Multi-specific constructs and uses thereof

Abstract

Provided herein are bispecific and multispecific antibodies comprising an agonist anti--4-1BB single domain antibody capable of effecting effector function mediated tumor suppression. In addition, these agonist antibodies bind to 4-1BB in the absence of cells expressing tumor antigens, but are not able to activate 4-1BB signaling. However, in the presence of tumor antigen-expressing cells, these antibodies can trigger tumor antigen-dependent 4-1BB signaling, resulting in a potent immune response to tumor cells that express tumor antigens.

Description

Technical Field

[0001] This application relates to multispecific molecules, e.g., anti-tumor antigen / anti-4-1BB bispecific antibodies and their uses, which uses include treating a disease or condition. Background Art

[0002] 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule expressed after activation of immune cells, including innate and adaptive immune cells. 4-1BB plays an important role in regulating the activity of immune cells. Agonists of 4-1BB enhance immune cell proliferation, survival, cytokine secretion, and the cytolytic activity of CD8 T cells. Many studies have shown that activation of 4-1BB enhances the immune response to eliminate tumors in mice, indicating that 4-1BB is 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

[0004] Prior to the present disclosure, the conventional wisdom was that agonistic anti-4-1BB antibodies should not possess ADCC function. This was at least because FcγR binding that induces ADCC could deplete 4-1BB+ cells, thereby weakening the agonistic effect of the antibody and causing toxicity (e.g., hepatotoxicity) due to FcγR involvement. For example, the leading anti-4-1BB antibody drug candidate, urelumab, includes an IgG4 Fc with no effector function or limited effector function.

[0005] In an unexpected finding, the inventors of the present invention demonstrated that when a proprietary anti-4-1BB single-domain antibody (represented by SEQ ID NO:27) is used in bispecific form together with a second antibody moiety targeting a tumor-associated antigen (TAA), the effector function using WTIgG1 actually enhances 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 included wild-type (WT) IgG1 Fc and the other had a silent mutation (N297A or NA). As Figure 7B shown, although both the WT (with effector function) and NA (without effector function) versions exhibited good anti-tumor activity, the WT version achieved significantly higher and more durable tumor inhibition.

[0006] Without being bound by a particular theory, this unique effector function-aiding property of the bispecific antibody may be attributed to the activity or form (e.g., single-domain) of the anti-4-1BB antibody of the present invention.

[0007] Thus, according to one embodiment of the present disclosure, there is provided a multispecific construct, such as 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 activation activity of the second antibody portion is dependent 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 functions.

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

[0010] In some embodiments, the second antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the second antibody portion is selected from full-length antibodies, half-antibodies, single-chain half-antibodies, Fab, Fab', F(ab') 2 , scFv and sdAb. In some embodiments, the second antibody portion is 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 monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27. In some embodiments, CDR1, CDR2 and CDR3 are according to the Kabat numbering scheme. In some embodiments, the sdAb comprises: (1) sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24; (2) sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25; and (3) sdAb-CDR3, which comprises 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 that has at least about 80% sequence identity with SEQ ID NO:27.

[0011] In some embodiments, the multispecific construct is a bispecific antibody or a 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 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, after the first antibody portion binds to the tumor antigen, the activation of 4 - 1BB by the second antibody portion is enhanced by at least 10 - fold. In some embodiments, the activation of 4 - 1BB by the second antibody portion results in an increase in IFNγ level, IL - 2 level, or NFκB signal transduction. In some embodiments, the first antibody portion has an approximate binding affinity of about 10 -7 M to about 10 -13 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 portion is a Fab' that is fused to the N - terminus of an IgG Fc domain, and the second antibody portion is an sdAb that is fused to the C - terminus of the IgG Fc domain. In some embodiments, the second antibody portion 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. In some embodiments, the second tumor antigen is the same as the tumor antigen but has a different epitope.

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

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

[0019] In some embodiments, the first antibody portion is a Fab' that is fused to the N-terminus of an IgG Fc domain, and the second antibody portion is a sdAb that is fused to the C-terminus of the IgG Fc domain, the third antibody portion is a scFv that is fused to the N-terminus of a paired IgG Fc domain, and the second antibody portion is a sdAb that is 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 portion is fused to the IgG Fc domain via a linker, and the second antibody portion is fused to the paired IgG Fc domain via a linker. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:23.

[0020] In some embodiments, the third antibody portion is fused to the N-terminus of the paired IgG Fc domain via a tether. The tether comprises, from the N-terminus to the C-terminus, a hinge variant of the short linker "AA" and / or "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 that has at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:285, and the paired Fc domain comprises the amino acid sequence of SEQ ID NO:288 or a variant thereof that has at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown 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 that has at least about 80% sequence identity (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more than 99%) with the sequence shown in SEQ ID NO: 286, and the paired Fc domain comprises the amino acid sequence of SEQ ID NO: 289 or a variant thereof that has at least about 80% sequence identity (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more than 99%) with the sequence shown in SEQ ID NO: 289.

[0023] In another aspect, the present disclosure provides a pharmaceutical composition comprising a multispecific construct as described herein and a pharmaceutically acceptable carrier.

[0024] In another aspect, the present disclosure provides a nucleic acid encoding a multispecific construct as described herein.

[0025] In another aspect, the present disclosure provides a vector comprising a nucleic acid as described herein.

[0026] In another aspect, the present disclosure provides a host cell comprising a nucleic acid as described herein or a vector as described herein.

[0027] In another aspect, the present disclosure provides 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 as described herein or a pharmaceutical composition as described herein. In some embodiments, the disease or condition is cancer.

[0028] In another aspect, the present disclosure provides 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 an antigen-binding fragment thereof that is specific for the human mucin 16 (MUC16) protein, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3, and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are selected from:

[0030] (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),

[0031] (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),

[0032] (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),

[0033] (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),

[0034] (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),

[0035] (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),

[0036] (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),

[0037] (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),

[0038] (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),

[0039] (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),

[0040] (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),

[0041] (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

[0042] (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).

[0043] 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 SEQ ID NO: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 SEQ ID NO:63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161, and 169.

[0044] 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 SEQ ID NO: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 SEQ ID NO:64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162, and 170.

[0045] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise

[0046] (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;

[0047] (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;

[0048] (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;

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (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;

[0056] (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;

[0057] (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

[0058] (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.

[0059] In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0061] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a first antibody portion and a second antibody portion, the first antibody portion being specific for the human MUC16 protein and the second antibody portion being specific for a second protein, wherein the first antigen-binding portion comprises the antibodies or antigen-binding fragments thereof provided herein.

[0062] 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 antibody portion and the second antibody portion are fused to each other via a linker. In some embodiments, the second antibody portion comprises the HCDR1 of SNCMG (SEQ ID NO:24), the HCDR2 of VICTGGGSPSYADSVKG (SEQ ID NO:25), and the 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 with the amino acid sequence of SEQ ID NO:27.

[0063] In one aspect, the present disclosure provides a composition comprising the antibodies or antigen-binding fragments thereof provided herein or the bifunctional molecules provided herein and a pharmaceutically acceptable carrier.

[0064] In one aspect, the present disclosure provides an isolated cell comprising one or more polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein or the bifunctional molecules provided herein.

[0065] In one aspect, the present disclosure provides a polynucleotide encoding one or more chains of the antibodies or antigen-binding fragments thereof provided herein or the bifunctional molecules provided herein.

[0066] 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 the antibodies or antigen-binding fragments thereof provided herein or the bifunctional molecules provided herein.

[0067] In some embodiments, the cancer is selected from ovarian cancer, prostate cancer, urinary tract cancer, 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.

[0068] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figures 1A - 1C Illustrate the binding affinity of the CLDN6 x 4-1BB bispecific antibody (BsAb) disclosed herein to CLDN6.

[0070] Figures 2A - 2C Illustrate the binding affinity of the CLDN6 x 4-1BB BsAb disclosed herein to cells expressing CLDN6.

[0071] Figures 3A - 3D Illustrate the binding affinity of the CLDN6 x 4-1BB BsAb disclosed herein to 4-1BB.

[0072] Figures 4A - 4B Illustrate the binding of the CLDN6 x 4-1BB BsAb disclosed herein to soluble 4-1BB and cells expressing 4-1BB.

[0073] Figures 5A - 5D Illustrate the CLDN6-dependent 4-1BB activation of the CLDN6 x 4-1BB BsAb disclosed herein.

[0074] Figures 6A - 6H Illustrate the activation of PBMC by the CLDN6 x 4-1BB BsAb disclosed herein.

[0075] Figures 7A - 7B Illustrate tumor suppression in hu4-1BB mice after treatment with the CLDN6 x 4-1BB BsAb disclosed herein.

[0076] Figures 8A - 8B Illustrate liver function after treatment with the CLDN6 X 4-1BB BsAb of the present application.

[0077] Figures 9A - 9C Illustrate the binding activity of 13 selected monoclonal antibodies to human MUC16 protein measured by ELISA.

[0078] Figure 10 Illustrate the binding activity of 13 selected monoclonal antibodies to cynomolgus monkey MUC16 protein measured by ELISA.

[0079] Figures 11A - 11C Illustrate the cell-based binding of 13 selected monoclonal antibodies to human MUC16-positive and -negative cells.

[0080] Figures 12A - 12C Describe the cell-based binding activities of 13 selected monoclonal antibodies to human MUC16 in OVCAR3, SNU216, and MUC16-overexpressing HEK293 cells, respectively.

[0081] Figures 13A - 13D Describe the cell-based binding activities of 5 selected monoclonal antibodies to human MUC16 in the OVCAR3 cell line in the presence or absence of CA125.

[0082] Figures 14A - 14B Describe the kinetics of the binding activities of 3 selected monoclonal antibodies to human MUC16.

[0083] Figures 15A - 15E Describe MUC16-dependent 4-1BB activation in a 4-1BB NFκB reporter gene assay in cell lines with different MUC16 expression levels.

[0084] Figures 16A - 16F Describe MUC16-dependent 4-1BB activation in cell lines with different MUC16 expression levels, which induces T cell co-stimulatory activity in PBMCs to release human IFNγ and IL-2 cytokines.

[0085] Figures 17A - 17F Describe MUC16-dependent 4-1BB activation in cell lines with different MUC16 expression levels, which induces T cell co-stimulatory activity in CD8+ T cells to release human IFNγ and IL-2 cytokines.

[0086] Figures 18A - 18B Show the cell binding activity of the bispecific anti-ROR1 / anti-4-1BB antibody measured by FACS.

[0087] Figures 19A - 19C Show ROR1-dependent 4-1BB activation of the bispecific anti-ROR1 / anti-4-1BB antibody in a reporter gene assay.

[0088] Figures 20A - 20C Show the epitope grouping of the anti-ROR1 antibody. A. Epitope grouping workflow; B-C. Epitope grouping results of 3C5 and 8F5, respectively.

[0089] Figures 21A - 21C Show ROR1-dependent 4-1BB activation of the bi-paratopic anti-ROR1 / anti-4-1BB antibody in a reporter gene assay.

[0090] Figures 22A - 22DShows the cellular binding activity of anti-ROR1 mAb to ROR1 measured by FACS before and after humanization, and (C-D) ROR1-dependent 4-1BB activation in a reporter gene assay of bispecific anti-ROR1 / anti-4-1BB antibody before and after humanization.

[0091] Figures 23A - 23F Shows the cellular binding activity of bispecific and bisepitopic anti-ROR1 / anti-4-1BB antibodies measured by FACS using ROR1-positive and -negative tumor cell lines; and (D-F) ROR1-dependent 4-1BB activation of bispecific and bisepitopic anti-ROR1 / anti-4-1BB antibodies in a reporter gene assay.

[0092] Figures 24A - 24C Shows the SPR results of humanized bispecific and bisepitopic anti-ROR1 / anti-4-1BB antibodies.

[0093] Figures 25A - 25B Shows the in vivo efficacy study of humanized bispecific and bisepitopic anti-ROR1 / anti-4-1BB antibodies. (A) Study design; (B) Antitumor efficacy in 4-1BB knock-in mice.

[0094] Figures 26A - 26E Shows ROR1-dependent 4-1BB activation of affinity-matured bispecific anti-ROR1 / anti-4-1BB antibody in a reporter gene assay.

[0095] Figures 27A - 27C Shows ROR1-dependent 4-1BB activation of affinity-matured bisepitopic anti-ROR1 / anti-4-1BB antibody in a reporter gene assay.

[0096] Figures 28A - 28B Shows ROR1-dependent 4-1BB-induced cytokine release of affinity-matured bispecific and bisepitopic anti-ROR1 / anti-4-1BB antibodies in a reporter gene assay.

[0097] Figures 29A - 29C Shows the in vivo efficacy study of affinity-matured bisepitopic anti-ROR1 / anti-4-1BB antibody. (A) Study design; (B) In vivo antitumor efficacy in 4-1BB knock-in C57 mice inoculated with MC38 cells expressing hROR1; (C) Summary of tumor growth inhibition (TGI) for each treatment group. Detailed Description

[0098] Definition

[0099] 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, provided that they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0100] 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 may 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, and heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by 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 the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified into their respective classes based on the amino acid sequence of their heavy-chain constant regions. The five main 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. Several of the main antibody classes are divided into subclasses, such as 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).

[0101] The term "half-antibody" as used herein refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. It will be readily understood by those skilled in the art that a half-antibody can encompass fragments thereof and can also have an antigen-binding domain consisting of a single variable domain, such as those derived from camelids. In certain embodiments, the half-antibody comprises Fab′ and Fc domains. The Fab' is fused to the N-terminus of the Fc domain.

[0102] As used herein, the term "single-chain half antibody" refers to a single-chain polypeptide comprising a VL domain, an optionally present CL domain, a linker, a VH domain, an optionally present CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the domains are in the following positions relative to each other in the direction from the N-terminus to the C-terminus: VL-linker-VH-hinge-CH2-CH3, VL-linker-VH-partial hinge-CH2-CH3, VL-linker-VH-hinge variant-CH2-CH3, or VL-CL-linker-VH-CH1-hinge-CH2-CH3.

[0103] 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) can confer antigen binding. In other words, a single variable domain does not need to interact with another variable domain in order to recognize a target antigen. Examples of single-domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks) and those derived recombinantly from human and murine antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO 2005 / 035572; WO 03 / 035694; FebsLett (1994) 339:285-290; WO00 / 29004; WO 02 / 051870). When an sdAb contains only a heavy chain, it can be used interchangeably with "VHH" or "single heavy-chain variable domain antibody" or "nanobody".

[0104] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, which includes, for example, diabodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain Fv (scFv), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising one or more CDRs, single-domain antibody (e.g., camelized single-domain antibody), nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody that are grafted into framework regions from one or more different human antibodies.

[0105] "Fab" with respect to an antibody refers to the monovalent antigen-binding fragment of the antibody, which is composed of one light chain (variable and constant regions) bound by a disulfide bond to the variable region and the first constant region of one heavy chain. Fab can be obtained by digesting the antibody with papain at residues near the N-terminus of the disulfide bond between the hinge regions of the heavy chains.

[0106] "Fab'" refers to a Fab fragment that includes a portion of the hinge region and can be obtained by digesting the antibody with pepsin at residues near the C-terminus of the disulfide bond between the hinge regions of the heavy chains, and thus it differs from Fab in terms of a small number of residues in the hinge region, including one or more cysteines.

[0107] "F(ab) 2 " refers to a dimer of Fab′ that comprises two light chains and a portion of two heavy chains.

[0108] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that comprises VH and VL antibody domains joined 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 scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0109] As used herein, the term "CDR" or "complementary determining region" means the non - contiguous antigen - binding sites found within the variable regions of heavy - and light - chain polypeptides. These specific regions have been described in Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); Kabat et al., U.S. 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 M.P. et al., Dev. Comp. Immunol., 27:55 - 77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657 - 670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared to each other. However, the use of any of these definitions to refer to the CDRs of an antibody or a graft - antibody or its variants is intended to fall within the scope of the term as defined and used herein. The amino acid residues covering the CDRs defined by each of the above - cited references are listed 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 this application and may be included in one or more of the claims herein.

[0110] Table 1: CDR Definitions

[0111]

[0112] 1Residue numbering follows the nomenclature of Kabat et al., as described above.

[0113] 2 Residue numbering follows the nomenclature of Chothia et al., as described above.

[0114] 3 Residue numbering follows the nomenclature of MacCallum et al., as described above.

[0115] 4 Residue numbering follows the nomenclature of Lefranc et al., as described above.

[0116] 5 Residue numbering follows the nomenclature of Honeger and Plückthun, as described above.

[0117] The expression "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system for the heavy chain variable domain or light chain variable domain used in the compilation of antibodies in Kabat et al. (as described above). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to shortening or insertion in the FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include an amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues of a given antibody can be determined by aligning the antibody sequence with the homologous region of the "standard" Kabat numbering sequence.

[0118] Unless otherwise specified herein, the numbering of residues in the immunoglobulin heavy chain is the numbering of the EU index in Kabat et al. as described above, with minor modifications. Briefly, we added 5 more residues in the hypervariable loop before heavy chain CDR1. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0119] "Framework" or "FR" residues are those variable domain residues other than the CDR residues as defined herein.

[0120] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient hypervariable regions (HVRs) are replaced by residues from the hypervariable regions (donor antibody) of a non-human species (such as mouse, rat, rabbit, or non-human primate) having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Additionally, humanized antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies will essentially comprise all of at least one and usually two variable domains, in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are those of human immunoglobulin sequences. Optionally, the humanized antibody will also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. For more 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).

[0121] "Human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or prepared using any technique for preparing human antibodies as disclosed herein. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. A variety of techniques known in the art, including phage display libraries, can be used to generate human antibodies. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also useful for preparing human monoclonal antibodies are 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). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been inactivated, such as immunized xenomice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE TM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding the production of human antibodies by human B cell hybridoma technology.

[0122] For the polypeptide and antibody sequences identified herein, "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, after aligning the sequences to account for any conservative substitutions as part of sequence identity. The alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes herein, the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004) is used to generate the % amino acid sequence identity values.

[0123] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in two DNA molecules is occupied by adenine, then the two 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 times 100. For example, if 6 of 10 positions in two sequences match or are homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, the comparison is made when the two sequences are aligned to give maximal homology.

[0124] The term "constant domain" refers to the part of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another part of the immunoglobulin (the variable domain that contains the antigen-binding site). The constant domain contains the C H 1, C H 2 and C H 3 domains (collectively referred to as C H ) and the CHL (or C L ) domain of the light chain.

[0125] The "light chain" of an antibody (immunoglobulin) from any mammalian species can be divided into one of two distinct types based on the amino acid sequence of its constant domain, called kappa ("κ") and lambda ("λ").

[0126] The "CH1 domain" (also referred to as "C1" of the "H1" domain) typically extends from approximately amino acid 118 to approximately amino acid 215 (EU numbering system).

[0127] 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)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the inter - heavy - chain S - S bond in the same positions.

[0128] The "CH2 domain" (also referred to as the "C2" domain) of the human IgG Fc region typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is unique in that it does not pair tightly with another domain. Instead, two N - linked branched glycans are inserted between the two CH2 domains in a complete native IgG molecule. It is hypothesized that the sugars can provide an alternative for domain - domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161 - 206(1985).

[0129] The "CH3 domain" (also referred to as the "C3" domain) contains a segment of residues C - terminal to the CH2 domain in the Fc region (i.e., from approximately amino acid residue 341 of the antibody sequence to the C - terminus, typically at amino acid residue 446 or 447 in IgG).

[0130] 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 typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C - terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of an antibody or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of a full - length antibody can contain a population of antibodies with all K447 residues removed, a population of antibodies with no K447 residues removed, and a population of antibodies that is a mixture of antibodies with and without the K447 residue. Suitable native - sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0131] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. In addition, preferred FcRs are FcRs that bind IgG antibodies (gamma receptors) and include the FcγRI, FcγRII, and FcγRIII subclasses of receptors (including allelic variants and alternatively spliced forms of these receptors). The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. 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 that will be identified in the future) are encompassed by the term "FcR" as used herein.

[0132] As used herein, the term "epitope" refers to a specific atom or group of amino acids on an antigen that binds to an antibody or antibody portion. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0133] As used herein, a first antibody or fragment thereof "competes" with a second antibody or fragment thereof for binding to a target antigen when, in the presence of the first antibody or fragment thereof at equimolar concentration, the first antibody or fragment thereof inhibits the binding of the second antibody or fragment thereof to the target antigen by at least about 50% (such as any one of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa. High-throughput methods for "binning" antibodies based on cross-competition of antibodies are described in PCT Publication No. WO 03 / 48731.

[0134] As used herein, the terms "specifically bind", "specifically recognize", and "is specific for" refer to a measurable and reproducible interaction, such as the binding between a target and an antibody or antibody portion, which can determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody portion that specifically recognizes a target (which can be an epitope) is an antibody or antibody portion that has a higher affinity for binding to that target, a stronger avidity, binds more readily and / or has a longer duration of binding compared to its binding to other targets. In some embodiments, the degree of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (K -5 M, ≤ 10 -6 M, ≤ 10 - 7 M, ≤ 10 -8 M, ≤ 10 -9 M, ≤ 10 -10 M, ≤ 10 -11 M or ≤ 10 -12 M of dissociation constant (K D ). In some embodiments, the antibody specifically binds an epitope on a protein, and the epitope 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 blotting, ELISA-assay, RIA-assay, ECL-assay, IRMA-assay, EIA-assay, BIACORE TM -assay, and peptide scanning.

[0135] An "isolated" antibody (or construct) is an antibody that has been identified, separated, and / or recovered from the components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is not associated with all of the other components from its production environment.

[0136] An "isolated" nucleic acid molecule encoding the coding constructs, antibodies or antigen-binding fragments thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it was originally associated in its production environment. Preferably, the isolated nucleic acid is not associated with all of the components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are in a form different from that found in nature. Thus, an isolated nucleic acid molecule is different from the nucleic acid encoding the polypeptides and antibodies described herein that occurs naturally in a cell. Isolated nucleic acids include nucleic acid molecules contained in a cell that originally contained the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0137] As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a DNA of a presequence or secretory leader that is expressed as a preprotein involved in polypeptide secretion is operably linked to the DNA 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 its position facilitates 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. However, enhancers do not have to be contiguous. The ligation is accomplished by ligation at appropriate restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0138] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of the host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0139] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cells include the original subject cells and their progeny.

[0140] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells that have had exogenous nucleic acid introduced into them, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the original transformed cell and its derived progeny, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0141] As used herein, "treatment" or "treating" refers to a method for obtaining a beneficial or desired result, including a clinical result. For the 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 caused by a disease, reducing the severity of a disease, stabilizing a disease (e.g., preventing or delaying the worsening of a disease), preventing or delaying the spread of a disease (e.g., metastasis), preventing or delaying the recurrence of a disease, delaying or slowing the progression of a disease, improving the disease state, providing remission (partial or complete) of a disease, reducing the dosage of one or more other drugs required to treat a disease, delaying the progression of a disease, improving or enhancing the quality of life, increasing body weight, and / or prolonging survival. "Treatment" also includes alleviating the pathological consequences of cancer (e.g., tumor volume). The methods of this application cover any one or more of these aspects of treatment.

[0142] In the context of cancer, the term "treatment" includes any one or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and improving one or more symptoms associated with the disease.

[0143] The term "inhibition" or "inhibit" refers to a decrease or cessation of any phenotypic characteristic, or a decrease in the incidence, degree, or likelihood of that characteristic. "Reduce" or "inhibit" means a decrease, reduction, or blockade of activity, function, and / or amount as compared to a reference. In certain embodiments, "reduce" or "inhibit" means an overall decrease of 20% or more. In another embodiment, "reduce" or "inhibit" means an overall decrease of 50% or more. In yet another embodiment, "reduce" or "inhibit" means an overall decrease of 75%, 85%, 90%, 95% or more.

[0144] The term "agonizing" refers to an increase or enhancement of any phenotypic trait or an increase or enhancement in the incidence, degree, or likelihood of that trait. "Increase" or "enhancement" is a decrease, reduction, or inhibition of activity, function, and / or amount compared to a reference. In certain embodiments, "increase" or "enhancement" means an overall increase of at least about 1-fold or more in, for example, activity, function, and / or amount. In another embodiment, "increase" or "enhancement" means an overall increase of at least about 5-fold in, for example, activity, function, and / or amount compared to a reference. In yet another embodiment, "increase" or "enhancement" means an overall increase of 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 in, for example, activity, function, and / or amount compared to a reference, including any range between these values, or an increase of more than about 100-fold compared to a reference.

[0145] As used herein, "reference" means any sample, standard, or level used for comparison purposes. A reference can be obtained from healthy and / or non-diseased samples. In some instances, a reference can be obtained from an untreated sample. In some instances, a reference is obtained from an undiseased or untreated sample of an individual. In some instances, a reference is obtained from one or more healthy individuals who are not a particular individual or patient.

[0146] As used herein, "delaying the development of a disease" means postponing, hindering, slowing, retarding, stabilizing, inhibiting, and / or deferring the development of a disease (such as cancer). The length of such delay can vary depending on the history of the disease and / or the individual receiving treatment. It will be apparent to those skilled in the art that a sufficient or significant delay can actually encompass prevention, as the individual does not develop the disease. For example, advanced cancer (such as the development of metastases) can be delayed.

[0147] As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in an individual who may be predisposed to that disease but has not been diagnosed with the disease.

[0148] As used herein, "inhibiting" a function or activity means reducing that function or activity when compared to a condition that is otherwise identical except for the condition or parameter of interest or when compared to another condition. For example, an antibody that inhibits tumor growth will reduce the growth rate of the tumor compared to the growth rate of the tumor in the absence of the antibody.

[0149] As used herein, "based on" includes assessing, determining, or measuring a characteristic of an individual as described herein (and preferably selecting an individual suitable for receiving treatment). When the abnormal state of Claudin-18 is "used as a basis" for selecting, assessing, measuring, or determining a treatment method as described herein, the Claudin-6 abnormality and the obtained state (including the presence, absence, expression level, activity level, and / or phosphorylation level of Claudin-6) determined before and / or during treatment are used by a clinician to evaluate any of the following: (a) the possible or probable suitability of the individual for initially receiving treatment; (b) the possible or probable unsuitability of the individual for initially receiving treatment; (c) responsiveness to treatment; (d) the possible or probable suitability of the individual for continuing to receive treatment; (e) the possible or probable unsuitability of the individual for continuing to receive treatment; (f) adjusting the dose; or (g) predicting the likelihood of clinical benefit.

[0150] The terms "subject", "individual", and "patient" are used interchangeably herein and 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.

[0151] It should be understood that the embodiments of the present application described herein include embodiments of "consisting of" and / or "consisting essentially of".

[0152] As used herein, the reference to "about" a value or parameter includes (and describes) variations to that value or parameter itself. For example, the description of "about X" includes the description of "X".

[0153] As used herein, the reference to "not" a value or parameter generally means and describes "except for" that value or parameter. For example, a method not for treating cancer type X means that the method is for treating other types of cancer other than type X.

[0154] The term "about X-Y" as used herein has the same meaning as "about X to about Y".

[0155] As used herein and in the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents.

[0156] Antibody binding affinity

[0157] The binding specificity of the antibody portion of the multispecific construct described herein can be determined experimentally by methods known in the art. Such methods include but are not limited to Western blotting, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -testing and peptide scanning.

[0158] In some embodiments, the binding affinity is measured by the dissociation constant K D The dissociation constant can be determined by 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 from ForteBio), mesoscale discovery assay (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.

[0159] In some embodiments, the K of the binding between the antibody portion and 4-1BB D is from about 10 -7 M to about 10 -12 M, about 10 - 7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -7 M to about 10 -11 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M, or about 10 -7 M to about 10 -9 M. In some embodiments, the K of the binding between the antibody portion and 4-1BB D is stronger than about 10 -7 M, 10 -8M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 or 10 -13 Any one of M. In some embodiments, 4-1BB is a human antigen.

[0160] In some embodiments, the K of the binding between the antibody portion and 4-1BB on is about 10 3 M -1 s -1 to about 10 8 M -1 s -1 , about 10 3 M -1 s -1 to about 10 4 M -1 s -1 , about 10 4 M -1 s -1 to about 10 5 M -1 s -1 , about 10 5 M -1 s -1 to about 10 6 M -1 s -1 , about 10 6 M -1 s -1 to about 10 7 M -1 s -1 or about 10 7 M -1 s -1 to about 10 8 M -1 s -1 。In some embodiments, the K of the binding between the antibody portion and 4-1BB on is about 10 3 M -1 s -1 to about 10 5 M -1 s -1 、about 10 4 M -1 s -1 to about 10 6 M -1 s -1 、about 10 5 M -1 s -1 to about 10 7 M-1 s -1 , about 10 6 M -1 s -1 to about 10 8 M -1 s -1 , about 10 4 M -1 s -1 to about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 to about 10 8 M -1 s -1 In some embodiments, the K of the binding between the antibody portion and 4-1BB is on No more than 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.

[0161] In some embodiments, the K of the binding between the antibody portion and 4-1BB is off About 1s -1 to about 10 -6 s -1 , about 1s -1 to about 10 -2 s -1 , about 10 -2 s -1 to about 10 -3 s -1 , about 10 -3 s -1 to about 10 -4 s -1 , about 10 -4 s -1 to about 10 -5 s -1 , about 10 -5 s-1 to about 10 -6 s -1 、about 1 s -1 to about 10 -5 s -1 、about 10 -2 s -1 to about 10 -6 s -1 、about 10 -3 s -1 to about 10 -6 s -1 、about 10 -4 s -1 to about 10 -6 s -1 、about 10 -2 s -1 to about 10 -5 s -1 ,or about 10 -3 s -1 to about 10 -5 s -1 。In some embodiments, the K of the binding between the antibody portion and 4-1BB 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.

[0162] In some embodiments, the K of the binding between the antibody portion and the tumor antigen D is about 10 -7 M to about 10 -12 M、about 10 -7 M to about 10 -8 M、about 10 -8 M to about 10 -9 M、about 10 -9 M to about 10 -10 M、about 10 -10 M to about 10 -11 M、about 10 -11 M to about 10 -12 M、about 10 -7 M to about 10 -12 M、about 10 -8 M to about 10 -12 M、about 10-9 from M to about 10 -12 M, about 10 -10 from M to about 10 -12 M, about 10 -7 from M to about 10 -11 M, about 10 -8 from M to about 10 -11 M, about 10 -9 from M to about 10 -11 M, about 10 -7 from M to about 10 -10 M, about 10 -8 from M to about 10 -10 M, or about 10 -7 from M to about 10 -9 M. In some embodiments, the K of the binding between the antibody portion and the tumor antigen D is stronger than about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M of any one. In some embodiments, the tumor antigen is a human antigen.

[0163] In some embodiments, the K of the binding between the antibody portion and the tumor antigen on is about 10 3 M -1 s -1 to about 10 8 M -1 s -1 、about 10 3 M -1 s -1 to about 10 4 M -1 s -1 、about 10 4 M -1 s -1 to about 10 5 M -1 s -1 、about 10 5 M -1 s -1 to about 10 6 M -1 s -1 、about 10 6 M - 1 s -1 to about 10 7 M -1 s -1 ,or about 10 7 M-1 s -1 to about 10 8 M -1 s -1 。In some embodiments, the K of the binding between the antibody portion and the tumor antigen on is about 10 3 M -1 s -1 to about 10 5 M -1 s -1 、about 10 4 M -1 s -1 to about 10 6 M -1 s -1 、about 10 5 M -1 s -1 to about 10 7 M - 1 s -1 、about 10 6 M -1 s -1 to about 10 8 M -1 s -1 、about 10 4 M -1 s -1 to about 10 7 M -1 s -1 ,or about 10 5 M -1 s -1 to about 10 8 M -1 s -1 。In some embodiments, the K of the binding between the antibody portion and the tumor antigen on is not more than 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 of any one. In some embodiments, the tumor antigen is a human antigen.

[0164] In some embodiments, the K of the binding between the antibody portion and the tumor antigen off is about 1 s -1 to about 10 -6 s -1 、about 1 s -1 to about 10 -2 s -1 、about 10 -2 s -1 to about 10 -3 s -1 、about 10 -3 s -1 to about 10 -4 s -1 、about 10 -4 s -1 to about 10 -5 s -1 、about 10 - 5 s -1 to about 10 -6 s -1 、about 1 s -1 to about 10 -5 s -1 、about 10 -2 s -1 to about 10 -6 s -1 、about 10 -3 s -1 to about 10 -6 s -1 、about 10 -4 s -1 to about 10 -6 s -1 、about 10 -2 s -1 to about 10 -5 s -1 or about 10 -3 s -1 to about 10 -5 s -1 。In some embodiments, the K of the binding between the antibody portion and the tumor antigen 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 -1Any of the above. In some embodiments, the tumor antigen is a human antigen.

[0165] Chimeric or humanized antibodies

[0166] In some embodiments, one or more antibody portions of the multispecific constructs 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, the 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, the chimeric antibody is a "class switched" antibody in which the class or subclass has been altered relative to the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0167] In some embodiments, the chimeric antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental 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. The humanized antibody optionally further comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), such as to restore or improve antibody specificity or affinity.

[0168] Reviews of humanized antibodies and methods of making them are found, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, 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 “guided selection” method of FR shuffling).

[0169] 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 from consensus sequences of human antibodies from specific 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 (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions from screening 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)).

[0170] Human antibody

[0171] In some embodiments, one or more antibody portions of the multispecific constructs of the present application are human antibodies (referred to as human domain antibodies or human DAbs). A variety of techniques known in the art can be used to generate human antibodies. General descriptions of human antibodies are 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.

[0172] Human antibodies (e.g., human DAA) can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or which is present extrachromosomally or is randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically 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 the XENOMOUSE TM technology; U.S. Patent No. 5,770,429, which describes ; U.S. Patent No. 7,041,870, which describes the K-M technology, and U.S. Patent Application Publication No. US2007 / 0061900), which describes technology. The human variable regions of the intact antibodies produced from these animals can be further modified, for example, by combining with different human constant regions.

[0173] Human antibodies, such as human DAA, can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., 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 by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described 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).

[0174] Human antibodies, such as human DAA, can also be generated by isolating the selected Fv clone variable domain sequences from a phage display library derived from humans. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0175] Anti-MUC16 antibody

[0176] MUC16 is a tumor-associated antigen polypeptide that is expressed by epithelial cells in the human ocular surface epithelium, bronchus, fallopian tube, and uterus mucosa. One function of MUC16 is thought to provide a protective lubricating barrier against particulate and infectious agents at the mucosal surface. The highly polymorphic MUC16 consists of three domains: an N-terminal domain rich in Ser / Thr, a repeat domain containing partially conserved tandem repeats of 11 to over 60, each averaging 156 amino acids, and a C-terminal non-repeating domain containing a transmembrane sequence and a short cytoplasmic tail. MUC16 is heavily O-glycosylated and N-glycosylated. It has been reported that MUC16 is highly overexpressed in certain types of human cancerous ovarian, breast, and pancreatic tumors, respectively, compared to the corresponding normal human ovarian, breast, and pancreatic tissues. Due to its overexpression in certain human tumors, MUC16 polypeptides and nucleic acids encoding the polypeptides are targets for quantitative and qualitative comparison of various mammalian tissue samples. The unique expression profile of MUC16 polypeptides and the nucleic acids encoding the polypeptides can be used for the diagnosis and treatment of certain types of cancerous tumors in mammals.

[0177] The present disclosure provides antibodies, including antibodies or antigen-binding fragments thereof, that have binding specificity for human MUC16 protein. As demonstrated in the experimental examples, 13 anti-human MUC16 antibodies were obtained, which have high binding affinity for human MUC16 protein. Antibody clones D57, B218, C25, and D100 were selected for further bispecific antibody construction. Human antibodies bind human MUC16 with high affinity, and bispecific antibodies effectively induce MUC16-dependent 4-1BB activation in T cells.

[0178] According to one embodiment of the present disclosure, there is provided an antibody or an antigen-binding fragment thereof, which includes a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain and the light chain variable domain have the CDR regions of the antibodies prepared in the experimental examples. The CDRs and variable regions (Kabat numbering) are summarized in Table 4 of the examples.

[0179] In some embodiments, VH CDR1, CDR2, and CDR3 are selected from any set of VH CDR1, CDR2, and CDR3 shown in Table 1, and VL CDR1, CDR2, and CDR3 are selected from any set of VL CDR1, CDR2, and CDR3 shown in Table 1. In some embodiments, VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 are selected from those derived from the same antibody in the examples.

[0180] In some embodiments, at least one, or two, or three, or four, or five, or six of the above VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 are modified by adding, deleting, or substituting one, two, or three amino acids or a combination thereof.

[0181] The CDRs, heavy chain variable regions, and light chain variable regions of the present disclosure can be further modified. In some embodiments, the modified heavy chain variable region 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.

[0182] In some embodiments, the modification is a substitution at no more than one hot spot position in each CDR. In some embodiments, the modification is a substitution at one, two, or three such hot spot positions. In one embodiment, the modification is a substitution at one of the hot spot positions. In some embodiments, such substitutions are conservative substitutions.

[0183] Those of ordinary skill in the art will also understand that the antibodies disclosed herein can be modified such that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar, e.g., having a specific percentage identity to the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0184] 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.

[0185] In certain embodiments, the present disclosure provides a bifunctional molecule comprising a first antigen-binding portion and a second portion, the first antigen-binding portion being specific for a human MUC16 protein and the second portion being specific for a second protein, wherein the first antigen-binding portion comprises an anti-MUC16 antibody or a fragment thereof as provided herein.

[0186] In certain embodiments, the second portion is an antibody or an antigen-binding fragment thereof. In certain embodiments, the second portion is specific for an immune checkpoint. In certain embodiments, the second portion is specific for other tumor antigens.

[0187] In certain embodiments, the second portion is a 4-1BB antigen-binding portion.

[0188] In certain embodiments, the first antigen-binding portion provided herein has a full-length antibody / IgG form, and the second antigen-binding portion provided herein has a single-domain antibody (sdAb) form.

[0189] In certain embodiments, the sdAb is fused to the N-terminus or C-terminus of the IgG. In certain embodiments, the sdAb is fused to the N-terminus of the variable region of the IgG heavy chain. In certain embodiments, the sdAb is fused to the C-terminus of the constant region of the IgG heavy chain (IgG(CH)) that includes the Fc domain.

[0190] In certain embodiments, the multispecific construct provided herein includes a first polypeptide and a second polypeptide. The first polypeptide comprises, from the N-terminus to the C-terminus: VH(MUC16)-IgG(CH)-VHH(4-1BB), and the second polypeptide comprises, from the N-terminus to the C-terminus: VL(MUC16)-constant region of the IgG light chain (IgG(CL)). The first polypeptide and the second polypeptide are paired by VH(MUC16)-VL(MUC16) pairing.

[0191] In certain embodiments, the multispecific construct provided herein includes two first polypeptides and two second polypeptides. The first polypeptide comprises, from the N-terminus to the C-terminus: VH(MUC16)-IgG(CH)-VHH(4-1BB), and the second polypeptide comprises, from the N-terminus to the C-terminus: VL(MUC16)-constant region of the IgG light chain (IgG(CL)). The first polypeptide and the second polypeptide are paired by VH(MUC16)-VL(MUC16) pairing. The two first polypeptides can be paired by IgG(CH) pairing.

[0192] The first antigen-binding portion and the second antigen-binding portion are fused by a linker.

[0193] Substitution, insertion, and deletion variants

[0194] In some embodiments, antibody variants comprising one or more amino acid substitutions are included in the multispecific constructs or antibodies described herein. Sites of interest for substitution mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred substitutions". More substantial changes are provided in Table 2 under the heading "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 screened to obtain the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0195] Table 2. Amino acid substitutions

[0196]

[0197] 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 affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0198] A non-conservative substitution involves replacing a member of one of these categories with a member of another category.

[0199] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have an improvement (e.g., an enhancement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be readily generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for a particular biological activity (e.g., binding affinity).

[0200] Changes (e.g., substitutions) can be made in the HVRs, e.g., to improve antibody affinity. Such changes can be made in HVR “hotspots” (i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation) (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in the SDRs (α-CDRs), where the binding affinity of the resulting variant VH or VL is tested. Affinity maturation has been described, e.g., by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)) by constructing secondary libraries and reselecting therefrom for affinity maturation. 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 having the desired affinity. Another method of introducing diversity involves HVR-directed methods, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, e.g., using alanine-scan mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0201] In some embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, so long as these changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) can be made in the HVRs, which do not significantly reduce binding affinity. Such changes can be outside of HVR “hotspots” or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0202] A useful method for identifying residues or regions in an antibody that can serve as mutagenesis targets is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial replacement. Optionally or additionally, the crystal structure of the antigen-antibody complex identifies the 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 possess the desired properties.

[0203] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include fusions of the antibody N-terminus or C-terminus with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

[0204] Glycosylation variants

[0205] 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 degree of glycosylation of the constructs. Addition or deletion of glycosylation sites of the antibody can be conveniently achieved by altering the amino acid sequence, thereby creating or removing one or more glycosylation sites.

[0206] In the case where the antibody portion contains an Fc region, the carbohydrate linked thereto can be altered. Native antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally linked via an N-linkage to Asn297 in the C H 2 domain. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can 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, the oligosaccharide in the antibody portion can be modified to create antibody variants with certain improved properties.

[0207] In some embodiments, the antibody portion has a carbohydrate structure lacking fucose linked (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 within the sugar chain at Asn297 relative to the sum of all sugar structures linked to Asn297 (e.g., complex, hybrid, and high mannose structures) measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the antibody, 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 can have improved ADCC function. See, for example, U.S. Patent Publication No. US 2003 / 0157108 (Presta, L.); US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 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 afucosylated antibodies include Lec13 CHO cells that lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines such as α-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).

[0208] In some embodiments, the antibody portion has bisected oligosaccharides, e.g., wherein the biantennary oligosaccharides linked to the antibody Fc region are bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharides linked to the Fc region are also provided. Such antibody variants can have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0209] Fc domain

[0210] In some embodiments, the first antibody portion or the second antibody portion comprises an Fc region (also referred to herein as "Fc fragment"). In some embodiments, the Fc region is an Fc domain, i.e., an Fc region having some or all effector functions, including, e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In some embodiments, the Fc domain is derived from IgG1 or IgG3.

[0211] In some embodiments, the effector functions of the Fc region (such as ADCC and / or CDC) are maintained or improved.

[0212] In some embodiments, one or more amino acid modifications can be introduced into the Fc domain, resulting in Fc domain variants. The Fc domain variants can comprise a human Fc domain sequence (e.g., derived from the human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, the Fc domain variants alter one or more functional and / or pharmacokinetic properties of the antibody.

[0213] In some embodiments, the Fc domain has partial but not all effector functions, making it a desirable candidate in applications where the in vivo half-life of the antibody portion is important but certain effector functions (e.g., CDC and ADCC) are unnecessary or detrimental.

[0214] 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 the antibody has FcγR binding (and thus potentially ADCC activity) and / or retains the ability to bind FcRn. The major cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The 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 for assessing the ADCC activity of a molecule of interest are described in U.S. Patent 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); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI of flow cytometry TM Non-radioactive cytotoxicity assay CellTechnology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Available effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Optionally or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model as disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0215] Antibodies with reduced effector function include those having one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581). In some embodiments, the Fc region of the multispecific construct does not contain mutations that reduce its effector function, such as one or more of the mutations described herein. In some embodiments, the Fc region of the multispecific construct contains one or more of these mutations.

[0216] Describes certain antibody variants with improved or diminished binding to FcR. (See, e.g., U.S. Patent 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 the multispecific construct does not contain variants with improved or diminished binding to FcR. In some embodiments, the Fc region of the multispecific construct contains variants with improved binding to FcγRI. In some embodiments, the Fc region of the multispecific construct contains variants with improved binding to FcγRII. In some embodiments, the Fc region of the multispecific construct contains variants with improved binding to FcγRIII. In some embodiments, the Fc region of the multispecific construct contains variants with diminished binding to FcγRI. In some embodiments, the Fc region of the multispecific construct contains variants with diminished binding to FcγRII. In some embodiments, the Fc region of the multispecific construct contains variants with diminished binding to FcγRIII.

[0217] In some embodiments, the Fc domain is derived from human IgG1. In some embodiments, the Fc domain derived from human IgG1 does not contain the L234A mutation and / or the L235A mutation. In some embodiments, the Fc domain derived from human IgG1 contains the L234A mutation and / or the 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 Fc domain derived from human IgG4 contains the S228P, F234A, and / or L235A mutations. In some embodiments, the Fc domain derived from human IgG4 does not contain the S228P, F234A, and / or L235A mutations.

[0218] In some embodiments, the multispecific construct contains an Fc domain with one or more amino acid substitutions that improve ADCC. In some embodiments, one or more of the substitutions are at positions 298, 333, and / or 334 in the Fc region (EU numbering of the residues).

[0219] In some embodiments, alterations are made in the Fc domain that result in altered (i.e., improved or diminished) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0220] In some embodiments, the multispecific construct comprises a variant Fc domain that contains one or more amino acid substitutions that alter the half-life and / or alter the binding to the neonatal Fc receptor (FcRn). Antibodies with an extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the 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 contain an Fc region with one or more substitutions therein that alter the binding of the Fc region to FcRn. These Fc variants include variants with substitutions at one or more positions in the Fc region residues, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0221] 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, which relate to other examples of Fc region variants.

[0222] In some embodiments, the multispecific construct comprises an Fc domain that contains 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 SEQ ID NO: 46-56. In some embodiments, the multispecific construct comprises an Fc domain that contains an amino acid sequence selected from any one of SEQ ID NO: 46-56.

[0223] Cysteine-modified antibody variants and heterodimers

[0224] In some embodiments, it may be desirable to create cysteine-modified antibody moieties, such as "thioMAb", in which one or more residues of one or more antibody moieties in the multispecific constructs herein are replaced with cysteine residues. In certain embodiments, the residues to be replaced occur at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, to create an immunoconjugate. In some embodiments, any one or more of the following residues may be replaced with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibody moieties can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0225] heterodimer

[0226] Generally, two identical Fc regions form a homodimer. However, by mutating one or both chains, e.g., by knob-into-hole (KIH), disulfide bonds (-S-S-), or by hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility, two different Fc regions can form a heterodimer.

[0227] As used herein, the term "knob-into-hole" or "KIH" technology refers to a technique for guiding the pairing of two polypeptides in vitro or in vivo by introducing a protrusion (knob) into one polypeptide and a groove (hole) into the other polypeptide at the interface where the two polypeptides interact. For example, KIH has been introduced at the Fc:Fc binding interface, CL:CH1 interface, or VH / VL interface of antibodies (see, e.g., US2011 / 0287009, US2007 / 0178552, WO 96 / 027011, WO 98 / 050431, Zhu et al, 1997, Protein Science 6:781-788, and WO2012 / 106587). In some embodiments, KIH drives the pairing of two different heavy chains during the preparation of a multispecific antibody. For example, a multispecific antibody having KIH in its Fc region can further comprise a single variable domain linked to each Fc region, or can further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The KIH technique can also be used to pair two different receptor extracellular domains or to pair any other polypeptide sequences comprising different target recognition sequences (e.g., including affibodies, peptibodies, and other Fc fusions).

[0228] As used herein, the term "knob mutation" 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.

[0229] As used herein, the term "hole mutation" refers to a mutation that introduces a groove (hole) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0230] 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).

[0231] In some embodiments, the club mutations in the IgG1 constant region are S354C and T366W (EU numbering). In some embodiments, the socket mutations in the IgG1 constant region comprise one or more mutations selected from Y349C, T366S, L368A, and Y407V (EU numbering).

[0232] Bispecific antibodies can also be prepared by engineering electrostatic steering effects to produce antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); by cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al, Science, 229:81 (1985)); by using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J. Immunol, 148(5):1547-1553 (1992)); by using the "diabody" technology to produce bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by using single-chain Fv (sFv) dimers (see, e.g., Gruber et al, J. Immunol, 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0233] In some embodiments, the Fc domain provided herein comprises a club mutation and the paired Fc domain comprises one or more socket mutations, or vice versa.

[0234] In some embodiments, the multispecific construct comprises an Fc domain that comprises the amino acid sequence of any one of SEQ ID NOs: 285-286 and 288-289.

[0235] In some embodiments, the multispecific construct comprises an Fc domain and a paired Fc domain, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:285 or a variant thereof, the variant having at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:285, and the paired Fc domain comprises the amino acid sequence of SEQ ID NO:288 or a variant thereof, the variant having at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:288.

[0236] In some embodiments, the multispecific construct comprises an Fc domain and a paired Fc domain, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:286 or a variant thereof, the variant having at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:286, and the paired Fc domain comprises the amino acid sequence of SEQ ID NO:289 or a variant thereof, the variant having at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:289.

[0237] In some embodiments, the Fc domain and / or the paired Fc domain provided herein is an IgG Fc domain. In some embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4.

[0238] Anti-4-1BB antibody portion

[0239] The anti-4-1BB antibody portion of the multispecific constructs described in the present 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 that has four extracellular cysteine-rich domains (“CRD”, 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). Compared to other TNFRs, h4-1BB exists as a disulfide-linked dimer, and dimerization may occur through an unpaired cysteine (Cys 121 ) found within CRD4 of h4-1BB. In some embodiments, h4-1BB comprises the sequence shown 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.

[0240] The anti-4-1BB antibody portion can be in any suitable form known in the art. In some embodiments, the anti-4-1BB antibody portion is selected from full-length antibodies, Fab, Fab', F(ab') 2 , scFv, and sdAb. In some embodiments, the anti-4-1BB antibody portion comprises a single-domain antibody that binds to 4-1BB.

[0241] Exemplary anti-4-1BB antibody portions

[0242] In some embodiments, the anti-4-1BB antibody portion comprises a single-domain antibody (sdAb) that 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 having the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant having at least about 80% (including, for example, any one of at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:27.

[0243] In some embodiments, the anti-4-1BB antibody portion comprises a single-domain antibody (sdAb) that comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, where the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence shown in SEQ ID NO:27, and where the 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) that comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, where the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence shown in SEQ ID NO:27, and where the 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) that comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, where the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain having the amino acid sequence shown in SEQ ID NO:27, and where the CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme.

[0244] In some embodiments, the affinity of such an anti-4-1BB antibody portion for 4-1BB (e.g., human 4-1BB) is comparable (e.g., the same) to the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB.

[0245] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) that comprises: a) sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; b) sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and c) sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3.

[0246] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) that comprises a) sdAb-CDR1 comprising the amino acid sequence of SEQ ID NO:24, sdAb-CDR2 comprising the amino acid sequence of SEQ ID NO:25, and sdAb-CDR3 comprising the amino acid sequence of SEQ ID NO:26.

[0247] In some embodiments, the anti-4-1BB antibody portion comprises a single domain antibody (sdAb) that comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:27. In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (such as human 4-1BB) that is comparable (such as the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB.

[0248] Tumor antigen

[0249] The tumor antigen specifically bound by the first portion (such as the first antibody portion) can be any suitable tumor antigen known in the art. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6.

[0250] In some embodiments, the tumor antigen is selected from the group consisting of 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-1-4, APRIL, MAGE3, folate receptor alpha, folate receptor beta, GPC2, CD70, BAFF-R, and TROP-2.

[0251] 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 proliferation. MUC16 is thought to be involved in cell-cell interactions, which promote metastasis through binding to mesothelin. MUC16 can 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, at UniProtKB Q8WX17.

[0252] In some embodiments, the first antibody portion binds to MUC16. Any anti-MUC16 antibody that binds to MUC16 and triggers activation of 4-1BB by the second antibody portion can be used in the present invention. Suitable anti-MUC16 antibodies include, but are not limited to, those described in 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 Biol., 2007; 39(1):1943-1954), WO 2002 / 092836, WO 2020 / 102555, WO 2020 / 227538, WO 2016 / 149368, US Patent Publication 2021 / 0309758, US Patent Publication 2020 / 0317810, US Patent 10,941,208, WO 2007 / 001851, US Patent No. 7,078,188, WO 2007 / 001851, WO 2019 / 213747, and WO 2008 / 141044, which are incorporated herein by reference in their entirety. In some embodiments, the anti-MUC16 antibody is selected from the group consisting of 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 carboxyl-terminal region of MUC16. In some embodiments, the anti-MUC16 antibody binds to the juxtamembrane domain, cytoplasmic tail region, or C-terminal region of the mucin repeat domain.

[0253] 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 type of transmembrane protein that is involved in the hydrolysis of extracellular nucleotides and has been found to be expressed in several cancers and cancer cells, including neoplastic mast cells, acute basophilic leukemia, colon cancer, renal cell carcinoma, hepatocellular carcinoma, and tumor cell cholangiocytes. Exemplary protein sequences of ENPP3 can be found, for example, at UniProtKB O14638.

[0254] In some embodiments, the first antibody portion binds to ENPP3. Any anti-ENPP3 antibody in which binding of the first antibody portion to ENPP3 triggers activation of 4-1BB by the second antibody portion 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. Publication No. 2010 / 0099111, U.S. Patent No. 8,562,989, U.S. Publication No. 2016 / 0176977, and U.S. Publication No. 2019 / 0092874, which are hereby incorporated by reference in their entirety.

[0255] In some embodiments, the tumor antigen is receptor tyrosine kinase transmembrane receptor (ROR1). ROR1 is also known as NTRKR1. ROR1 is thought to play a role in tumor cell survival, proliferation, migration, and chemotaxis and has been 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. Exemplary protein sequences of ROR2 can be found, for example, at UniProtKB Q01973.

[0256] In some embodiments, the first antibody portion binds to ROR1. Any anti-ROR1 antibody in which binding of the first antibody portion to ROR1 triggers activation of 4-1BB by the second antibody portion 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, WO 2010 / 124188, WO 2016 / 187220, WO 2012 / 045085, WO 2017 / 072361, WO 2019 / 008377, WO 2019 / 005636, WO 2014 / 031174, and WO 2017 / 127664, which are hereby incorporated by reference in their entirety. In some embodiments, the first antibody portion is cirmtuzumab.

[0257] In some embodiments, the tumor antigen is SLC7A11. SLC7A11 is a cysteine / glutamate transporter, also known as CCBR1, xCT, and solute carrier family 7 member 11. Overexpression of SLC7A11 is thought to promote tumor growth (in part by inhibiting ferroptosis). An exemplary protein sequence of SLC7A11 can be found, for example, at UniProtKB Q9UPY5.

[0258] In some embodiments, the first antibody portion binds to SLC7A11. Any anti-SLC7A11 antibody in which binding of the first antibody portion to SLC7A11 triggers activation of 4-1BB by the second antibody portion can be used in the present invention. Suitable anti-SLC7A11 antibodies include, but are not limited to, those described in WO 2018 / 204278 and WO 2020 / 227640, which are incorporated herein by reference in their entireties.

[0259] In some embodiments, the tumor antigen is delta-like 3 (DLL3). DLL3 is a member of the delta protein ligand family and functions as a Notch ligand. High DLL3 expression has been observed in several cancer types, particularly in neuroendocrine-related tumors, and has been studied as a potential target in several cancer types, including small cell lung cancer, non-small cell lung cancer, and large cell neuroendocrine carcinoma. High expression of DLL3 is part of... An exemplary protein sequence of DLL3 can be found, for example, at UniProtKB Q9NYJ7.

[0260] In some embodiments, the first antibody portion binds to DLL3. Any anti-DLL3 antibody in which binding of the first antibody portion to DLL3 triggers activation of 4-1BB by the second antibody portion can be used in the present invention. Suitable anti-DLL3 antibodies include, but are not limited to, those described in Saunders et al. (Sci Transl Med., 2015; 7(302):302ra136), WO 2017 / 031458, WO 2019 / 217145, WO 2013 / 126746, WO 2015 / 127407, WO 2011 / 093097, and WO 2021 / 007371, which are incorporated herein by reference in their entireties. In some embodiments, the anti-DLL3 antibody is rovalpituzumab.

[0261] In some embodiments, the tumor antigen is B7H4, also known as V-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 contribute to tumor evasion of the immune system. Exemplary protein sequences of B7H4 can be found, for example, at UniProtKB Q7Z7D3.

[0262] In some embodiments, the first antibody portion binds to B7H4. Any anti-B7H4 antibody that triggers activation of 4-1BB upon binding to B7H4 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, WO 2012 / 145568, WO 2013 / 067492, and WO 2014 / 100483, which are incorporated herein by reference in their entirety. In some embodiments, the antibody is Alsevalimab (FP150).

[0263] 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. Exemplary protein sequences of EPHA2 can be found, for example, at UniProtKB P29317.

[0264] In some embodiments, the first antibody portion binds to EPHA2. Any anti-EPHA2 antibody that binds to EPHA2 and triggers activation of 4-1BB by the second antibody portion can be used in the present invention. Suitable anti-EPHA2 antibodies include, but are not limited to, those described in 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, WO 2004 / 014292, U.S. Patent Publication 2007 / 0086943, U.S. Patent Publication 2010 / 0298545, WO 2016 / 081601, U.S. Patent No. 7,659,374, WO 2006 / 023403, U.S. Patent No. 10,406,225, and U.S. Patent Publication 2016 / 0031987, which are hereby incorporated by reference in their entirety.

[0265] 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 whose extracellular domain contains two CUB domains. CD318 phosphorylation has been observed in many cancer types, including pre-invasive cancers, invasive cancers, and tumor metastases. An exemplary protein sequence of CD318 can be found, for example, at UniProtKB Q9H5V8.

[0266] In some embodiments, the first antibody portion binds to CD318. Any anti-CD318 antibody that binds to CD318 and triggers activation of 4-1BB by the second antibody portion can be used in the present invention. Suitable anti-CD318 antibodies include, but are not limited to, those described in WO 2011 / 023389, WO 2018 / 112334, WO 2019 / 084319, WO 2021 / 132427, and WO 2011 / 023390, which are hereby incorporated by reference in their entirety.

[0267] In some embodiments, the tumor antigen is Claudin 6 (CLDN6). Claudin 6 (CLDN6) is a member of the claudin protein family and, as a tight junction molecule, plays an important role in intercellular adhesion in epithelial or endothelial cell sheets. It encodes a tetraspanin protein that is 220 amino acids in size and has a molecular weight of 23,292 Da. CLDN6 has been identified as the origin of cell adhesion signaling and is involved in the regulation of nuclear receptor activity by targeting molecules of the nuclear receptor superfamily and regulating their gene expression (Sugimoto et al. (2019). “Cell adhesion signals regulate the nuclear receptor activity.” Proc. Natl. Acad. Sci. U.S.A. 116, 24600–24609). CLDN6 appears to be significantly upregulated in 20 human cancers (Zhang et al. (2021) Front. Cell. Dev. Biol. 9:726656). In some embodiments, CLDN6 is human CLDN6 (“hCLDN6”). In some embodiments, hCLDN6 comprises the amino acid sequence shown in SEQ ID NO:40 or a variant thereof (e.g., a post-translationally modified variant and / or a conformational variant).

[0268] In some embodiments, the first antibody portion binds to CLDN6. Any anti-CLDN6 antibody that triggers activation of 4-1BB upon binding to CLDN6 can be used in the present invention. Suitable anti-CLDN6 antibodies include, but are not limited to, any of the anti-CLDN6 antibodies described herein, U.S. Patent No. 9,274,119, WO 2012 / 156018, WO 2019 / 056023, and U.S. Patent No. 10,053,511, all of which are incorporated herein by reference in their entirety.

[0269] 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 inhibiting the adaptive immune system. PD-L1 binds to PD-1 expressed on T cells, B cells, and myeloid cells. Overexpression of PD-L1 on tumor cells is thought to contribute to cancer evasion of the immune system. An exemplary protein sequence of PD-L1 can be found, for example, at UniProtKB Q9NZ17.

[0270] In some embodiments, the first antibody portion binds to PD-L1. Any anti-PD-L1 antibody that binds to PD-L1 and triggers activation of 4-1BB by the second antibody portion can be used in the present invention. Suitable anti-PD-L1 antibodies include, but are not limited to, Atezolizumab, Avelumab, Durvalumab, Atezolizumab (e.g., Tencentriq ), Avelumab (e.g., Bavencio ), and Durvalumab (e.g., Imfinzi TM ).

[0271] Multispecific construct

[0272] In one aspect, the present disclosure provides a multispecific construct comprising: a first portion (such as 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 activation of 4-1BB by the second antibody portion. In some embodiments, the second antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB.

[0273] In some embodiments, the present disclosure provides a multispecific construct 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 triggers activation of 4-1BB by the second antibody portion. In some embodiments, after binding of the first antibody portion to the tumor antigen, 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 between these values. In some embodiments, the multispecific construct activates 4-1BB signal transduction in the absence of binding to the tumor antigen. In some embodiments, the second portion does not activate 4-1BB signal transduction in the absence of binding to the tumor antigen.

[0274] In some embodiments of the multispecific constructs of the present application, the second antibody moiety 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 monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27. 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 monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27, and CDR1, CDR2, and CDR3 are according to the Kabat numbering scheme.

[0275] In some embodiments, the sdAb comprises sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3.

[0276] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant having at least about 80% (including, for example, at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more than 99%) sequence identity to the sequence shown in SEQ ID NO:27. In some embodiments, such an sdAb has an affinity for 4-1BB (such as human 4-1BB) comparable to (such as the same as) the affinity of the sdAb comprising SEQ ID NO:27 for 4-1BB.

[0277] In some embodiments, the tumor antigen specifically bound by the first antibody moiety can be any suitable tumor antigen known in the art. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6.

[0278] In some embodiments, the present application provides a multispecific construct that binds to both MUC16 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies comprising 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.

[0279] In some embodiments, the present application provides a multispecific construct that binds to both ENPP3 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies 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.

[0280] In some embodiments, the present application provides a multispecific construct that binds 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.

[0281] In some embodiments, the present application provides a multispecific construct that binds to both SLC7A11 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies 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.

[0282] 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 comprising 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.

[0283] 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 comprising 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.

[0284] In some embodiments, the present application provides a multispecific construct that binds to both EPHA2 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies that comprise 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.

[0285] In some embodiments, the present application provides a multispecific construct that binds to both CD318 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies that comprise 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.

[0286] In some embodiments, the present application provides a multispecific construct that binds to both CLDN6 and 4-1BB. In some embodiments, the multispecific constructs described herein are bispecific antibodies that comprise 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.

[0287] In some embodiments, the multispecific construct is bispecific and comprises a third antibody portion that specifically binds to the same tumor antigen as the first antibody portion but to a different epitope.

[0288] In some embodiments, the anti-4-1BB antibody portion comprises an sdAb that comprises: an sdAb-CDR1 that comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in the sdAb-CDR1; an sdAb-CDR2 that comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in the sdAb-CDR2; and an sdAb-CDR3 that comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising up to about 3 (such as any one of about 1, 2, 3) amino acid substitutions in the sdAb-CDR3. In some embodiments, the anti-4-1BB antibody portion comprises an sdAb that comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant having at least about 80% sequence identity to SEQ ID NO:27 (such as at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% sequence identity, including any range between these values).

[0289] In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific or bispecific or trispecific antibodies) that comprise an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific antibodies) that comprise an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, and 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 bispecific and thus comprises a third antibody portion that specifically binds the same tumor antigen as the first antibody portion but to a different epitope. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half antibody, single-chain half antibody, and full-length antibody. The form of the third antibody portion may be the same as or different from that of the first antibody portion. For example, both the first antibody portion and the third antibody portion may be in the form of full-length antibodies. For another example, the first antibody portion is a full-length antibody and the third antibody portion is an scFv, or vice versa. For another example, the first antibody portion is a half antibody and the third antibody portion is a single-chain half antibody, or vice versa.

[0290] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7M to about 10 -13 The affinity of M binds to a tumor antigen. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to the tumor antigen triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an 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 (such as a bispecific or bivalent or trispecific antibody) comprising an anti-MUC16 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific construct described herein is a multispecific construct (such as a bispecific antibody) comprising an anti-MUC16 antigen antibody portion and an anti-4-1BB antibody portion, and 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 bivalent and thus comprises a second anti-MUC16 portion that specifically binds to the same tumor antigen as the first anti-MUC16 portion but targets a different epitope. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. The form of the second anti-MUC16 portion may be the same as or different from that of the first anti-MUC16 portion. For example, both the first anti-MUC16 portion and the third anti-MUC16 portion may be in the form of full-length antibodies. For another example, the first anti-MUC16 portion is a full-length antibody and the second anti-MUC16 portion is an scFv, or vice versa. For 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.

[0292] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to MUC16. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to MUC16 triggers the activation of 4-1BB by the second antibody portion, such as activation 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 (such as bispecific or bivalent or trispecific antibodies) that comprise an anti-ENPP3 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific antibodies) that comprise an anti-ENPP3 antigen antibody portion and an anti-4-1BB antibody portion, and 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 bivalent and thus comprises a second anti-ENPP3 portion that specifically binds the same tumor antigen as the first anti-ENPP3 portion but to a different epitope. The form of the second anti-ENPP3 portion may be the same as or different from the first anti-ENPP3 portion. For example, both the first anti-ENPP3 portion and the third anti-ENPP3 portion may be in the form of full-length antibodies. For another example, the first anti-ENPP3 portion is a full-length antibody and the second anti-ENPP3 portion is a scFv, or vice versa. For 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.

[0294] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to ENPP3. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to ENPP3 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0295] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-ROR1 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 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, and 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 bivalent and thus comprises a second anti-ROR1 portion that specifically binds the same tumor antigen as the first anti-ROR1 portion but to a different epitope. The form of the second anti-ROR1 portion can be the same as or different from that of the first anti-ROR1 portion. For example, both the first anti-ROR1 portion and the third anti-ROR1 portion can be in the form of full-length antibodies. For another example, the first anti-ROR1 portion is a full-length antibody and the second anti-ROR1 portion is an scFv, or vice versa. For 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.

[0296] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to ROR1. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to ROR1 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an 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 (such as a bispecific or bivalent or trispecific antibody) comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific construct described herein is a multispecific construct (such as a bispecific antibody) comprising an anti-SLC7A11 antigen antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion comprises an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-SLC7A11 portion that specifically binds to the same tumor antigen as the first anti-SLC7A11 portion but targets a different epitope. The form of the second anti-SLC7A11 portion may be the same as or different from that of the first anti-SLC7A11 portion. For example, both the first anti-SLC7A11 portion and the third anti-SLC7A11 portion may be in the form of full-length antibodies. For another example, the first anti-SLC7A11 portion is a full-length antibody and the second anti-SLC7A11 portion is an scFv, or vice versa. For yet another example, the first anti-SLC7A11 portion is a half-antibody and the second anti-SLC7A11 portion is a single-chain half-antibody, or vice versa.

[0298] 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 at about 10 -7 M to about 10 -13The affinity of M binds to SLC7A11. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to SLC7A11 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0299] In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific or bispecific or trispecific antibodies) comprising an anti-DLL3 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific antibodies) comprising an anti-DLL3 antigen antibody portion and an anti-4-1BB antibody portion, and 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 bispecific and thus comprises a second anti-DLL3 portion that specifically binds to the same tumor antigen as the first anti-DLL3 portion but targets a different epitope. The form of the second anti-DLL3 portion may be the same as or different from that of the first anti-DLL3 portion. For example, both the first anti-DLL3 portion and the third anti-DLL3 portion may be in the form of full-length antibodies. For another example, the first anti-DLL3 portion is a full-length antibody and the second anti-DLL3 portion is an scFv, or vice versa. For 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.

[0300] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to DLL3. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to DLL3 triggers the activation of 4-1BB by the second antibody portion, such as activation 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 constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-B7H4 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 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, and 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 bivalent and thus comprises a second anti-B7H4 portion that specifically binds the same tumor antigen as the first anti-B7H4 portion but to a different epitope. The form of the second anti-B7H4 portion can be the same as or different from that of the first anti-B7H4 portion. For example, both the first anti-B7H4 portion and the third anti-B7H4 portion can be in the form of full-length antibodies. For another example, the first anti-B7H4 portion is a full-length antibody and the second anti-B7H4 portion is an scFv, or vice versa. For yet another example, the first anti-B7H4 portion is a half-antibody and the second anti-B7H4 portion is a single-chain half-antibody, or vice versa.

[0302] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to B7H4. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to B7H4 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0303] In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific or bispecific or trispecific antibodies) comprising an anti-EPHA2 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific antibodies) comprising an anti-EPHA2 antigen antibody portion and an anti-4-1BB antibody portion, and 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 bispecific and thus comprises a second anti-EPHA2 portion that specifically binds to the same tumor antigen as the first anti-EPHA2 portion but targets a different epitope. The form of the second anti-EPHA2 portion may be the same as or different from that of the first anti-EPHA2 portion. For example, both the first anti-EPHA2 portion and the third anti-EPHA2 portion may be in the form of full-length antibodies. For another example, the first anti-EPHA2 portion is a full-length antibody and the second anti-EPHA2 portion is an scFv, or vice versa. For 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.

[0304] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to EPHA2. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to EPHA2 triggers the activation of 4-1BB by the second antibody portion, such as activation 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 constructs described herein are multispecific constructs (such as bispecific or bivalent or trispecific antibodies) comprising an anti-CD318 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific antibodies) comprising an anti-CD318 antigen antibody portion and an anti-4-1BB antibody portion, and 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 bivalent and thus comprises a second anti-CD318 portion that specifically binds the same tumor antigen as the first anti-CD318 portion but to a different epitope. The form of the second anti-CD318 portion may be the same as or different from that of the first anti-CD318 portion. For example, both the first anti-CD318 portion and the third anti-CD318 portion may be in the form of full-length antibodies. For another example, the first anti-CD318 portion is a full-length antibody and the second anti-CD318 portion is an scFv, or vice versa. For 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.

[0306] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CD318. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to CD318 triggers the activation of 4-1BB by the second antibody portion, such as activation 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 constructs described herein are multispecific constructs (such as bispecific or bivalent or trispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, and the anti-4-1BB antibody portion specifically binds to the CRD 3 / 4 region of 4-1BB. In some embodiments, the multispecific constructs described herein are multispecific constructs (such as bispecific antibodies) comprising an anti-CLDN6 antigen antibody portion and an anti-4-1BB antibody portion, and 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 bivalent and thus comprises a second anti-CLDN6 portion that specifically binds the same tumor antigen as the first anti-CLDN6 portion but to a different epitope. The form of the second anti-CLDN6 portion may be the same as or different from that of the first anti-CLDN6 portion. For example, both the first anti-CLDN6 portion and the third anti-CLDN6 portion may be in the form of full-length antibodies. For another example, the first anti-CLDN6 portion is a full-length antibody and the second anti-CLDN6 portion is an scFv, or vice versa. For 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.

[0308] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CLDN6. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the first antibody portion (and / or the third antibody portion) and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from an IgG1 or IgG3 domain). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to CLDN6 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0309] In some embodiments, the multispecific construct is bispecific and thus comprises a second anti-CLDN6 portion that specifically binds the same tumor antigen as the first anti-CLDN6 portion but to a different epitope. The form 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 anti-CLDN6 portion and the third anti-CLDN6 portion can be in the form 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.

[0310] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) that comprise an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the amino acid sequences of sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant having at least about 80% sequence identity with SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) that is comparable (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-tumor antigen portion that specifically binds the same tumor antigen as the first anti-tumor antigen portion but to a different epitope. The form of the second anti-tumor antigen portion may be the same as or different from that of the first anti-tumor antigen portion. For example, both the first anti-tumor antigen portion and the third anti-tumor antigen portion may be in the form of full-length antibodies. For 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. For 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.

[0311] In some embodiments, the first antibody portion (and / or the third antibody portion) is present at about 10 -7 M to about 10 -13The affinity of M binds to tumor antigens. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, diabody, single-chain diabody, and full-length antibody. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third binding portion) to the tumor antigen triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0312] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to that of the 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 bivalent and thus comprises a second anti-MUC16 portion that specifically binds the same tumor antigen as the first anti-MUC16 portion but to a different epitope. The form of the second anti-MUC16 portion may be the same as or different from that of the first anti-MUC16 portion. For example, both the first anti-MUC16 portion and the third anti-MUC16 portion may be in the form of a full-length antibody. 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.

[0313] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to MUC16. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to MUC16 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0314] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to 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 bivalent and thus comprises a second anti-ENPP3 portion that specifically binds the same tumor antigen as the first anti-ENPP3 portion but to a different epitope. The form 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 anti-ENPP3 portion and the third anti-ENPP3 portion can be in the form of a full-length antibody. For another example, the first anti-ENPP3 portion is a full-length antibody and the second anti-ENPP3 portion is an scFv, or vice versa. For 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.

[0315] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to ENPP3. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to ENPP3 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0316] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) that comprise an anti-ROR1 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) that is comparable (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-ROR1 portion that specifically binds the same tumor antigen as the first anti-ROR1 portion but to a different epitope. The form of the second anti-ROR1 portion can be the same as or different from the first anti-ROR1 portion. For example, both the first anti-ROR1 portion and the third anti-ROR1 portion can be in the form of full-length antibodies. For another example, the first anti-ROR1 portion is a full-length antibody and the second anti-ROR1 portion is an scFv, or vice versa. For 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.

[0317] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to ROR1. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to ROR1 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0318] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-SLC7A11 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant having at least about 80% sequence identity with 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to that of the 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 bivalent and thus comprises a second anti-SLC7A11 portion that specifically binds the same tumor antigen as the first anti-SLC7A11 portion but to a different epitope. The form of the second anti-SLC7A11 portion can be the same as 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 form of full-length antibodies. For another example, the first anti-SLC7A11 portion is a full-length antibody and the second anti-SLC7A11 portion is an scFv, or vice versa. For yet another example, the first anti-SLC7A11 portion is a half antibody and the second anti-SLC7A11 portion is a single-chain half antibody, or vice versa.

[0319] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to SLC7A11. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to SLC7A11 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0320] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to that of the 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 bivalent and thus comprises a second anti-DLL3 portion that specifically binds the same tumor antigen as the first anti-DLL3 portion but to a different epitope. The form of the second anti-DLL3 portion may be the same as or different from that of the first anti-DLL3 portion. For example, both the first anti-DLL3 portion and the third anti-DLL3 portion may be in the form 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.

[0321] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to DLL3. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to DLL3 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0322] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant having at least about 80% sequence identity with 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to that of the 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 bivalent and thus comprises a second anti-B7H4 portion that specifically binds the same tumor antigen as the first anti-B7H4 portion but to a different epitope. The form of the second anti-B7H4 portion can be the same as or different from that of the first anti-B7H4 portion. For example, both the first anti-B7H4 portion and the third anti-B7H4 portion can be in the form 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, the first anti-B7H4 portion is a half-antibody and the second anti-B7H4 portion is a single-chain half-antibody, or vice versa.

[0323] In some embodiments, the first antibody portion (and / or the third antibody portion) is present at about 10 -7 M to about 10 -13The affinity of M binds to B7H4. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to B7H4 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0324] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to that of the 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 bivalent and thus comprises a second anti-EPHA2 portion that specifically binds the same tumor antigen as the first anti-EPHA2 portion but to a different epitope. The form of the second anti-EPHA2 portion can be the same as 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 form of full-length antibodies. For another example, the first anti-EPHA2 portion is a full-length antibody and the second anti-EPHA2 portion is an scFv, or vice versa. For 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.

[0325] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to EPHA2. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to EPHA2 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0326] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) that comprise an anti-CD318 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomeric variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to 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 bivalent and thus comprises a second anti-CD318 portion that specifically binds the same tumor antigen as the first anti-CD318 portion but to a different epitope. The form of the second anti-CD318 portion may be the same as or different from that of the first anti-CD318 portion. For example, both the first anti-CD318 portion and the third anti-CD318 portion may be in the form 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, the first anti-CD318 portion is a half-antibody and the second anti-CD318 portion is a single-chain half-antibody, or vice versa.

[0327] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CD318. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, half-antibody, single-chain half-antibody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first antibody portion and the second antibody portion are linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to CD318 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0328] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-CLDN6 antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3, and the sdAb-CDR1, sdAb-CDR2, and sdAb-CDR3 comprise the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a single monomer variable antibody domain comprising the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, the variant 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 more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) comparable (e.g., the same) to 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 bivalent and thus comprises a second anti-CLDN6 portion that specifically binds the same tumor antigen as the first anti-CLDN6 portion but to a different epitope. The form 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 anti-CLDN6 portion and the third anti-CLDN6 portion can be in the form 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 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.

[0329] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CLDN6. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, diabody, single-chain diabody, and full-length antibody. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to CLDN6 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0330] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bispecific 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bispecific and thus comprises a second anti-tumor antigen portion that specifically binds the same tumor antigen as the first anti-tumor antigen portion but to a different epitope. The form 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 anti-tumor antigen portion and the third anti-tumor antigen portion can be in the form of full-length antibodies. For 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. For yet another example, the first anti-tumor antigen portion is a diabody and the second anti-tumor antigen portion is a single-chain diabody, or vice versa.

[0331] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to tumor antigens. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, diabody, and full-length antibodies. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion (and / or the third antibody portion) to the tumor antigen triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0332] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or bivalent 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-MUC16 portion that specifically binds the same tumor antigen as the first anti-MUC16 portion but to a different epitope. The form of the second anti-MUC16 portion may be the same as or different from that of the first anti-MUC16 portion. For example, both the first anti-MUC16 portion and the third anti-MUC16 portion may be in the form of full-length antibodies. For another example, the first anti-MUC16 portion is a full-length antibody and the second anti-MUC16 portion is an scFv, or vice versa. For yet another example, the first anti-MUC16 portion is a diabody and the second anti-MUC16 portion is a single-chain diabody, or vice versa.

[0333] In some embodiments, the first antibody moiety (and / or the third antibody moiety) binds to MUC16 with an affinity of from about 10 -7 M to about 10 -13 M. In some embodiments, the first antibody moiety is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first binding moiety (and / or the third antibody moiety) to MUC16 triggers activation of 4-1BB by the second antibody moiety, such as activation enhanced by at least 10-fold, or activation that results in an increase in IFNγ, IL-2, or NFκB production or activity.

[0334] In some embodiments, the multispecific construct described herein is a multispecific construct comprising an anti-ENPP3 antibody moiety and an anti-4-1BB antibody moiety (e.g., a bispecific or bivalent or trispecific antibody), wherein the anti-4-1BB antibody moiety comprises an sdAb and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising up to about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising up to about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising up to about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody moiety is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-ENPP3 moiety that specifically binds the same tumor antigen as the first anti-ENPP3 moiety but to a different epitope. The form 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 anti-ENPP3 moiety and the third anti-ENPP3 moiety can be in the form of full-length antibodies. 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.

[0335] In some embodiments, the first antibody portion (and / or the third antibody portion) binds to ENPP3 with an affinity of about 10 -7 M to about 10 -13 M. In some embodiments, the first antibody portion is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first binding portion (and / or the third antibody portion) to ENPP3 triggers activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in IFNγ, IL-2, or NFκB production or activity.

[0336] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-ROR1 portion that specifically binds the same tumor antigen as the first anti-ROR1 portion but to a different epitope. The form of the second anti-ROR1 portion may be the same as or different from the first anti-ROR1 portion. For example, both the first anti-ROR1 portion and the third anti-ROR1 portion can be in the form of full-length antibodies. 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.

[0337] In some embodiments, the first antibody portion (and / or the third antibody portion) binds to ENPP3 with an affinity of about 10-7 M to about 10 -13 The affinity of M binds to ROR1. In some embodiments, the first antibody moiety is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding moiety (and / or the third antibody moiety) to ROR1 triggers the activation of 4-1BB by the second antibody moiety, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0338] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-SLC7A11 antibody moiety and an anti-4-1BB antibody moiety, wherein the anti-4-1BB antibody moiety comprises an sdAb and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody moiety is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-SLC7A11 moiety that specifically binds the same tumor antigen as the first anti-SLC7A11 moiety but to a different epitope. The form of the second anti-SLC7A11 moiety may be the same as or different from that of the first anti-SLC7A11 moiety. For example, both the first anti-SLC7A11 moiety and the third anti-SLC7A11 moiety may be in the form of full-length antibodies. 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 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.

[0339] In some embodiments, the first antibody moiety (and / or the third antibody moiety) is at about 10-7 M to about 10 -13 The affinity of M binds to SLC7A11. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to SLC7A11 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0340] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bispecificity 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bispecificity, and thus comprises a second anti-DLL3 portion that specifically binds the same tumor antigen as the first anti-DLL3 portion but to a different epitope. The form of the second anti-DLL3 portion may be the same as or different from that of the first anti-DLL3 portion. For example, both the first anti-DLL3 portion and the third anti-DLL3 portion may be in the form of full-length antibodies. For another example, the first anti-DLL3 portion is a full-length antibody and the second anti-DLL3 portion is an scFv, or vice versa. For 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.

[0341] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to DLL3. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to DLL3 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0342] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising up to about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising up to about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising up to about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-B7H4 portion that specifically binds the same tumor antigen as the first anti-B7H4 portion but to a different epitope. The form of the second anti-B7H4 portion may be the same as or different from the first anti-B7H4 portion. For example, both the first anti-B7H4 portion and the third anti-B7H4 portion may be in the form of full-length antibodies. 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 another example, the first anti-B7H4 portion is a half-antibody and the second anti-B7H4 portion is a single-chain half-antibody, or vice versa.

[0343] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to B7H4. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to B7H4 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0344] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-EPHA2 portion that specifically binds the same tumor antigen as the first anti-EPHA2 portion but to a different epitope. The form of the second anti-EPHA2 portion can be the same as or different from the first anti-EPHA2 portion. For example, both the first anti-EPHA2 portion and the third anti-EPHA2 portion can be in the form of full-length antibodies. For another example, the first anti-EPHA2 portion is a full-length antibody and the second anti-EPHA2 portion is an scFv, or vice versa. For 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.

[0345] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to EPHA2. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to EPHA2 triggers the activation of 4-1BB by the second antibody portion, for example, an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0346] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-CD318 portion that specifically binds the same tumor antigen as the first anti-CD318 portion but to a different epitope. The form of the second anti-CD318 portion may be the same as or different from the first anti-CD318 portion. For example, both the first anti-CD318 portion and the third anti-CD318 portion may be in the form of full-length antibodies. For another example, the first anti-CD318 portion is a full-length antibody and the second anti-CD318 portion is an scFv, or vice versa. For 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.

[0347] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CD318. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to CD318 triggers the second antibody portion to activate 4-1BB, for example, an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0348] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or bivalent 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 and the sdAb comprises: sdAb-CDR1, which comprises the amino acid sequence of SEQ ID NO:24 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR1; sdAb-CDR2, which comprises the amino acid sequence of SEQ ID NO:25 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR2; and sdAb-CDR3, which comprises the amino acid sequence of SEQ ID NO:26 or a variant thereof, the variant comprising at most about 3 (any one of about 1, 2, 3) amino acid substitutions in sdAb-CDR3. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-CLDN6 portion that specifically binds the same tumor antigen as the first anti-CLDN6 portion but to a different epitope. The form 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 in the form of full-length antibodies. For another example, the first anti-CLDN6 portion is a full-length antibody and the second anti-CLDN6 portion is an scFv, or vice versa. For 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.

[0349] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CLDN6. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to CLDN6 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0350] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent 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 and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has at least about 80% sequence identity with SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-tumor antigen portion that specifically binds the same tumor antigen as the first anti-tumor antigen portion but to a different epitope. The form of the second anti-tumor antigen portion can be the same as or different from the first anti-tumor antigen portion. For example, both the first anti-tumor antigen portion and the third anti-tumor antigen portion can be in the form of full-length antibodies. 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.

[0351] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to tumor antigens. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to the tumor antigen triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0352] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or bivalent or trispecific antibody) comprising an anti-MUC16 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant 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%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-MUC16 portion that specifically binds the same tumor antigen as the first anti-MUC16 portion but to a different epitope. The form of the second anti-MUC16 portion may be the same as or different from that of the first anti-MUC16 portion. For example, both the first anti-MUC16 portion and the third anti-MUC16 portion can be in the form of full-length antibodies. For another example, the first anti-MUC16 portion is a full-length antibody and the second anti-MUC16 portion is an scFv, or vice versa. For 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.

[0353] In some embodiments, the first antibody portion (and / or the third antibody portion) binds to MUC16 with an affinity of from about 10 -7 M to about 10 -13 M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, binding of the first binding portion to MUC16 triggers activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0354] In some embodiments, the multispecific construct described herein is a multispecific construct comprising an anti-ENPP3 antigen antibody portion and an anti-4-1BB antibody portion (e.g., a bispecific or bivalent or trispecific antibody), wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant 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%, or 99% or more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) that is comparable (e.g., the same) to the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-ENPP3 portion that specifically binds the same tumor antigen as the first anti-ENPP3 portion but to a different epitope. The form of the second anti-ENPP3 portion may be the same as or different from that of the first anti-ENPP3 portion. For example, both the first anti-ENPP3 portion and the third anti-ENPP3 portion may be in the form of full-length antibodies. 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.

[0355] In some embodiments, the first antibody portion (and / or the third antibody portion) binds to ENPP3 with an affinity of about 10 -7 M to about 10 -13 M. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to ENPP3 triggers the activation of 4-1BB by the second antibody portion, such as an activation that is enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0356] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-ROR1 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has at least about 80% sequence identity with SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more than 99% sequence identity, including any range between these values). In some embodiments, such an anti-4-1BB antibody portion has an affinity for 4-1BB (e.g., human 4-1BB) that is comparable (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-ROR1 portion that specifically binds the same tumor antigen as the first anti-ROR1 portion but to a different epitope. The form of the second anti-ROR1 portion can be the same as or different from the first anti-ROR1 portion. For example, both the first anti-ROR1 portion and the third anti-ROR1 portion can be in the form of full-length antibodies. For another example, the first anti-ROR1 portion is a full-length antibody and the second anti-ROR1 portion is an scFv, or vice versa. For 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.

[0357] In some embodiments, the first antibody portion (and / or the third antibody portion) binds to ENPP3 with an affinity of about 10-7 M to about 10 -13 The affinity of M binds to ROR1. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to ROR1 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0358] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) comprising an anti-SLC7A11 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has at least about 80% sequence identity with SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-SLC7A11 portion that specifically binds the same tumor antigen as the first anti-SLC7A11 portion but to a different epitope. The form of the second anti-SLC7A11 portion can be the same as or different from the first anti-SLC7A11 portion. For example, both the first anti-SLC7A11 portion and the third anti-SLC7A11 portion can be in the form of full-length antibodies. For another example, the first anti-SLC7A11 portion is a full-length antibody and the second anti-SLC7A11 portion is an scFv, or vice versa. For another example, the first anti-SLC7A11 portion is a half-antibody and the second anti-SLC7A11 portion is a single-chain half-antibody, or vice versa.

[0359] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10-7 M to about 10 -13 The affinity of M binds to SLC7A11. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to SLC7A11 triggers the activation of 4-1BB by the second antibody portion, such as activation enhanced by at least 10-fold, or activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0360] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific antibodies) that comprise an anti-DLL3 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has 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%, or 99% or more 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 (e.g., the same) to the affinity of an anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-DLL3 portion that specifically binds the same tumor antigen as the first anti-DLL3 portion but to a different epitope. The form of the second anti-DLL3 portion can be the same as or different from the first anti-DLL3 portion. For example, both the first anti-DLL3 portion and the third anti-DLL3 portion can be in the form of full-length antibodies. For another example, the first anti-DLL3 portion is a full-length antibody and the second anti-DLL3 portion is an scFv, or vice versa. For 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.

[0361] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to DLL3. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to DLL3 triggers the second antibody portion to activate 4-1BB, for example, an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0362] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or bivalent or trispecific antibody) comprising an anti-B7H4 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, the variant 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%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-B7H4 portion that specifically binds the same tumor antigen as the first anti-B7H4 portion but to a different epitope. The form of the second anti-B7H4 portion may be the same as or different from the first anti-B7H4 portion. For example, both the first anti-B7H4 portion and the third anti-B7H4 portion may be in the form of full-length antibodies. For another example, the first anti-B7H4 portion is a full-length antibody and the second anti-B7H4 portion is an scFv, or vice versa. For yet another example, the first anti-B7H4 portion is a half-antibody and the second anti-B7H4 portion is a single-chain half-antibody, or vice versa.

[0363] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to B7H4. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to B7H4 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0364] In some embodiments, the multispecific construct described herein is a multispecific construct (e.g., a bispecific or bivalent or trispecific antibody) comprising an anti-EPHA2 antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-4-1BB antibody portion comprises an sdAb and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has at least about 80% sequence identity with SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-EPHA2 portion that specifically binds to the same tumor antigen as the first anti-EPHA2 portion but to a different epitope. The form of the second anti-EPHA2 portion may be the same as or different from that of the first anti-EPHA2 portion. For example, both the first anti-EPHA2 portion and the third anti-EPHA2 portion may be in the form of full-length antibodies. For another example, the first anti-EPHA2 portion is a full-length antibody and the second anti-EPHA2 portion is an scFv, or vice versa. For 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.

[0365] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to EPHA2. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to EPHA2 triggers the activation of 4-1BB by the second antibody portion, for example, an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0366] In some embodiments, the multispecific constructs described herein are multispecific constructs (e.g., bispecific or bivalent or trispecific 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 and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has at least about 80% sequence identity with SEQ ID NO:27 (e.g., at least about 80%, 85%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bivalent and thus comprises a second anti-CD318 portion that specifically binds the same tumor antigen as the first anti-CD318 portion but to a different epitope. The form of the second anti-CD318 portion can be the same as or different from the first anti-CD318 portion. For example, both the first anti-CD318 portion and the third anti-CD318 portion can be in the form of full-length antibodies. For another example, the first anti-CD318 portion is a full-length antibody and the second anti-CD318 portion is an scFv, or vice versa. For 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.

[0367] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CD318. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to CD318 triggers the second antibody portion to activate 4-1BB, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0368] 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 and the sdAb comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof that has 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%, or 99% or more 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 (e.g., the same) to the affinity of the anti-4-1BB antibody portion comprising SEQ ID NO:27 for 4-1BB. In some embodiments, the first antibody portion is not an sdAb. In some embodiments, the multispecific construct is bispecific and thus comprises a second anti-CLDN6 portion that specifically binds the same tumor antigen as the first anti-CLDN6 portion but to a different epitope. The form 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 anti-CLDN6 portion and the third anti-CLDN6 portion can be in the form of full-length antibodies. For another example, the first anti-CLDN6 portion is a full-length antibody and the second anti-CLDN6 portion is an scFv, or vice versa. For 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.

[0369] In some embodiments, the first antibody portion (and / or the third antibody portion) is at about 10 -7 M to about 10 -13The affinity of M binds to CLDN6. In some embodiments, the first antibody portion (and / or the third antibody portion) is selected from scFv, Fab, and full-length antibodies. In some embodiments, the multispecific construct comprises an Fc domain (such as 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 linked by an Fc domain (such as an Fc domain derived from IgG1, IgG2, IgG3, or IgG4). In some embodiments, the binding of the first binding portion to CLDN6 triggers the activation of 4-1BB by the second antibody portion, such as an activation enhanced by at least 10-fold, or an activation that results in an increase in the production or activity of IFNγ, IL-2, or NFκB.

[0370] In some embodiments, there is provided a multispecific construct (such as a bispecific antibody) that comprises an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (such as a full-length antibody) that specifically binds to a tumor antigen (such as a human tumor antigen), and the anti-4-1BB antibody portion specifically binds to 4-1BB, and wherein the anti-4-1BB antibody portion is fused to the N-terminus of one or both heavy chains of the anti-tumor antigen antibody (such as scFv, Fab, or full-length anti-tumor antigen antibody). In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a second antibody portion (such as, sdAb), an optionally present first linker, an optionally present Fc domain, an optionally present second linker, and the heavy chain of the first antibody portion. In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a second antibody portion (such as sdAb), an optionally present first linker, and the heavy chain of the first antibody portion (such as a full-length antibody, such as a full-length antibody comprising an Fc domain).

[0371] In some embodiments, there is provided a multispecific construct (e.g., a bispecific antibody) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., scFv, Fab or full-length antibody) that specifically binds to a tumor antigen, and the anti-4-1BB antibody portion specifically binds to 4-1BB, and 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 MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a heavy chain of a first antibody portion (e.g., sdAb, scFv or Fab), an optionally present first linker, an optionally present Fc domain, an optionally present second linker, and a second antibody portion (e.g., sdAb). In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a heavy chain of a first antibody portion (e.g., a full-length antibody, such as a full-length antibody comprising an Fc domain), an optionally present first linker, and a second antibody portion (e.g., sdAb).

[0372] In some embodiments, there is provided a multispecific construct (e.g., a bispecific antibody) comprising an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., scFv, Fab or full-length antibody) that specifically binds to a tumor antigen, and the anti-4-1BB antibody portion specifically binds to 4-1BB, and wherein 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 MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a second antibody portion (e.g., sdAb), an optionally present first linker, an optionally present Fc domain, an optionally present second linker, and a light chain of a first antibody portion. In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a second antibody portion (e.g., sdAb), an optionally present first linker, and a light chain of a first antibody portion (e.g., a full-length antibody, such as a full-length antibody comprising an Fc domain).

[0373] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen, and 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 MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a light chain of a first antibody portion (e.g., sdAb, scFv, or Fab), an optionally present first linker, an optionally present Fc domain, an optionally present second linker, and a second antibody portion (e.g., sdAb). In some embodiments, the multispecific construct comprises, from the N-terminus to the C-terminus: a light chain of a first antibody portion (such as a full-length antibody comprising an Fc domain), an optionally present first linker, and a second antibody portion (e.g., sdAb).

[0374] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., a full-length antibody) that specifically binds to a tumor antigen (e.g., a human tumor antigen), and wherein the anti-4-1BB antibody portion comprises a single-domain antibody that binds to 4-1BB (e.g., human 4-1BB), and wherein the single-domain antibody is fused to the N-terminus of one or both heavy chains of the anti-tumor antigen antibody (e.g., scFv, Fab, or full-length anti-CLDN6 antibody). In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen, and wherein the anti-4-1BB antibody portion comprises a single-domain antibody that binds to 4-1BB, and wherein the single-domain antibody 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-CLDN6 antibody).

[0375] In some embodiments, a multispecific construct (e.g., a bispecific antibody) is provided that comprises an anti-tumor antigen antibody portion and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen, and the anti-4-1BB antibody portion comprises 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., scFv, Fab, or full-length anti-CLDN6 antibody). In some embodiments, the tumor antigen is selected from 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 and an anti-4-1BB antibody portion, wherein the anti-tumor antigen antibody portion comprises an antibody (e.g., scFv, Fab, or full-length antibody) that specifically binds to a tumor antigen, and the anti-4-1BB antibody portion 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., scFv, Fab, or full-length anti-tumor antigen antibody).

[0376] 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 (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 MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, n is 0 - 8. In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGSGGGGS (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).

[0377] The multispecific constructs of the present disclosure may also include a third antibody portion that binds the same antigen as one of the other two portions, but at a different site, as a bispecific. For example, the present disclosure provides bispecific (trispecific) constructs that include a first antibody portion and a third antibody portion that both bind the same tumor antigen. The first antibody portion and the third antibody portion are the same antigen but have different epitopes. In certain embodiments, the first antibody portion and the third antibody portion provided herein each have a full-length antibody / IgG form, and the second antibody portion provided herein as an anti-4-1BB antigen-binding portion has a single-domain antibody (sdAb) / VHH / nanobody form.

[0378] In certain embodiments, the first antibody portion provided herein has an IgG form, and the third antibody portion provided herein has a scFv form, and the anti-4-1BB antigen-binding portion provided herein has a single-domain antibody (sdAb) / VHH / nanobody form. 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.

[0379] In certain embodiments, the first antibody portion provided herein has a full-length antibody / IgG form, the third antibody portion provided herein has a single-chain half-antibody form, and the anti-4-1BB antigen-binding portion provided herein has a single-domain antibody (sdAb) / VHH / nanobody form. 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 this case, the first antibody portion and the third antibody portion can form a heterodimer by pairing through the Fc region.

[0380] The heterodimer paired through the Fc region can be achieved by forming a knobs-into-holes (KIH), a disulfide bond (-S-S-), or by hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

[0381] In some embodiments, provided herein is a multispecific construct 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 triggers activation of 4-1BB by the second antibody portion. In some embodiments, the tumor antigen is selected from MUC16, ENPP3, ROR1, SLC7A11, DLL3, B7H4, EPHA2, CD318, and CLDN6. In some embodiments, after binding of the first antibody portion to the tumor antigen, activation of 4-1BB by the second antibody portion 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. In some embodiments, the multispecific construct does not activate 4-1BB signaling in the absence of binding to an antigen. In some embodiments, the second portion activates 4-1BB signaling in the absence of binding to the tumor antigen. Activation of 4-1BB signal transduction is the intended mechanism of agonist antibodies such as utomilumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB portion of some antibodies of the present disclosure does not require such activity. Indeed, in some embodiments, it is preferred that the anti-4-1BB portion of the present antibody cannot independently activate 4-1BB in the absence of tumor antigen binding. As shown in the experimental examples, interestingly, when the anti-tumor antigen portion binds to the tumor antigen on the cell, such binding can trigger activation of 4-1BB signal transduction.

[0382] Compared to known anti-4-1BB agonist antibodies that are typically associated with dose-limiting on-target hepatotoxicity, the antibodies of the present disclosure are expected to be safer. Since tumor antigens are not expressed under healthy conditions, the antibodies of the present disclosure are not expected to trigger a cytotoxic immune response because they cannot activate 4-1BB signaling. Instead, in tumor tissues where tumor antigens are expressed and / or accessible, the present antibodies can elicit a potent immune response against tumor cells. Thus, unlike those anti-4-1BB antibodies currently in clinical development that suffer from on-target / innate toxicity, the antibodies of the present disclosure combine potency and safety in the treatment of cancer.

[0383] 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 activation of the immune system by the second antibody portion. 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 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 triggers the second antibody portion to increase NFκB signaling.

[0384] 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 activation of NFκB signaling by the second antibody portion.

[0385] In some embodiments, activation of NFκB signaling is assessed by measuring changes in the expression of downstream targets of NFκB signal transduction, such as cytokines, growth factors, adhesion molecules, and / or anti-apoptotic genes. In some embodiments, changes in the expression of downstream targets of NFκB signal transduction are measured by determining the RNA transcript expression levels of the downstream targets of NFκB signal transduction. Suitable methods for measuring RNA transcript levels in a sample are known in the art and include, for example, by 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, changes in the expression of downstream targets of NFκB signal transduction are measured by determining the protein expression levels of the downstream targets of NFκB signal transduction. Suitable methods for measuring protein expression in a sample are known in the art and include, for example, immunoassays (e.g., Meso Scale Discovery or MSD assay), immunohistochemistry (IHC), PET imaging, Western blot, enzyme-linked immunosorbent assay (ELISA), 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, such as 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.

[0386] In some embodiments, after the first antibody portion binds to a tumor antigen, the activation of NFκB signaling by the second antibody portion 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. In some embodiments, in the absence of binding to an antigen, the multispecific construct does not activate NFκB signal transduction.

[0387] 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 activation of the second antibody portion to increase the levels of one or more cytokines.

[0388] In some embodiments, the levels of one or more cytokines are measured by determining the RNA transcript expression levels of one or more cytokines. Suitable methods for measuring RNA transcript levels in a sample are known in the art and include, for example, by Northern blot analysis, nuclease protection assays, in situ hybridization, PCR analysis (e.g., qPCR, RT-PCR, RT-qPCR, etc.), and next-generation sequencing (e.g., RNAseq). In some embodiments, the transcript expression levels of a biomarker are measured by RT-PCR, in situ hybridization, and / or RNAseq.

[0389] In some embodiments, the levels of one or more cytokines are measured by determining the protein expression levels of one or more cytokines. Suitable methods for measuring protein expression in a sample are known in the art and include, for example, immunoassays (e.g., Meso Scale Discovery or MSD assays), immunohistochemistry (IHC), PET imaging, Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and mass spectrometry. In some embodiments, the protein expression levels of a biomarker are measured by immunoassay, Western blot, ELISA, IHC, and / or flow cytometry.

[0390] In some embodiments, after the first antibody portion binds to a tumor antigen, the levels of one or more cytokines are increased 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. In some embodiments, in the absence of binding to an antigen, the multispecific construct does not result in an increase in the levels of one or more cytokines. In some embodiments, the cytokines are IFNγ and / or IL-12.

[0391] 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 activation of the second antibody portion to increase the level of IFNγ. In some embodiments, after binding of the first antibody portion and / or the third antibody portion to the tumor antigen, the level of IFNγ 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. In some embodiments, in the absence of binding to the antigen, the multispecific construct does not result in an increase in the level of IFNγ.

[0392] 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 activation of the second antibody portion to increase the level of IL-2. In some embodiments, after binding of the first antibody portion and / or the third antibody portion to the tumor antigen, the level of IL-2 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. In some embodiments, in the absence of binding to the antigen, the multispecific construct does not result in an increase in the level of IL-2.

[0393] In some embodiments, the levels of IFNγ, IL-2, and / or NFκB signal transduction 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 tissue and / or fluid form that is known or believed to contain diseased cells and / or the target 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 or expected 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 contains one or more cancer cells.

[0394] Methods for obtaining suitable tissue and / or fluid samples (e.g., methods suitable for obtaining representative samples from a particular type, location, diseased tissue, etc.) are well known to those of ordinary skill in the art and include, 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, excisional biopsy, etc.), various other surgical tissue (e.g., tumor tissue) biopsy and / or excision strategies, and fluid collection (e.g., collecting urine, blood, serum, plasma, sputum, etc.).

[0395] Nucleic acid

[0396] Also covered are nucleic acid molecules encoding the multispecific constructs or various antibody moieties described herein. In some embodiments, a nucleic acid (or a set of nucleic acids) is provided that encodes one or more polypeptides of a multispecific construct or various antibody moieties. In some embodiments, a nucleic acid (or a set of nucleic acids) is provided that encodes a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a bispecific antibody, or a trispecific antibody) or a polypeptide moiety thereof.

[0397] Also covered herein is an isolated host cell that contains a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a bispecific antibody, or a trispecific antibody), a nucleic acid encoding the polypeptide component of the multispecific construct, or a vector containing a nucleic acid encoding the polypeptide component of the multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a bispecific antibody, or a trispecific antibody) described herein.

[0398] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions to a nucleic acid sequence encoding a multispecific construct (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a bispecific antibody, or a trispecific antibody) or various antibody moieties described herein.

[0399] This application also provides a vector into which the nucleic acid of this application is inserted.

[0400] The nucleic acid can be cloned into a variety of types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0401] In addition, the expression vector can be provided to the cell in the form of a viral vector. 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 that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain a functional origin of replication, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers in at least one organism (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0402] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties that are not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure can comprise a flexible linker sequence or can be modified to add a functional moiety (e.g., PEG, drug, toxin, or label).

[0403] The antibodies, variants, or derivatives of the present disclosure include modified derivatives, i.e., by covalently linking any type of molecule to the antibody such that the covalent linkage does not prevent the antibody from binding to the epitope. For example, but not limited to, the antibody can be modified, for example, by glycosylation, acetylation, polyethylene glycolylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, ligation to a cell ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-canonical amino acids.

[0404] In some embodiments, the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biologic response modifier, pharmaceutical agent, or PEG.

[0405] The antibody can be conjugated or fused to a therapeutic agent, which can include a detectable label, such as a radiolabel, immunomodulator, hormone, enzyme, oligonucleotide, photoactive therapeutic agent or diagnostic agent, cytotoxic agent (which can be a drug or toxin), ultrasound enhancer, non-radiolabel, combinations thereof, and other such substances known in the art.

[0406] Methods of Treatment

[0407] The present disclosure also provides methods for treating a disease or condition in an individual. The methods include administering to the individual (e.g., a mammal, such as a human) a multispecific construct described herein (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody). In some embodiments, the individual is a mammal (e.g., a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, 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.

[0408] In some embodiments of the methods, 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 lymphoma (NHL).

[0409] Compositions, kits, and articles of manufacture

[0410] The present disclosure also provides compositions (e.g., formulations) that include any of the multispecific constructs described herein (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a diabody, or a trispecific antibody), a nucleic acid encoding any of the multispecific constructs or a portion thereof, a vector that includes a nucleic acid encoding one of the multispecific constructs, or a host cell that includes the nucleic acid or the vector.

[0411] Suitable formulations of the multispecific constructs described herein (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a diabody, or a trispecific antibody) can be obtained by mixing the multispecific construct having the desired purity with a pharmaceutically acceptable carrier, excipient, or stabilizer, if present (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0412] The present disclosure also provides kits that include any of the multispecific constructs described herein (e.g., an anti-tumor antigen / anti-4-1BB bispecific antibody, a diabody, or a trispecific antibody). The kits can be used in any of the treatment methods described herein.

[0413] The kits of the present application are in a suitable package. Suitable packages include, but are not limited to, vials, bottles, cans, flexible packages (e.g., sealed Mylar or plastic bags), etc. The kits can optionally provide additional components, such as buffers and explanatory information.

[0414] Accordingly, the present application also provides a finished product. The finished product may include a container and a label or a packaging insert attached to or associated with the container. Suitable containers include vials (such as sealed vials), bottles, cans, flexible packages, etc. Generally, the container contains the composition and may have a sterile access port (for example, the container may be an intravenous infusion bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0415] Those skilled in the art will recognize that there can be various embodiments within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention, but should not be construed as limiting its scope in any way.

[0416] Embodiment

[0417] The following examples are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the present invention. They are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to indicate that the following experiments are all the experiments conducted or the only experiments. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. The following examples are only intended as pure examples of the present application and should not be regarded as limiting the present application in any way. The following examples and detailed description are provided as examples only, not as limitations.

[0418] Example 1: Generation of CLDN6 X 4-1BB Bispecific Antibody

[0419] Design and generate the exemplary CLDN6 x 4-1BB bispecific antibodies shown in Table 3 below.

[0420] Table 3. Exemplary CLDN6 x 4-1BB Bispecific Antibodies

[0421]

[0422]

[0423]

[0424]

[0425] Example 2. Antigen-Binding Activity of CLDN6 x 4-1BB BsAb

[0426] 2.1 Binding Affinity of CLDN6 x 4-1BB BsAb to CLDN6

[0427] The binding affinities of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT (prepared as in Example 1) to human CLDN6 were measured by surface plasmon resonance (SPR). As Figures 1A - 1C shown, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bind to CLDN6 virus-like particles (VLPs), with K D values of 1.61×10 -9 M and 2.36×10 -9 M, respectively.

[0428] 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. Then, a phycoerythrin (PE)-conjugated anti-human IgG antibody was added after washing, and the cells were further incubated at 4°C for 30 minutes. The mean fluorescence intensity (MFI) of PE was evaluated by FACS. As Figure 2A shown, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bind to CLDN6-expressing cells in a concentration-dependent manner.

[0429] OVCAR3 and OV90 are human ovarian cancer cell lines with endogenous CLDN6 expression levels. As Figure 2B and 2C shown, both CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT are able to bind to OVCAR3 and OV90. Overall, the BsAb containing the anti-CLDN6-1 antibody portion has a binding affinity to human CLDN6 comparable to that of the parental CLDN6-1 antibody to human CLDN6. The binding signal is closely related to the CLDN6 expression level on the surfaces of OVCAR3 and OV90 cells.

[0430] 2.2 Binding Affinity of CLDN6x4-1BB BsAb to 4-1BB

[0431] The binding affinities of CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT to human 4-1BB were measured by SPR. As Figures 3A - 3D shown, CLDN6-1x4-1BB NA and CLDN6-1x4-1BB WT bind to monomeric human 4-1BB, with KD are 1.640×10 -8 M and 1.573×10 -8 M, respectively. The affinity of the parental anti-4-1BB sdAb antibody conjugated with the IgG1 Fc fragment (4-1BB sdAb-Fc) for 4-1BB was measured in parallel and found to have a K -9 of 4.393×10 D M, indicating that the 4-1BB antibody portion in the BsAb has an affinity for 4-1BB comparable to that of 4-1BB sAb-Fc for 4-1BB.

[0432] The binding of CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT to soluble recombinant human 4-1BB was analyzed by ELISA. As Figure 4A shown, both CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT bound to recombinant human 4-1BB in a concentration-dependent manner, with EC50 values of 0.129 nM and 0.078 nM, respectively. These EC50 values were comparable to those of 4-1BB sdAb-Fc. In addition, the binding of CLDN6-1 x 4-1BB NA and CLDN6-1x 4-1BB WT to HEK293 cells expressing 4-1BB was evaluated by FACS. As Figure 4B shown, both CLDN6-1 x 4-1BB NA and CLDN6-1x4-1BB WT were able to bind to 4-1BB, with EC50 values of 0.406 nM and 0.347 nM, respectively. These EC50 values were comparable to those of 4-1BB sdAb-Fc.

[0433] Example 3. Functional activity of CLDN6-1 x 4-1BB BsAb

[0434] 3.1 Cell line-based functional characterization of CLDN6 x 4-1BB BsAb

[0435] To test the ability of the CLDN6 x 4-1BB bispecific antibody to activate 4-1BB signaling, the GloResponse TM NFκB-luc2 / 4-1BB Jurkat cell line stably expressing 4-1BB and the NFκB luciferase reporter gene was used as the effector cell, and cells expressing CLDN6 (CHO-K1 CLDN6, OVCAR3, or OV90) were used as the target cells. The RKO colon cancer cells that do not express CLDN6 were used as a negative control.

[0436] Briefly, the GloResponse TMNFκB-luc2 / 4-1BB Jurkat cells (at a density of 5.0 x 10 4 cells per well) were mixed with 5.0 x 10 4 target cells in a white 96-well plate. The antibodies were serially diluted and added to the plate. Luminescence was measured after incubation at 37 °C for 6 hours. As Figures 5A to 5D shown, urelumab triggered 4-1BB activation regardless of CLDN6 expression, while the 4-1BB sdAb-Fc of the present application did not have agonist activity under the same experimental settings, although it was able to bind to 4-1BB. Similarly, CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT induced NFκB activity in the presence of all CLDN6-expressing target cells and was independent of the CLDN6 expression level. In contrast, when RKO cells that do not express CLDN6 were used as target cells, CLDN6-1 x 4-1BB NA and CLDN6-1 x 4-1BB WT led to significantly lower 4-1BB activation compared to urelumab, as Figure 5D shown.

[0437] 3.2 Activity of CLDN6 x 4-1BB BsAb in promoting immune responses of human peripheral blood mononuclear cells (PBMCs)

[0438] 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 the 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 homogeneous HTRF assay.

[0439] As Figures 6A - 6F shown, when PBMCs were co-cultured with CLDN6-expressing target cells, CLDN6-1 X 4-1BB NA and CLDN6-1 X 4-1BB WT stimulated the production of IL-2 and IFNγ. However, in the presence of RKO that does not express CLDN6, CLDN6-1X 4-1BB NA and CLDN6-1 X 4-1BB WT did not stimulate PBMCs to produce IL-2 or IFNγ, as Figure 6G and 6H shown, indicating that the activity of CLDN6-1 X 4-1BB NA and CLDN6-1X4-1BB WT is dependent on the presence of tumor antigens. In contrast, 4-1BBsdAb-Fc was inactive in this assay.

[0440] Example 4. Tumor growth inhibition by CLDN6-1 x 4-1BB BsAb

[0441] CT26 cells endogenously expressing CLDN6 were subcutaneously implanted into BALB / c humanized 4-1BB mice. When the tumors grew to the average value of 100 mm 3 , the mice were intraperitoneally injected with (a) human IgG1, (b) CLDN6-1 x 4-1BB NA (2 mg / kg), (c) CLDN6-1 x 4-1BB WT (2 mg / kg), or (d) a combination of the parental CLDN6-1 antibody and 4-1BB sAb-Fc (1.8 mg / kg and 0.7 mg / kg). The treatment was performed twice a week for a total of 6 doses. Tumor growth was monitored by volume measurement. As Figure 7A and 7B shown, both CLDN6-1 x 4-1BB WT and CLDN6-1 x 4-1BB NA exhibited anti-tumor activity, with CLDN6-1 x 4-1BB WT achieving an even stronger activity and a tumor growth inhibition (TGI) of 75%.

[0442] Example 5. Hepatic toxicity evaluation of CLDN6 X 4-1BB BsAb

[0443] The main problem with t4-1BB agonist antibody therapy is dose-limiting hepatic toxicity, as observed in the clinical development of urelumab. The most common adverse events are elevated alanine transaminase (ALT), elevated aspartate transaminase (AST), and fatigue. Therefore, the hepatic toxicity of CLDN6-1 x 4-1BB WT and CLDN6-1 x 4-1BB NA was further evaluated.

[0444] Briefly, after treating CLDN6-1 x 4-1BB WT or CLDN6-1 x 4-1BBNA twice a week at different doses, blood samples were collected from hu4-1BB mice for ALT and AST measurements. As Figures 8A - 8B shown, no significant increase in ALT and AST was observed, indicating a low risk of hepatic toxicity induced by other 4-1BB agonist antibodies.

[0445] Example 6. Generation of MUC16 antibody

[0446] Panning based on phage ELISA and phage FACS (OVCAR3 cells)

[0447] A human naïve Fab phage library was panned with the MUC16 antigen (human MUC16 AA13789-14451) to obtain 13 unique hits C25, C73, D1, D20, D30, D41, D46, D57, D79, D100, B76, B195, and B218 (Table 4).

[0448] Table 4. Sequences of anti-MUC16 monoclonal antibodies (mAbs)

[0449]

[0450]

[0451]

[0452]

[0453] Example 7. Binding activity of MUC16 mAbs

[0454] The binding activity of MUC16 mAbs was detected by ELISA using different fragments of human MUC16 antigen ( Figures 9A - 9C ). Similar to M16-2 but different from M16-1, all 13 mAbs bound to the AA13789-14197 domain of human MUC16. M16-1 and M16-2 are reference antibodies, and their sequences are listed in amino acid sequence table A.

[0455] Cross-species testing showed that, except for D46, the other 12 mAbs were able to cross-react with cyno MUC16 ( Figure 10 ).

[0456] OVCAR3 has the highest endogenous MUC16 expression level. In the cell-binding assay, all 13 mAbs (human IgG1 Fc) were able to bind to OVCAR3 cells ( Figures 11A - 11B ). In contrast, the 13 mAbs (human IgG1 Fc) did not bind to MUC16-negative ES-2 cells ( Figure 11C ).

[0457] For five selected clones C25, D30, D100, D57, and B218, a cell-based binding assay was performed ( Figures 12A - 12C ). All mAbs showed efficient binding to hu MUC16 in cells, while h3A5 did not bind to HEK293-MUC16(13810-14507) ( Figures 12A - 12C ). Among the three human MUC16-positive cell lines, OVCAR3 has the highest endogenous MUC16 expression level. M16-2 and 3A5 are reference antibodies, and their sequences are listed in amino acid sequence table A.

[0458] In another cell-based binding assay, soluble CA125 (cancer antigen 125, an extracellular shedding protein encoded by the MUC16 gene and routinely used as a serum marker for monitoring ovarian cancer patients) from patient ascites was present or absent. After adding soluble CA125, the binding ability of 5 mAbs to OVCAR3 did not significantly decrease, indicating that the mAbs bind to epitopes different from CA125. However, after adding 5000 U / mL soluble CA125, the binding of the reference 3A5 to OVCAR3 decreased significantly ( Figures 13A - 13D ).

[0459] The kinetics of the binding assay showed the KD values of D57, B218, and C25 respectively ( Figures 14A - 14B ).

[0460] Example 8. MUC16 x 4-1BB bispecific antibody

[0461] Design and generate the exemplary MUC16 x 4-1BB bispecific antibodies shown in Table 5 below.

[0462] Table 5. Exemplary MUC16 x 4-1BB bispecific antibodies

[0463]

[0464]

[0465]

[0466] In the 4-1BB NFκB reporter gene assay, D57-4B and B218-4B BsAbs can stimulate 4-1BB signals in the presence of MUC16 high (+++, OVCAR3), medium (++, SNU216), and low (+, HCC827) expressing cells, with stronger signals than urelumab ( Figures 15A - 15C ). In contrast, in negative cells (ES-2), urelumab can stimulate 4-1BB activation, while the BsAbs hardly induce activation signals ( Figure 15D ).

[0467] In another 4-1BB BsAb activity assay for measuring PBMC cytokine release in MUC16 high (+++, OVCAR3), medium (++, SNU216), and low (+, HCC827) expressing cells, D57-4B showed the strongest T cell co-stimulation activity (inducing human IFNγ and IL-2 cytokine release), and B218-4B showed T cell co-stimulation activity comparable to urelumab ( Figures 16A - 16F ).

[0468] In a further 4-1BB BsAb activity assay for measuring CD8+ T cell cytokine release in cells with high (+++, OVCAR3), medium (++, SNU216), and low (+, HCC827) expression of MUC16, D57-4B showed the strongest CD8+ T cell co-stimulatory activity (inducing human IFNγ and IL-2 cytokine release), and B218-4B showed CD8+ T cell co-stimulatory activity comparable to urelumab ( Figures 17A - 17F ).

[0469] Example 9. Generation of Bispecific and Bifunctional ROR1 x 4-1BB Antibodies

[0470] The construction of ROR1 x 4-1BB bispecific and bifunctional antibodies is shown in Table 6A below.

[0471] Table 6A. Designed Bispecific and Trispecific Antibody Sequences

[0472]

[0473]

[0474]

[0475]

[0476] Example 9. Binding Activity of Bispecific and Bifunctional ROR1 x 4-1BB Antibodies

[0477] 9.1 Chimeric Antibodies

[0478] Binding Affinity

[0479] FACS binding activity showed that ROR1 x 4-1BB BsAb binds to tumor cells A549 endogenously expressing ROR1 and HEK293 cells overexpressing 4-1BB in a dose-dependent manner ( Figures 18A - 18B ).

[0480] Epitope Binning

[0481] In the epitope binning of anti-ROR1 mAbs, 3C5 and 8F2 showed that the binding epitopes to the ROR1 antigen were non-competitive or non-overlapping, as detected by Octet (see Figures 20A - 20C ).

[0482] Based on the non-overlapping binding epitopes of 3C5 and 8F2 to ROR1, a bispecific ROR1 x 4-1BB antibody (Table 6A) was designed. The design hypothesis was that an antibody targeting the antigen with bispecificity could cause more 4-1BB aggregation and induce stronger activation of the 4-1BB pathway.

[0483] Reporter gene assay

[0484] In this assay, Jurkat cells co-expressing 4-1BB and NFKB-luciferase reporter genes were used as the reporter cell line, and ROR1-positive or -negative cell lines were used as target cells. The reporter cells and target cells were co-cultured with serially diluted ROR1 x 4-1BB BsAb or anti-4-1BB urelumab for 6 hours, and then the 4-1BB NFKB activation signal was read out using One-Glo TM reagent. In this experiment, 4B-3C5 and 4B-8F2 BsAb showed strong ROR1-dependent 4-1BB activation ( Figures 19A - 19C ).

[0485] A similar reporter gene assay was performed using the bispecific ROR1 x 4-1BB antibody. The results showed that the bispecific ROR1 x 4-1BB TsAb in the 4-BiR1 form showed the best 4-1BB activation (see Figures 21A - 21C ).

[0486] 9.2 Humanized antibodies

[0487] Humanized anti-ROR1 antibodies were used to design mono-topic (bispecific) and bispecific ROR1 x 4-1BB TsAb (see Table 6B).

[0488] Table 6B. Humanized bispecific and bispecific antibodies

[0489]

[0490]

[0491]

[0492] Cell binding affinity and reporter gene assay

[0493] Cell binding of the humanized anti-ROR1 mAb was measured by FACS in a similar manner as above, and the ROR1-dependent 4-1BB activation of the humanized ROR1 x 4-1BB BsAb was tested. 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 ) In contrast, urelumab does not activate 4-1BB in ROR1-positive tumor cell lines within the corresponding concentration range.

[0494] Comparison among the humanized antibodies showed that the bispecific antibody 4-BiR1 had higher cellular binding to ROR1 ( Figures 23A - 23C ) and higher ROR1-dependent 4-1BB activation ( Figures 23D - 23F ) in ROR1-positive tumor cell lines compared to 4B-3C5 and 4B-8F5, while 4B-3C5 and 4B-8F5 showed similar cellular binding affinities and ROR1-dependent 4-1BB activation. In ROR1-negative tumor cell lines, no ROR1-independent 4-1BB activation was observed for all tested antibodies.

[0495] SPR

[0496] Surface plasmon resonance assays showed that the humanized antibody 4B-h3C5 had a K D value of 7.93E-08 M for the binding affinity to ROR1, 4B-h8F5 had a K D value of 2.37E-07 M for the binding affinity to ROR1, and 4B-hBiR1 had a K D value of 1.21E-08 M for the binding affinity to ROR1 ( Figures 24A - 24C ).

[0497] In vivo efficacy

[0498] A syngeneic model (4-1BB humanized mouse (Biocytogen)) inoculated with the ROR1-MC38 cell line was used to evaluate the in vivo efficacy of the monospecific and bispecific ROR1×4-1BB. All tested humanized antibodies showed significant inhibition of tumor growth ( Figures 25A - 25B ).

[0499] 9.3 Affinity maturation of antibodies

[0500] Given the low yield of 4-hBiR1, potential PTMs in the anti-ROR1 sequence, and the low affinities of h3C5 and h8F2, antibodies were engineered with affinity maturation (Tables 6C and 6D).

[0501] Table 6C. Designed bispecific antibody sequences (affinity matured)

[0502]

[0503]

[0504]

[0505]

[0506] Table 6D. Designed bispecific antibody sequences (affinity matured)

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514] Reporter gene assay

[0515] Similar reporter gene assays were performed using the affinity matured bispecific or bis complementary site ROR1 x 4-1BB antibodies. The results showed that the ROR1-dependent 4-1BB activation of the affinity matured bispecific antibody was better compared to the humanized form ( Figures 26A - 26E ). The ROR1-dependent 4-1BB activation of the affinity matured bis complementary site antibody was significantly better compared to the humanized bispecific form and the benchmark ROR1×4-1BB BsAb (clone ID: BA6(NA)×1A10 M12) from patent WO2021 / 101346A1 ( Figures 27A - 27C ).

[0516] Cytokine release assay

[0517] 4B-2773, 4B-27, and 4B-73 were selected as representatives of the engineered single epitope and bis complementary site ROR1 x 4-1BB antibodies to be compared with 4B-hBiR1 and the benchmark (clone ID: BA6(NA)x1A10 M12) in the cytokine release assay. 1 μg / ml anti-CD3 (OKT3) was coated on the plate, and the MDA-MB-231 cell line was used as the target cell. PBMC and serially diluted antibodies were co-cultured for 72 hours. IFN-γ and IL-2 induced by 4-1BB were detected. The results showed that all the tested engineered ROR1 x 4-1BB antibodies released higher cytokines than the benchmark and urelumab ( Figures 28A - 28B ).

[0518] In vivo efficacy

[0519] A syngeneic model using ROR1-MC38 cell line (4-1BB humanized mice (Biocytogen)) was used to evaluate the in vivo efficacy of engineered single epitope and dual epitope ROR1×4-1BB. All tested humanized antibodies showed significant inhibition of tumor growth ( Figures 29A - 29C ).

[0520] The scope of the present disclosure is not limited by the specific embodiments described, which are merely separate examples of various aspects of the present disclosure, and any composition or method that is functionally equivalent is within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0521] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0522] The present invention is described in terms of specific embodiments discovered or proposed by the inventors, which include preferred modes of practicing the present invention. Those skilled in the art will appreciate that, given the present disclosure, many modifications and variations can be made to the specific embodiments exemplified without departing from the intended scope of the present invention. For example, due to codon redundancy, changes can be made to the underlying DNA sequence without affecting the protein sequence. Additionally, considering biological functional equivalence, changes can be made to the protein structure without affecting the type or degree of biological action. All such modifications are intended to be included within the scope of the appended claims.

[0523] Summary Table A of Amino Acid Sequences

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532] Summary Table B of Amino Acid Sequences (Anti-ROR1)

[0533]

[0534]

[0535]

[0536]

[0537]

Claims

1. A multispecific construct 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, wherein binding of the first antibody portion to the tumor antigen triggers activation of 4-1BB by the second antibody portion.

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

3. The multispecific construct of claim 1 or 2, wherein the first antibody moiety is selected from a full-length antibody, a half-antibody, a single-chain half-antibody, a Fab, a Fab′, an F(ab′) 2 and an scFv.

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

5. The multispecific construct according to any one of claims 1-4, wherein the second antibody moiety is selected from a full-length antibody, a single-chain half-antibody, Fab, Fab′, F(ab′) 2 , scFv, and sdAb.

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

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

27.

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

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

26.

10. The multispecific construct of any one of claims 1-9, wherein the second antibody portion comprises the amino acid sequence of SEQ ID NO:27 or a variant thereof, and the variant has at least about 80% sequence identity with SEQ ID NO:

27.

11. The multispecific construct of any one of claims 1-10, wherein the multispecific construct is a bispecific antibody or a bispecific binding fragment.

12. The multispecific construct of claim 11, wherein the Fc domain is derived from any one selected from 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 with any one of SEQ ID NO:46-56, 285-286, and 288-289.

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

16. A multispecific construct according to any one of claims 1-15, wherein activation of 4-1BB by the second antibody moiety results in an increase in IFNγ level, IL-2 level or NFκB signal transduction.

17. A multispecific construct according to any one of claims 1-16, wherein the first antibody portion has a binding affinity of from about 10 -7 M to about 10 -13 M.

18. A multispecific construct according to any one of claims 1-17, wherein the first antibody moiety is fused to the C-terminus of the second antibody moiety.

19. A multispecific construct according to any one of claims 1-17, wherein the first antibody moiety is fused to the N-terminus of the second antibody moiety.

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

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

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

23. A multispecific construct according to any one of claims 1-22, further comprising a third antibody moiety that specifically binds to a second tumor antigen.

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

25. The multispecific construct of claim 23, wherein the third antibody moiety is selected from the group consisting of a full-length antibody, a half antibody, a single-chain half antibody, Fab, Fab′, F(ab′) 2 and scFv.

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

27. The multispecific construct of claim 26, wherein 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.

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

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

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

31. A nucleic acid encoding a multispecific construct of any one of claims 1-29.

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

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

34. 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 of any one of claims 1-29, or the pharmaceutical composition of claim 30.

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

36. Use of a multispecific construct of any one of claims 1-29 in the manufacture of a medicament for treating a disease or condition in a subject in need thereof.

37. An antibody or antigen-binding fragment thereof that is specific for human mucin 16 (MUC16) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from: (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).

38. The antibody or antigen-binding fragment thereof of claim 37, which comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161 and 169, or a peptide having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:63, 71, 79, 87, 95, 103, 121, 129, 137, 145, 153, 161 and 169.

39. The antibody or antigen-binding fragment thereof of claim 37, which comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162 and 170, or a peptide having at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO:64, 72, 80, 88, 96, 104, 122, 130, 138, 146, 154, 162 and 170.

40. The antibody or antigen-binding fragment thereof according to any one of claims 37-39, which comprises (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.

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

42. The antibody or antigen-binding fragment thereof according to any one of claims 37 - 39, which further comprises 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 and a second antibody portion, wherein the first antibody portion is specific for the human MUC16 protein and the second antibody portion is specific for a second protein, and wherein the first antigen-binding portion comprises the antibody or antigen-binding fragment thereof according to any one of claims 37 - 42.

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

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

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

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

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

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

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

27.

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

52. An isolated cell comprising one or more polynucleotides encoding an antibody or an antigen - binding fragment thereof according to any one of claims 37 - 42, or a bifunctional molecule according to any one of claims 43 - 50.

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

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

55. The method of claim 54, wherein the cancer is selected from ovarian cancer, prostate cancer, urinary tract cancer, 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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