti-4-1BB antibody portion that includes a single domain antibody that binds to 4-1BB, wherein the single domain antibody is fused to the N-terminus of one or both light chains of the anti-tumor antigen antibody (e.g., an scFv, Fab, or full-length anti-CLDN6 antibody). In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-tumor antigen antibody portion that comprises an antibody that specifically binds to a tumor antigen (e.g., an scFv, Fab, or full-length antibody) and an anti-4-1BB antibody portion that comprises a single domain antibody that binds to 4-1BB, wherein the single domain antibody is fused to the C-terminus of one or both light chains of the anti-tumor antigen antibody (e.g., an scFv, Fab, or full-length anti-tumor antigen antibody).

[0327] In some embodiments, the anti-4-1BB antibody portion is fused to the anti-tumor antigen antibody portion via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker has a length of about 4 to about 50 amino acids. In some embodiments, the linker is selected from the group consisting of (GS)n, (GGGS)n (SEQ ID NO: 290), (GGGGS)n (SEQ ID NO: 287), and (GSGGS)n (SEQ ID NO: 254). In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, n is 0 to 8. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGGSGGGGGS (SEQ ID NO: 23). In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 284) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 171).

[0328] The multispecific construct of the present disclosure may also include a third antibody portion that binds to the same antigen as one of the other two portions, but at a different site biparatopically. For example, the present disclosure provides a biparatopic (trispecific) construct comprising a first antibody portion and a third antibody portion that both bind to the same tumor antigen. The first antibody portion and the third antibody portion are antigens with the same but different epitopes. In certain embodiments, the first antibody portion and the third antibody portion provided herein each have a full-length antibody / IgG format, and the second antibody portion, which is an anti-4-1BB antigen-binding portion provided herein, has a single domain antibody (sdAb) / VHH / nanobody format.

[0329] In certain embodiments, the first antibody portion provided herein has an IgG format, the third antibody portion provided herein has an scFv format, and the anti-4-1BB antigen-binding portion provided herein has a single domain antibody (sdAb) / VHH / nanobody format. In certain embodiments, the third antibody portion is fused to the N-terminus of the first antibody portion, and the second antibody portion is fused to the C-terminus of the first antibody portion. The first antibody portion and the third antibody are fused via a linker. The first antibody portion and the second antibody are fused via a linker.

[0330] In certain embodiments, the first antibody portion provided herein has a full-length antibody / IgG format, the third antibody portion provided herein has a single-chain half antibody format, and the anti-4-1BB antigen-binding portion provided herein has a single-domain antibody (sdAb) / VHH / nanobody format. In certain embodiments, the second antibody portion is fused to the C-terminus of the first antibody portion and the third antibody portion, respectively. The second antibody portion and the third antibody are fused via a linker. The first antibody portion and the second antibody are fused via a linker. In such cases, the first antibody portion and the third antibody portion can form a heterodimer through Fc region pairing.

[0331] Heterodimers via Fc region pairing can be achieved by forming knobs-into-holes (KIH), disulfide bonds (-SS-), or through hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

[0332]

[0010] In some embodiments, provided herein are multispecific constructs comprising a first antibody moiety that specifically binds to a tumor antigen and a second antibody moiety that specifically binds to 4-1BB, wherein binding of the first antibody moiety to the tumor antigen induces the second antibody moiety to activate 4-1BB. In some embodiments, the tumor antigen is selected from the group consisting of MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, activation of 4-1BB by the second antibody moiety is enhanced by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold after binding of the first antibody moiety to the tumor antigen. In some embodiments, the multispecific construct does not activate 4-1BB signaling in the absence of antigen binding. In some embodiments, the second moiety activates 4-1BB signaling in the absence of tumor antigen binding. Activation of 4-1BB signaling is the predicted mechanism for agonist antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513). However, some anti-4-1BB moieties of the antibodies disclosed herein do not require such activity. In fact, in some embodiments, the anti-4-1BB moiety of the antibody of the present invention is preferably unable to independently activate 4-1BB in the absence of tumor antigen binding. Interestingly, as experimental examples demonstrate, when the anti-tumor antigen moiety binds to a tumor antigen on a cell, such binding can induce 4-1BB signaling activation.

[0333] Compared to known anti-4-1BB agonist antibodies, which are generally associated with dose-limiting on-target liver toxicity, the antibodies of the present disclosure are intended to be much safer. Because tumor antigens are not expressed in healthy tissues, the antibodies of the present disclosure are not expected to induce cytotoxic immune responses, as they are unable to activate 4-1BB signaling. In contrast, in tumor tissues where tumor antigens are expressed and / or accessible, the antibodies of the present disclosure can initiate a potent immune response against tumor cells. Thus, unlike anti-4-1BB antibodies currently in clinical development that suffer from on-target / intrinsic toxicity, the antibodies disclosed herein can be both potent and safe when treating cancer.

[0334] In some embodiments, provided herein are multispecific constructs comprising a first antibody portion and / or a third antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion and / or the third antibody portion to the tumor antigen induces the second antibody portion to activate the immune system. In some embodiments, binding of the first antibody portion and / or the third antibody portion to the tumor antigen induces the second antibody portion to increase the level of one or more cytokines. In some embodiments, the cytokine is IFNγ or IL-2. In some embodiments, binding of the first antibody portion and / or the third antibody portion to the tumor antigen induces the second antibody portion to increase NFκB signaling.

[0335] In some embodiments, provided herein is a multispecific construct comprising a first antibody portion and / or a third antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion and / or the third antibody portion to the tumor antigen triggers the second antibody portion to activate NFκB signaling.

[0336] In some embodiments, the activation of NFκB signaling is evaluated by measuring the changes in the expression of downstream targets of NFκB signaling, such as cytokines, growth factors, adhesion molecules and / or anti-apoptotic genes.In some embodiments, the changes in the expression of downstream targets of NFκB signaling are measured by determining the level of RNA transcript expression of downstream targets of NFκB signaling.Suitable methods for measuring the RNA transcript level in a sample are known in the art, including, for example, Northern blot analysis, nuclease protection assay, in situ hybridization, PCR analysis (e.g., qPCR, RT-PCR, RT-qPCR, etc.) and next-generation sequencing (e.g., RNAseq).In some embodiments, the changes in the expression of downstream targets of NFκB signaling are measured by determining the level of protein expression of downstream targets of NFκB signaling. Suitable methods for measuring protein expression in a sample are known in the art, including, for example, immunoassays (e.g., Meso Scale Discovery or MSD assays), immunohistochemistry (IHC), PET imaging, Western blotting, enzyme-linked immunosorbent assays (ELISAs), flow cytometry, and mass spectrometry. In some embodiments, activation of NFκB signaling is assessed by measuring the activation of one or more components of the NFκB signaling cascade, for example, by measuring the levels of activated IκB kinase and / or IκBα. In some embodiments, activation of NFκB signaling is assessed by measuring the levels of cytoplasmic and / or nuclear NFκB.

[0337] In some embodiments, activation of NFκB signaling by the second antibody moiety is enhanced by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold following binding of the first antibody moiety to the tumor antigen. In some embodiments, the multispecific construct does not activate NFκB signaling in the absence of antigen binding.

[0338] In some embodiments, provided herein are multispecific constructs comprising a first antibody portion and / or a third antibody portion that specifically binds to a tumor antigen; and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion and / or the third antibody portion to the tumor antigen triggers activation of the second antibody portion such that the levels of one or more cytokines are increased.

[0339] In some embodiments, the level of one or more cytokines is measured by determining the level of RNA transcript expression of one or more cytokines.Suitable methods for measuring the RNA transcript level in a sample are known in the art, including, for example, Northern blot analysis, nuclease protection assay, in situ hybridization, PCR analysis (for example, qPCR, RT-PCR, RT-qPCR, etc.) and next-generation sequencing (for example, RNAseq).In some embodiments, the level of transcript expression of biomarker is measured by RT-PCR, in situ hybridization and / or RNAseq.

[0340] In some embodiments, the level of one or more cytokines is measured by determining the level of protein expression of one or more cytokines. Suitable methods for measuring protein expression in a sample are known in the art, including, for example, immunoassays (e.g., mesoscale discovery or MSD assays), immunohistochemistry (IHC), PET imaging, Western blotting, enzyme-linked immunosorbent assays (ELISA), flow cytometry and mass spectrometry. In some embodiments, the level of protein expression of biomarkers is measured by immunoassays, Western blotting, ELISA, IHC and / or flow cytometry.

[0341] In some embodiments, the level of one or more cytokines increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold after binding of the first antibody moiety to the tumor antigen. In some embodiments, in the absence of binding to the antigen, the multispecific construct does not result in an increase in the level of one or more cytokines. In some embodiments, the cytokines are IFNγ and / or IL-12.

[0342]

[0010] In some embodiments, provided herein are multispecific constructs comprising a first antibody portion and / or a third antibody portion that specifically binds to a tumor antigen and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion and / or the third antibody portion to the tumor antigen triggers activation of the second antibody portion to increase IFNγ levels. In some embodiments, the IFNγ level increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold after binding of the first antibody portion and / or the third antibody portion to the tumor antigen. In some embodiments, the multispecific construct does not result in an increase in IFNγ levels in the absence of antigen binding.

[0343]

[0010] In some embodiments, provided herein are multispecific constructs comprising a first antibody portion and / or a third antibody portion that specifically binds to a tumor antigen and a second antibody portion that specifically binds to 4-1BB, wherein binding of the first antibody portion and / or the third antibody portion to the tumor antigen triggers activation of the second antibody portion to increase IL-2 levels. In some embodiments, the IL-2 level increases by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold after binding of the first antibody portion and / or the third antibody portion to the tumor antigen. In some embodiments, in the absence of antigen binding, the multispecific construct does not result in an increase in IL-2 levels.

[0344] In some embodiments, levels of IFNγ, IL-2 and / or NFκB signaling are measured in one or more (e.g., one or more, two or more, three or more, four or more, etc.) samples obtained from a subject. Any suitable sample in the form of tissue and / or bodily fluid known or suspected to contain diseased cells and / or targets of interest can be used in the methods described herein, including, for example, sputum, pleural effusion, lymphatic fluid, bone marrow, blood, plasma, serum, urine, tissue samples (including samples known to or suspected to contain cancer cells), tumor samples, tumor biopsies, etc. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a serum sample. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample comprises one or more cancer cells.

[0345] Methods of obtaining suitable tissue and / or fluid samples (e.g., methods suitable for obtaining a representative sample from a particular type, location, diseased tissue, etc.) are well known to those of skill in the art, including, for example, by excision, bone marrow biopsy or aspiration, endoscopic biopsy or aspiration (e.g., cystoscopy, bronchoscopy, colonoscopy, etc.), needle biopsy or aspiration (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, image-guided biopsy, etc.), skin biopsy (e.g., shave biopsy, punch biopsy, incisional biopsy, excision biopsy, etc.), various other surgical tissue (e.g., tumor tissue) biopsy and / or resection strategies, and fluid collection (e.g., collection of urine, blood, serum, plasma, sputum, etc.). nucleic acid

[0346] Nucleic acid molecules encoding the multispecific constructs or various antibody portions described herein are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) encoding one or more polypeptides of the multispecific constructs or various antibody portions is provided. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, biparatopic antibody, or trispecific antibody) or polypeptide portion thereof is provided.

[0347] Also contemplated herein are isolated host cells comprising a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, biparatopic antibody, or trispecific antibody) described herein, a nucleic acid(s) encoding a polypeptide component of a multispecific construct, or a vector comprising a nucleic acid encoding a polypeptide component of a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, biparatopic antibody, or trispecific antibody).

[0348] The present application also includes variants to these nucleic acid sequences, for example, nucleotide sequences that hybridize under at least moderately stringent hybridization conditions to nucleic acid sequences encoding the multispecific constructs (e.g., anti-tumor antigen / anti-4-1BB bispecific, biparatopic, or trispecific antibodies) or various antibody portions described herein.

[0349] The present application also provides a vector into which the nucleic acid of the present application is inserted.

[0350] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0351] Furthermore, expression vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses.Generally, suitable vectors contain a replication origin functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, for example, WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0352] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure may comprise a flexible linker sequence or may be modified to attach a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0353] Antibodies, variants, or derivatives thereof of the present disclosure include derivatives modified by the covalent attachment of any type of molecule to the antibody, i.e., such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, and without limitation, antibodies can be modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, antibodies can contain one or more non-classical amino acids.

[0354] In some embodiments, the antibody may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0355] The antibody may be conjugated or fused to a therapeutic agent which may include a detectable label, for example, a radiolabel, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound-enhancing agent, a non-radioactive label, combinations thereof and other such agents known in the art. Treatment method

[0356] Also provided herein are methods of treating a disease or condition in an individual. These methods comprise administering a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody) described herein to an individual (e.g., a mammal, e.g., a human). In some embodiments, the individual is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.

[0357] In some embodiments of the method, the disease or condition is a proliferative disorder. In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the cancer is a solid tumor, melanoma, renal cancer, ovarian cancer, colorectal cancer, squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer, or non-Hodgkin's lymphoma (NHL). Compositions, Kits and Articles of Manufacture

[0358] Also provided herein are compositions (e.g., formulations) comprising any one of the multispecific constructs described herein (e.g., anti-tumor antigen / anti-4-1BB bispecific antibodies, biparatopic antibodies, or trispecific antibodies), nucleic acids encoding any of the multispecific constructs or portions thereof, vectors comprising a nucleic acid encoding one of the multispecific constructs, or host cells comprising the nucleic acid or vector.

[0359] Suitable formulations of the multispecific constructs described herein (e.g., anti-tumor antigen / anti-4-1BB bispecific, biparatopic, or trispecific antibodies) can be obtained by mixing the multispecific constructs having the desired degree of purity with pharmaceutically acceptable carriers, excipients, or stabilizers as needed (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0360] Kits containing any one of the multispecific constructs described herein (e.g., anti-tumor antigen / anti-4-1BB bispecific antibody, biparatopic antibody, or trispecific antibody) are also provided. The kits may be useful for any of the methods of treatment described herein.

[0361] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kits may optionally provide additional components, such as buffers and interpretive information.

[0362] Thus, the present application also provides an article of manufacture. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. Generally, the container holds the composition and may have a sterile access port (e.g., the container may be an intravenous infusion bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0363] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0364] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. The following examples are intended to be purely illustrative of the present application and therefore should not be construed as limiting the present application in any way. The following examples and detailed description are offered by way of illustration, not limitation. Example 1 Generation of CLDN6×4-1BB bispecific antibody

[0365] Exemplary CLDN6x4-1BB bispecific antibodies shown in Table 3 below were designed and generated. [Table 3-1] [Table 3-2] [Table 3-3] Example 2 Antigen binding activity of CLDN6×4-1BB BsAb 2.1 Binding affinity of CLDN6×4-1BB BsAb to CLDN6

[0366] The binding affinity of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT (as prepared in Example 1) to human CLDN6 was measured by surface plasmon resonance (SPR). As shown in Figures 1A to 1C, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT each had a binding affinity of 1.61 x 10 -9 M and 2.36 × 10 -9 K of M D and bound to CLDN6 virus-like particles (VLPs).

[0367] A CHO-K1 cell line stably expressing human CLDN6 (CHO-K1-CLDN6) was prepared to evaluate the binding ability of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to CLDN6. The parental CLDN6-1 antibody was used as a control. Briefly, CHO-K1-CLDN6 cells were incubated with different concentrations of BsAb in FACS buffer at 4°C for 30 minutes. Phycoerythrin (PE)-conjugated anti-human IgG antibody was then added after washing, and the cells were further incubated at 4°C for 30 minutes. The mean fluorescence intensity (MFI) of PE was assessed by FACS. As shown in Figure 2A, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bound to CLDN6-expressing cells in a concentration-dependent manner.

[0368] OVCAR3 and OV90 are human ovarian cancer cell lines with endogenous CLDN6 expression levels. As shown in Figures 2B and 2C, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT were able to bind to OVCAR3 and OV90. Overall, the binding affinity of the BsAb containing the anti-CLDN6-1 antibody moiety to human CLDN6 is comparable to that of the parent CLDN6-1 antibody to human CLDN6. The binding signal correlated well with the CLDN6 expression levels on the surface of OVCAR3 and OV90 cells. 2.2 Binding affinity of CLDN6×4-1BB BsAb to 4-1BB

[0369] The binding affinity of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to human 4-1BB was measured by SPR. As shown in Figures 3A to 3D, the binding affinity of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT was 1.640 x 10, respectively. -8 M and 1.573 x 10 -8 K of M D The affinity of the parent anti-4-1BB sdAb antibody conjugated to an IgG1 Fc fragment (4-1BB sdAb-Fc) for 4-1BB was measured in parallel and found to be 4.393 × 10 -9 K of M D This suggests that the affinity of the 4-1BB antibody portion in the BsAb for 4-1BB is comparable to that of the 4-1BB sdAb-Fc for 4-1BB.

[0370] The binding of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to soluble recombinant human 4-1BB was analyzed via ELISA. As shown in Figure 4A, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bound to recombinant human 4-1BB in a concentration-dependent manner with EC50s of 0.129 nM and 0.078 nM, respectively. These EC50s were comparable to the EC50 of 4-1BB sdAb-Fc. Furthermore, the binding of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to HEK293 cells expressing 4-1BB was assessed by FACS. As shown in Figure 4B, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT were able to bind to 4-1BB with EC50s of 0.406 nM and 0.347 nM, respectively, which were comparable to that of 4-1BB sdAb-Fc. Example 3 Functional activity of CLDN6-1×4-1BB BsAb 3.1 Cell line-based functional characterization of CLDN6x4-1BB BsAb

[0371] To test the ability of the CLDN6 x 4-1BB bispecific antibody to activate 4-1BB signaling, the GloResponse™ NFκB-luc2 / 4-1BB Jurkat cell line, stably expressing 4-1BB and an NFκB luciferase reporter, was used as effector cells, and CLDN6-expressing cells (CHO-K1 CLDN6, OVCAR3, or OV90) were used as target cells. RKO colon cancer cells, which do not express CLDN6, were used as a negative control.

[0372] Briefly, GloResponse™ NFκB-luc2 / 4-1BB Jurkat cells (5.0 × 10 per well) were cultured in a 2000-well plate. 4 The cell density was 5.0 × 10 cells in a white 96-well plate. 4The antibody was serially diluted and added to the plate. Luminescence was measured after 6 hours of incubation at 37°C. As shown in Figures 5A-5D, urelumab induced 4-1BB activation regardless of CLDN6 expression, whereas the present 4-1BB sdAb-Fc, despite its ability to bind to 4-1BB, did not have agonist activity under the same experimental setting. Similarly, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT induced NFκB activity in the presence of all CLDN6-expressing target cells, regardless of CLDN6 expression level. In contrast, when RKO cells, which do not express CLDN6, were used as targets, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT resulted in significantly lower 4-1BB activation compared to urelumab, as shown in Figure 5D. 3.2 Activity of CLDN6×4-1BB BsAb in promoting human peripheral blood mononuclear cell (PBMC) immune responses

[0373] Pre-activated human PBMCs were co-cultured with CLDN6-expressing cells or RKO at an effector-to-target (E:T) ratio of 10:1. Different concentrations of antibodies were added to the mixed cultures. After 48 hours, the levels of IL-2 or IFNγ in the culture medium were measured using a homogenous HTRF assay.

[0374] As shown in Figures 6A-6F, when PBMCs were cocultured with CLDN6-expressing target cells, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT stimulated IL-2 and IFNγ production. However, in the presence of RKO, which does not express CLDN6, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT did not stimulate IL-2 or IFNγ production from PBMCs, as shown in Figures 6G and 6H, suggesting that the activity of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT depends on the presence of tumor antigen. In contrast, 4-1BB sdAb-Fc was inactive in this assay. Example 4 Tumor growth inhibition by CLDN6-1×4-1BB BsAb

[0375] CT26 cells, which endogenously express CLDN6, were subcutaneously implanted into BALB / c humanized 4-1BB mice. Tumors grew to an average of 100 mm 3 When tumors reached tumor size (T1), mice were treated intraperitoneally with (a) human IgG1, (b) CLDN6-1x4-1BB NA (2 mg / kg), (c) CLDN6-1x4-1BB WT (2 mg / kg), or (d) a combination of the parental CLDN6-1 antibody and 4-1BB sdAb-Fc (1.8 mg / kg and 0.7 mg / kg). Treatments were administered twice weekly for a total of six doses. Tumor growth was monitored by volumetric measurement. As shown in Figures 7A and 7B, both CLDN6-1x4-1BB WT and CLDN6-1x4-1BB NA exhibited antitumor activity, with CLDN6-1x4-1BB WT achieving even stronger activity with 75% tumor growth inhibition (TGI). Example 5 Hepatotoxicity evaluation of CLDN6×4-1BB BsAb

[0376] A major concern with t4-1BB agonist antibody therapy is the dose-limiting liver toxicity observed in the clinical development of urelumab. The most common adverse events were elevated alanine transaminase (ALT), aspartate aminotransferase (AST), and fatigue. Therefore, the liver toxicity of CLDN6-1x4-1BB WT and CLDN6-1x4-1BB NA was further evaluated.

[0377] Briefly, blood samples were collected from hu4-1BB mice for ALT and AST measurements after treatment with different doses of CLDN6-1x4-1BB WT or CLDN6-1x4-1BB NA twice weekly. As shown in Figures 8A-8B, no significant elevations in ALT and AST were observed, suggesting little risk of liver toxicity commonly induced by other 4-1BB agonist antibodies. Example 6 Generation of MUC16 antibodies Phage ELISA and phage FACS (OVCAR3 cells) based panning

[0378] Panning was performed using the MUC16 antigen (human MUC16 AA13789-14451) against a human naive Fab phage library, yielding 13 unique hits: C25, C73, D1, D20, D30, D41, D46, D57, D79, D100, B76, B195, and B218 (Table 4). [Table 4-1] [Table 4-2] [Table 4-3] Example 7 Binding activity of MUC16 mAb

[0379] The binding activity of the MUC16 mAbs was tested via ELISA using different fragments of the human MUC16 antigen (Figures 9A-9C). Similar to M16-2, but unlike M16-1, all 13 mAbs bind to the AA13789-14197 domain of human MUC16. M16-1 and M16-2 are benchmark antibodies with the amino acid sequences listed in Table A.

[0380] Cross-species testing showed that, except for D46, the other 12 mAbs were able to cross-react with cyno MUC16 (FIG. 10).

[0381] OVCAR3 has the highest level of endogenous MUC16 expression. In cell binding assays, all 13 mAbs (human IgG1 Fc) bind to OVCAR3 cells (Figures 11A-11B). In contrast, none of the 13 mAbs (human IgG1 Fc) bind to MUC16-negative ES-2 cells (Figure 11C).

[0382] Cell-based binding assays were performed on five selected clones: C25, D30, D100, D57, and B218 (Figures 12A-12C). All mAbs showed effective binding to huMUC16 in cells, except for h3A5, which does not bind to HEK293-MUC16 (13810-14507) (Figures 12A-12C). Among the three human MUC16-positive cell lines, OVCAR3 has the highest level of endogenous MUC16 expression. M16-2 and 3A5 are benchmark antibodies with the amino acid sequences listed in Table A.

[0383] In another cell-based binding assay, soluble CA125 (cancer antigen 125, an extracellular shed protein encoded by the MUC16 gene, a serum marker routinely used to monitor patients with ovarian cancer) derived from patient ascites was either present or absent. The binding capacity of five mAbs to OVCAR3 was not significantly reduced by the addition of soluble CA125, indicating that the mAbs bind to epitopes distinct from CA125. However, the binding of the benchmark marker 3A5 to OVCAR3 is significantly reduced by the addition of 5000 U / mL of soluble CA125 (Figures 13A-13D).

[0384] The kinetics of the binding assays showed KD values ​​for D57, B218 and C25, respectively (Figures 14A-14B). Example 8 MUC16×4-1BB bispecific antibody

[0385] Exemplary MUC16x4-1BB bispecific antibodies shown in Table 5 below were designed and generated. [Table 5-1] [Table 5-2]

[0386] In the 4-1BB NFκB reporter assay, the D57-4B and B218-4B BsAbs were able to stimulate 4-1BB signaling in the presence of MUC16-high (+++, OVCAR3), medium (++, SNU216), and low (+, HCC827) expressing cells, with much stronger signals than urelumab (Figures 15A-15C). In contrast, in negative cells (ES-2), urelumab was able to stimulate 4-1BB activation, whereas the BsAbs induced little activation signal (Figure 15D).

[0387] In a separate 4-1BB BsAb activity assay measuring PBMC cytokine release in MUC16 high (+++, OVCAR3), medium (++, SNU216), and low (+, HCC827) expressing cells, D57-4B exhibited the strongest T cell costimulatory activity (inducing human IFNγ and IL-2 cytokine release), while B218-4B exhibited T cell costimulatory activity comparable to that of urelumab (Figures 16A-16F).

[0388] In further 4-1BB BsAb activity assays measuring CD8+ T cell cytokine release in MUC16 high (+++, OVCAR3), medium (++, SNU216), and low (+, HCC827) expressing cells, D57-4B exhibited the strongest CD8+ T cell costimulatory activity (inducing human IFNγ and IL-2 cytokine release), while B218-4B exhibited CD8+ T cell costimulatory activity comparable to that of urelumab (Figures 17A-17F). Example 9 Generation of bispecific and biparatopic ROR1x4-1BB antibodies

[0389] ROR1x4-1BB bispecific and biparatopic antibodies are constructed as shown in Table 6A below. [Table 6A-1] [Table 6A-2] [Table 6A-3] Example 9 Binding activity of bispecific and biparatopic ROR1×4-1BB antibodies 9.1 Chimeric antibodies binding affinity

[0390] FACS binding activity shows that ROR1x4-1BB BsAb binds to ROR1 endogenously expressing tumor cells A549 and 4-1BB overexpressing HEK293 cells in a dose-dependent manner (Figs. 18A-18B). Epitope binning

[0391] In epitope binning of anti-ROR1 mAbs, 3C5 and 8F2 showed non-competing or non-overlapping binding epitopes with the ROR1 antigen as detected by Octet (see Figures 20A-20C).

[0392] Based on the non-overlapping binding epitopes of 3C5 and 8F2 with ROR1, a biparatopic ROR1 x 4-1BB antibody (Table 6A) was designed based on the hypothesis that a biparatopic antigen-targeting antibody could result in more 4-1BB clustering and induce stronger 4-1BB pathway activation. Reporter gene assay

[0393] In this assay, Jurkat cells co-expressing 4-1BB and NFKB-luciferase reporter were used as the reporter cell line, and ROR1-positive or -negative cell lines were used as target cells. Reporter and target cells were co-cultured with gradient-diluted ROR1 × 4-1BB BsAb or anti-4-1BB urelumab for 6 hours, and then 4-1BB NFKB activation signals were read out using One-Glo™ reagent. 4B-3C5 and 4B-8F2 BsAbs demonstrated strong ROR1-dependent 4-1BB activation (Figures 19A-19C).

[0394] Similar reporter gene assays were performed using biparatopic ROR1x4-1BB antibodies, and the results show that the biparatopic ROR1x4-1BB TsAb in the 4-BiR1 format exhibits the best 4-1BB activation (see Figures 21A-21C). 9.2 Humanized Antibodies

[0395] Humanized anti-ROR1 antibodies were used to design monotopic (bispecific) and biparatopic ROR1x4-1BB TsAbs (see Table 6B). [Table 6B-1] [Table 6B-2] [Table 6B-3] Cell-binding affinity and reporter gene assays

[0396] Cell binding of the humanized anti-ROR1 mAb as measured by FACS and ROR1-dependent 4-1BB activation of the humanized ROR1 x 4-1BB BsAb were tested similarly. Compared with the chimeric antibody, the humanized mAb showed better or comparable cell binding (Figures 22A-22B), and the humanized BsAb showed better or comparable ROR1-dependent 4-1BB activation (Figures 22C-22D). However, the 4-1BB VHH control urelumab does not activate 4-1BB in ROR1-positive tumor cell lines at the corresponding concentration range.

[0397] Comparison between the humanized antibodies showed that the biparatopic antibody 4-BiR1 had higher cell binding of ROR1 (Figures 23A-23C) and higher ROR1-dependent 4-1BB activation (Figures 23D-23F) in ROR1-positive tumor cell lines compared with 4B-3C5 and 4B-8F5, whereas 4B-3C5 and 4B-8F5 showed similar cell binding affinity and ROR1-dependent 4-1BB activation. In ROR1-negative tumor cell lines, there was no ROR1-independent 4-1BB activation for all antibodies tested. SPR

[0398] Surface plasmon resonance assay revealed a K of 7.93E-08M D The humanized antibody 4B-h3C5 had a K value of 2.37E-07M. D The value of 4B-h8F5 is 1.21E-08M, and the K D The binding affinity of 4B-hBiR1 to ROR1 was shown in terms of α, β and β values ​​(Figures 24A to 24C). In vivo efficacy

[0399] The in vivo efficacy of monotopic and biparatopic ROR1x4-1BB was evaluated using a syngeneic model (4-1BB humanized mice (Biocytogen)) inoculated with the ROR1-MC38 cell line. All humanized antibodies tested demonstrated significant inhibition of tumor growth (Figures 25A-25B). 9.3 Antibody affinity maturation

[0400] Given the low yield of 4-hBiR1, the potential PTMs in the anti-ROR1 sequence, and the low affinity of h3C5 and h8F2, antibodies were engineered using affinity maturation (Tables 6C and 6D). [Table 6C-1] [Table 6C-2] [Table 6C-3] [Table 6D-1] [Table 6D-2] [Table 6D-3] [Table 6D-4] [Table 6D-5] [Table 6D-6] Reporter gene assay

[0401] Similar reporter gene assays were performed using affinity-matured bispecific or biparatopic ROR1x4-1BB antibodies. Results showed better ROR1-dependent 4-1BB activation for the affinity-matured bispecific antibody compared to the humanized version (Figures 26A-26E). Much better ROR1-dependent 4-1BB activation was observed for the affinity-matured biparatopic antibody compared to the humanized bispecific version and the benchmark ROR1x4-1BB BsAb (clone ID: BA6(NA)x1A10M12) from patent WO2021 / 101346A1 (Figures 27A-27C). Cytokine release assay

[0402] 4B-2773, 4B-27, and 4B-73 were selected as representatives of engineered monotopic and biparatopic ROR1x4-1BB antibodies to compare with 4B-hBiR1 and a benchmark antibody (clone ID: BA6(NA)x1A10M12) in a cytokine release assay. 1 μg / ml of anti-CD3 (OKT3) was coated onto plates, and MDA-MB-231 cell lines were used as target cells. PBMCs and gradient-diluted antibodies were co-cultured for 72 hours. IFN-γ and IL-2 induced by 4-1BB were detected. Results showed that all engineered ROR1x4-1BB antibodies tested released higher cytokines than the benchmark antibody and urelumab (Figures 28A-28B). In vivo efficacy

[0403] The in vivo efficacy of engineered monotopic and biparatopic ROR1x4-1BB antibodies was evaluated using a syngeneic model (4-1BB humanized mice (Biocytogen)) inoculated with the ROR1-MC38 cell line. All humanized antibodies tested demonstrated significant inhibition of tumor growth (Figures 29A-29C).

[0404] The present disclosure is not limited in scope by the specific embodiments described, which are intended as mere illustrations of individual aspects of the disclosure; any functionally equivalent compositions or methods are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure, provided they come within the scope of the appended claims and their equivalents.

[0405] All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0406] The present invention has been described in terms of specific embodiments discovered or proposed by the inventors to comprise preferred modes for carrying out the invention. In light of this disclosure, those skilled in the art will understand that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Furthermore, due to considerations of biological functional equivalence, changes can be made in the protein structure without affecting the type or amount of biological action. All such modifications are intended to be within the scope of the appended claims.

[0407] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 8-1] [Table 8-2] [Table 8-3]

Claims

1. (1) a first antibody portion that specifically binds to a tumor antigen; and (2) a second antibody portion that specifically binds to 4-1BB wherein binding of said first antibody portion to said tumor antigen induces said second antibody portion to activate 4-1BB.

2. The multispecific construct of claim 1 further comprising an Fc domain with maintained or improved effector function.

3. The first antibody portion may be a full-length antibody, a half antibody, a single-chain half antibody, Fab, Fab', F(ab') ２ and scFv.

4. The multispecific construct of any one of claims 1 to 3, wherein the second antibody portion specifically binds to the CRD3 / 4 region of 4-1BB.

5. The second antibody portion may be a full-length antibody, a single-chain half antibody, Fab, Fab', F(ab') ２ 5. The multispecific construct of claim 1 , wherein the construct is selected from the group consisting of: a scFv and a sdAb.

6. The multispecific construct of claim 5 , wherein the second antibody portion is an sdAb.

7. 7. The multispecific construct of claim 6, wherein said sdAb comprises sdAb-CDR1, sdAb-CDR2 and sdAb-CDR3 comprising the amino acid sequences of CDR1, CDR2 and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence set forth in SEQ ID NO:

27.

8. 8. The multispecific construct of claim 7, wherein the CDR1, CDR2 and CDR3 are according to the Kabat numbering scheme.

9. the sdAb (1) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO: 24; (2) sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (3) sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO: 26 7. The multispecific construct of claim 6, comprising:

10. 10. The multispecific construct of claim 1, wherein the second antibody portion comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% sequence identity to SEQ ID NO:

27.

11. 11. A multispecific construct according to any one of claims 1 to 10 which is a bispecific antibody or bispecific binding fragment.

12. 12. The multispecific construct of claim 11, wherein the Fc domain is derived from any one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

13. The multispecific construct of claim 12 , wherein the Fc domain is derived from IgG1.

14. The multispecific construct of claim 13, wherein the Fc domain comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 46-56, 285-286 and 288-289.

15. 15. The multispecific construct of any one of claims 1 to 14, wherein activation of 4-1BB by the second antibody moiety is enhanced by at least 10-fold after binding of the first antibody moiety to the tumor antigen.

16. The multispecific construct of any one of claims 1 to 15, wherein activation of the 4-1BB by the second antibody portion results in an increase in IFNγ levels, IL-2 levels or NFκB signaling.

17. The first antibody portion is 10 －７ M ~ about 10 －１３ 17. The multispecific construct of any one of claims 1 to 16, having a binding affinity of M.

18. 18. The multispecific construct of claim 1, wherein the first antibody portion is fused to the C-terminus of the second antibody portion.

19. 18. The multispecific construct of claim 1, wherein the first antibody portion is fused to the N-terminus of the second antibody portion.

20. 20. The multispecific construct of any one of claims 1 to 19, wherein the first antibody moiety and the second antibody moiety are fused to each other via a linker.

21. 18. The multispecific construct of any one of claims 13 to 17, wherein the first antibody moiety is a Fab' fused to the N-terminus of the IgG Fc domain and the second antibody moiety is an sdAb fused to the C-terminus of the IgG Fc domain.

22. 22. The multispecific construct of claim 21 , wherein the second antibody portion is fused to the IgG Fc domain via a linker.

23. 23. The multispecific construct of any one of claims 1 to 22, further comprising a third antibody portion that specifically binds to a second tumor antigen.

24. 24. The multispecific construct of claim 23, wherein the second tumor antigen has the same epitope as the tumor antigen but a different epitope.

25. The third antibody portion may be a full-length antibody, a half antibody, a single-chain half antibody, Fab, Fab', F(ab') ２ and scFv.

26. 24. The multispecific construct of claim 23, wherein the first antibody moiety is a Fab' fused to the N-terminus of the IgG Fc domain, the second antibody moiety is a sdAb fused to the C-terminus of the IgG Fc domain, and the third antibody moiety is a scFv fused to the N-terminus of the first antibody moiety.

27. 27. The multispecific construct of claim 26, wherein the second antibody portion is fused to the IgG Fc domain via a linker and the third antibody portion is fused to the first antibody portion via a linker.

28. 24. The multispecific construct of claim 23, wherein a first antibody moiety is a Fab' fused to the N-terminus of an IgG Fc domain, said second antibody moiety is an sdAb fused to the C-terminus of said IgG Fc domain, said third antibody moiety is an scFv fused to the N-terminus of a paired IgG Fc domain, and said second antibody moiety is an sdAb fused to the C-terminus of said paired IgG Fc domain, and said paired IgG Fc domain forms a heterodimer with said IgG Fc domain.

29. 29. The multispecific construct of claim 28, wherein the second antibody portion is fused to the IgG Fc domain via a linker and the second antibody portion is fused to the IgG'(CH) via a linker.

30. 30. A pharmaceutical composition comprising a multispecific construct according to any one of claims 1 to 29 and a pharmaceutically acceptable carrier.

31. 30. A nucleic acid encoding a multispecific construct according to any one of claims 1 to 29.

32. A vector comprising the nucleic acid of claim 31.

33. 33. A host cell comprising the nucleic acid of claim 31 or the vector of claim 32.

34. 31. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific construct of any one of claims 1 to 29 or a pharmaceutical composition of claim 30.

35. 35. The method of claim 34, wherein the disease or condition is cancer.

36. 30. Use of a multispecific construct according to any one of claims 1 to 29 in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

37. An antibody or antigen-binding fragment thereof having specificity for human mucin 16 (MUC16) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region including heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region including light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are: (a) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: DSRKYYYDSSGPALWGFDAFDI (SEQ ID NO: 59), LCDR1: RASQSISSYLN (SEQ ID NO: 60), LCDR2: AASSLQS (SEQ ID NO: 61), and LCDR3: QQSYSTLST (SEQ ID NO: 62); (b) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: EPPLSNYGDYATEQYYYGMDV (SEQ ID NO: 67), LCDR1: RASQSISSYLN (SEQ ID NO: 60), LCDR2: AASSLQS (SEQ ID NO: 61), and LCDR3: QQSYSTPLT (SEQ ID NO: 70); (c) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: APMVRGVPPTPYYYYYGMDV (SEQ ID NO: 75), LCDR1: RASQSVSNYLA (SEQ ID NO: 76), LCDR2: DASNRAT (SEQ ID NO: 77), and LCDR3: QQRSNWPS (SEQ ID NO: 78); (d) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: TPELLWFGELGGAYYFDY (SEQ ID NO: 83), LCDR1: RASESISSWLA (SEQ ID NO: 84), LCDR2: KASTLEN (SEQ ID NO: 85), and LCDR3: QQYRSHWSST (SEQ ID NO: 86); (e) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: ANFNIYYYYYGMDV (SEQ ID NO: 91), LCDR1: RSSQSLLHSNGYNYLD (SEQ ID NO: 92), LCDR2: LGSNRAS (SEQ ID NO: 93), and LCDR3: MQGTHWPRT (SEQ ID NO: 94); (f) HCDR1: SYEMN (SEQ ID NO: 97), HCDR2: RIKSKTDGGTTDYAAPV (SEQ ID NO: 98), HCDR3: DLAAVAGLFDY (SEQ ID NO: 99), LCDR1: QASQDISNYLN (SEQ ID NO: 100), LCDR2: DASNLET (SEQ ID NO: 101), and LCDR3: QQSYSTPWK (SEQ ID NO: 102); (g) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: RIIPIFGIANYAQKFQG (SEQ ID NO: 106), HCDR3: TGDYDILTGSYYYGMDV (SEQ ID NO: 107), LCDR1: RASQGIRNDLG (SEQ ID NO: 108), LCDR2: AASSLQS (SEQ ID NO: 61), and LCDR3: LQDYNYPFT (SEQ ID NO: 120); (h) HCDR1: DYYLS (SEQ ID NO: 123), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: GGPHYDFFWSGYTPGQHGGAFDI (SEQ ID NO: 125), LCDR1: RASQSVSSSYLA (SEQ ID NO: 126), LCDR2: GASSRAT (SEQ ID NO: 127), and LCDR3: QQRSNWRNT (SEQ ID NO: 128); (i) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: DSGSSITMVRGGDYYYMDV (SEQ ID NO: 133), LCDR1: RASQSVSSYLA (SEQ ID NO: 134), LCDR2: DASNRAT (SEQ ID NO: 77), and LCDR3: QQRSNWPPT (SEQ ID NO: 136); (j) HCDR1: YHAIS (SEQ ID NO: 139), HCDR2: GIIPILGTANYAQKFQG (SEQ ID NO: 140), HCDR3: GTTAARYYYYYYYMDV (SEQ ID NO: 141), LCDR1: QASQDISNYLN (SEQ ID NO: 100), LCDR2: DASNLET (SEQ ID NO: 101), and LCDR3: QQYDNLPLT (SEQ ID NO: 144); (k) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: SITDYYDSSGYYFRPHFNTGYYYGMDV (SEQ ID NO: 149), LCDR1: RASQGINNYLA (SEQ ID NO: 150), LCDR2: AASTLQS (SEQ ID NO: 151), and LCDR3: QQYDTFSET (SEQ ID NO: 152); (l) HCDR1: SYAIS (SEQ ID NO: 57), HCDR2: GIIPIFGTANYAQKFQG (SEQ ID NO: 58), HCDR3: GGPHYDFFWSGYTPGQHGGAFDI (SEQ ID NO: 125), LCDR1: RASQSISGWLA (SEQ ID NO: 158), LCDR2: RTSYLES (SEQ ID NO: 159), and LCDR3: QHYDTFSRA (SEQ ID NO: 160), or (m) HCDR1: YHAIS (SEQ ID NO: 139), HCDR2: SISSGGNTYYPDTVKGR (SEQ ID NO: 38), HCDR3: EGPDYGDYSWSMDYYYGMDV (SEQ ID NO: 165), LCDR1: RASQSVNSRYLA (SEQ ID NO: 166), LCDR2: GASTRAT (SEQ ID NO: 167), and LCDR3: QQYGTFSIT (SEQ ID NO: 168). An antibody or antigen-binding fragment thereof selected from the group consisting of:

38. 38. The antibody or antigen-binding fragment thereof according to claim 37, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161 and 169, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161 and 169.

39. 38. The antibody or antigen-binding fragment thereof of claim 37, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162 and 170, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162 and 170.

40. (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 64; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 71 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72; (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80; (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88; (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96; (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 103 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 104; (g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 121 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 122; (h) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 129 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130; (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 138; (j) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 145 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 146; (k) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 153 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 154; (l) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 161 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 162; or (m) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 169 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 170 40. The antibody or antigen-binding fragment thereof of any one of claims 37 to 39, comprising:

41. 40. The antibody or antigen-binding fragment thereof of any one of claims 37 to 39, wherein the antibody is a chimeric antibody or a humanized antibody.

42. 40. The antibody or antigen-binding fragment thereof of any one of claims 37 to 39, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

43. A bifunctional molecule comprising a first antibody portion having specificity for human MUC16 protein and a second antibody portion having specificity for a second protein, wherein the first antigen-binding portion comprises an antibody or antigen-binding fragment thereof described in any one of claims 37 to 42.

44. The bifunctional molecule of claim 43, wherein the second protein is 4-1BB.

45. 45. The bifunctional molecule of claim 43 or 44, wherein the first antibody portion is a full-length antibody.

46. 46. ​​The bifunctional molecule of any one of claims 43 to 45, wherein the second antibody portion is an sdAb.

47. 47. The bifunctional molecule of any one of claims 43 to 46, wherein the second antibody portion is fused to the C-terminus of the first antibody portion.

48. 48. The bifunctional molecule of any one of claims 43 to 47, wherein the first antibody portion and the second antibody portion are fused to each other via a linker.

49. 49. The bifunctional molecule of any one of claims 44 to 48, wherein the second antibody portion comprises an HCDR1 of SNCMG (SEQ ID NO: 24), an HCDR2 of VICTGGGSPSYADSVKG (SEQ ID NO: 25), and an HCDR3 of DLLRAGTPLSSYEFNY (SEQ ID NO: 26).

50. 50. The bifunctional molecule of claim 49, wherein the second antibody portion comprises the amino acid sequence of SEQ ID NO:27 or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

27.

51. A composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 37 to 42 or a bifunctional molecule described in any one of claims 43 to 50 and a pharmaceutically acceptable carrier.

52. An isolated cell comprising one or more polynucleotides encoding the antibody or antigen-binding fragment thereof described in any one of claims 37 to 42 or the bifunctional molecule described in any one of claims 43 to 50.

53. A polynucleotide encoding one or more chains of the antibody or antigen-binding fragment thereof of any one of claims 37 to 42 or the bifunctional molecule of any one of claims 43 to 50.

54. 52. A method of treating cancer in a patient in need thereof, comprising administering to the patient an antibody or antigen-binding fragment thereof described in any one of claims 37 to 42 or a bifunctional molecule described in any one of claims 43 to 50.

55. 55. The method of claim 54, wherein the cancer is selected from the group consisting of ovarian cancer, prostate cancer, cancer of the urinary tract, pancreatic cancer, lung cancer, breast cancer, bladder cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, and thyroid cancer.

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