Nectin4 targeting trispecific protein for treatment of cancer

Abstract

Provided herein are NECTIN4 binding proteins, pharmaceutical compositions comprising such proteins or fragments thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such NECTIN4 binding proteins. Also disclosed are methods of using the disclosed NECTIN4 binding proteins in the prevention, and / or treatment diseases, conditions and disorders.

Description

WSGR Docket No.47517-766.601 NECTIN4 TARGETING TRISPECIFIC PROTEIN FOR TREATMENT OF CANCER CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 478,343, filed January 3, 2023, which is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BACKGROUND OF THE DISCLOSURE

[0003] NECTIN4 is a protein that is a member of the nectin family, encoded by NECTIN4. NECTIN4 is a type I transmembrane polypeptide. It is involved in forming and maintaining adherens junctions and tight junctions. Studies have shown that aberrant NECTIN4 protein overexpression is associated with several carcinomas, such as lung, ovarian, pancreatic, bladder, and breast cancers. Carcinomas with high NECTIN4 expression is associated with increased disease recurrence, drug resistance, and is a poor prognostic factor for survival.

[0004] There is a need for a greater choice of treatment options which allows physicians to select the therapeutic with the best side effect profile for an individual patient. The present disclosure provides novel polypeptides and protein therapeutics useful in methods of treatment, particularly for treatment of conditions associated with abnormal expression of NECTIN4. SUMMARY OF THE DISCLOSURE

[0005] Provided herein is an NECTIN4 binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133- 198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264. In some embodiments, the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequenceWSGR Docket No.47517-766.601 selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264. In some embodiments, an amino acid sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66. In some embodiments, the NECTIN4 binding domain is part of a multispecific protein. In some embodiments, the multispecific protein further comprises a CD3 binding domain. In some embodiments, the multispecific protein comprises an active drug format. In some embodiments, the multispecific protein further comprises a bulk serum protein binding domain. In some embodiments, the bulk serum protein comprises a serum albumin protein. In some embodiments, the serum albumin protein comprises a human serum albumin protein. In some embodiments, the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 612. In some embodiments, the CD3 binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 613. In some embodiments, the multispecific protein comprises a sequence that is at least about 75% identical to the sequence as set forth in SEQ ID NOS: 265-329. In some embodiments, the bulk serum protein binding domain is a binding moiety comprising a linker and a masking moiety, wherein the masking moiety is capable of masking or masks the binding of the NECTIN4 binding domain or the CD3 binding domain, to their respective targets. In some embodiments, the multispecific protein comprises a non-cleavable prodrug format. In some embodiments, the masking moiety comprises a sequence selected from the group consisting of: SEQ ID NOS: 721- 765, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 721-765. In some embodiments, the linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770. In some embodiments, the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 613. In some embodiments, the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 330-347. In some embodiments, the active drug comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 330-347.

[0006] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of an NECTIN4 bindingWSGR Docket No.47517-766.601 domain according to descriptions herein, or a pharmaceutical composition comprising the same, to the subject. In some embodiments, the subject is human.

[0007] Provided herein is a conditionally active NECTIN4 binding protein comprising a binding moiety (M) which comprises a non-CDR loop, a cleavable linker (L), a first target antigen binding domain (T1), and a second target antigen binding domain (T2), wherein at least one of the first target antigen binding domain (T1) and the second target antigen binding domain (T2) comprises an NECTIN4 binding domain, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264, wherein the non-CDR loop is capable of binding to the NECTIN4 binding domain or the second target antigen binding domain, and wherein the binding moiety is capable of masking or masks the binding of the NECTIN4 binding domain or the second target antigen binding domain to its target. In some embodiments, the binding moiety comprises a masking moiety and wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOS: 721-765, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOS: 721-765. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 698-704, 718-720, and 766- 776, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 698-704, 718-720, and 766-776. In some embodiments, the binding moiety comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the second target antigen binding domain (T2) comprises a CD3 binding domain. In some embodiments, the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 613.

[0008] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a conditionally active chimeric antigen receptor according to descriptions herein, or a pharmaceutical composition comprising the same, to the subject. In some embodiments, the subject is human.WSGR Docket No.47517-766.601

[0009] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a conditionally active NECTIN4 binding protein according to descriptions herein, or a pharmaceutical composition comprising the same, to the subject. In some embodiments, the subject is human.

[0010] In some embodiments, the binding domain is a humanized antibody or an antigen binding fragment thereof. In some embodiments, the binding domain is a single domain antibody, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a Fab’, a non-Ig domain, a ligand, a knottin, or a small molecule entity. In some embodiments, binding domain comprises the single domain antibody. In some embodiments, the binding domain binds to NECTIN4 with a binding affinity (Kd) of about 0.001 nM to about 500 nM. In some embodiments, the binding domain binds to human NECTIN4, mouse NECTIN4, cynomolgus NECTIN4, or a combination thereof.

[0011] Provided herein is a multispecific protein comprising an NECTIN4 binding domain, wherein the NECTIN4 binding domain is according to descriptions herein. In some embodiments, the NECTIN4 binding domain according to descriptions herein (anti-NECTIN4 domain), and a CD3 binding domain (anti-CD3 domain). In some embodiments, the anti-NECTIN4 domain and the anti-CD3 domain are in an anti-NECTIN4:anti-CD3 orientation. In some embodiments, the anti-NECTIN4 domain and the anti-CD3 domain are in an anti-CD3: anti-NECTIN4 orientation. In some embodiments, the NECTIN4 binding domain according to descriptions herein (anti- NECTIN4 domain), the CD3 binding domain (anti-CD3 domain), and an albumin binding domain (anti-ALB domain). In some embodiments, the anti-CD3 domain comprises an amino acid as set forth in SEQ ID NO: 613. In some embodiments, the anti-ALB domain comprises an amino acid sequence as set forth in SEQ ID NO: 612. In some embodiments, the anti-NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ALB: anti-NECTIN4 orientation. In some embodiments, the anti-NECTIN4 domain, the anti-CD3 domain, and the anti- ALB domain are in an anti-NECTIN4: anti-ALB: anti-CD3 orientation. In some embodiments, the anti-NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti- NECTIN4: anti-CD3 orientation. In some embodiments, the anti-NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-NECTIN4: anti-ALB orientation. In some embodiments, the anti-NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-CD3: anti-NECTIN4 orientation. In some embodiments, the anti- NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-NECTIN4: anti- CD3: anti-ALB orientation.WSGR Docket No.47517-766.601

[0012] Provided here in is a multivalent protein comprising a sequence as set forth in any one of SEQ ID NOS: 265-329. Provided here in is a multivalent protein comprising a sequence as set forth in any one of SEQ ID NOS: 330-347.

[0013] Provided herein is an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 265-329. Provided herein is an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 330-347. Provided herein is an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 1-66.

[0014] Provided herein is a pharmaceutical composition comprising (i)(a) a NECTIN4 binding domain according to descriptions herein; (i)(b)a conditionally active NECTIN4 binding protein according to descriptions herein; (i)(c) a multispecific protein according to descriptions herein; (i)(d) a multivalent protein according to descriptions herein; or (i)(e) an active drug according to descriptions herein, and (ii) a pharmaceutically acceptable carrier.

[0015] Provided herein is a process for producing a NECTIN4 binding domain according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the NECTIN4 binding domain according to descriptions herein, under conditions allowing the expression of the NECTIN4 binding domain and recovering and purifying the produced protein from the culture.

[0016] Provided herein is a process for producing a multispecific protein according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multispecific NECTIN4 binding protein according to descriptions herein under conditions allowing the expression of the multispecific protein and recovering and purifying the produced protein from the culture.

[0017] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering an NECTIN4 binding domain according to descriptions herein, or a pharmaceutical composition according to descriptions herein, to the subject.

[0018] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the multispecific protein according to descriptions herein, a multivalent protein according to descriptions herein, an active drug according to descriptions herein, or a pharmaceutical composition according to descriptions herein, to the subject. In some embodiments, the subject is human. In some embodiments, the method further comprises administration of an agent in combination with an NECTIN4 binding domain according to descriptions herein, a multispecific protein according to descriptions herein, aWSGR Docket No.47517-766.601 multivalent protein according to descriptions herein, an active drug according to descriptions herein, or a pharmaceutical composition according to descriptions herein. In some embodiments, the NECTIN4 binding domain selectively binds to tumor cells expressing NECTIN4. In some embodiments, the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the tumorous disease comprises at least one of: a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a lung cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a bladder cancer, a prostate cancer, a cervical squamous cell carcinoma, a endocervical adenocarcinoma, a uterine corpus endometrial carcinoma, a gall bladder cancer, a head and neck cancer, e.g., a squamous cell carcinoma of head and neck, an esophageal cancer, a hepatocellular carcinoma, and a skin cancer, a combination thereof. In some embodiments, the method further comprises administration of an agent in combination with conditionally active chimeric antigen receptor according to descriptions herein, conditionally active NECTIN4 binding protein according to any one of descriptions herein, or a pharmaceutical composition comprising the same. In some embodiments, the NECTIN4 binding domain selectively binds to tumor cells expressing NECTIN4. In some embodiments, the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the tumorous disease is at least one of: a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a lung cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a bladder cancer, a prostate cancer, a cervical squamous cell carcinoma, endocervical adenocarcinoma, a uterine corpus endometrial carcinoma, a gall bladder cancer, a head and neck cancer, e.g., a squamous cell carcinoma of head and neck, an esophageal cancer, a hepatocellular carcinoma, and a skin cancer, or any combination thereof.

[0019] Provided herein is a process for producing a conditionally active chimeric antigen receptor according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the conditionally active chimeric antigen receptor according to descriptions herein, under conditions allowing the expression of the conditionally active chimeric antigen receptor and recovering and purifying the produced protein from the culture.

[0020] Provided herein is a process for producing a conditionally active NECTIN4 binding protein according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the conditionally active NECTIN4 binding protein according to descriptions herein, under conditions allowing the expression of the conditionally active NECTIN4 binding protein and recovering and purifying the produced protein from the culture.WSGR Docket No.47517-766.601

[0021] Provided herein is a process for producing a multivalent protein according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multivalent protein according to descriptions herein, under conditions allowing the expression of the multivalent protein and recovering and purifying the produced protein from the culture.

[0022] Provided herein is a process for producing an active drug according to descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the active drug according to descriptions herein, under conditions allowing the expression of the active drug and recovering and purifying the produced drug from the culture.

[0023] Provided herein is a pharmaceutical composition comprising: (i)(a) a conditionally active chimeric antigen receptor according to descriptions herein; or (i)(b) a conditionally active NECTIN4 binding protein according to descriptions herein, and (ii) a pharmaceutically acceptable carrier.

[0024] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the conditionally active chimeric antigen receptor according to descriptions herein, or the pharmaceutical composition according to descriptions herein, to the subject.

[0025] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering the conditionally active NECTIN4 binding protein according to descriptions herein, or the pharmaceutical composition according to descriptions herein, to the subject. In some embodiments, the subject is human. In some embodiments, the tumorous disease comprises at least one of: a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a lung cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a bladder cancer, a gall bladder cancer, a head and neck squamous cell cancer, a squamous cell carcinoma of head and neck, an esophageal cancer, a hepatocellular carcinoma, and a skin cancer.

[0026] Provided herein is a method of increasing a therapeutic index of an NECTIN4 binding domain, the method comprising conjugating the NECTIN4 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for the NECTIN4 binding domain, wherein the NECTIN4 binding domain is masked from binding its target by the binding moiety, wherein the NECTIN4 binding domain is able to bind its target upon cleavage of the cleavable linker. In some embodiments, the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3,WSGR Docket No.47517-766.601 wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67- 132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199- 264. In some embodiments, the NECTIN4 binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, wherein the multispecific protein further comprises a CD3 binding domain. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 613. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770. In some embodiments, the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the NECTIN4 binding domain conjugated to the binding moiety is part of a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor further comprises at least one of: a transmembrane domain, an intracellular signaling domain, and a costimulatory domain. In some embodiments, the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-WSGR Docket No.47517-766.601 198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66. In some embodiments, the NECTIN4 binding domain comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

[0027] Provided herein is a method of increasing a therapeutic index of an NECTIN4 binding protein comprising a first target antigen binding domain and a second target antigen binding domain, wherein at least one of the first and the second target antigen binding domain comprises an NECTIN4 binding domain, the method comprising conjugating the first or the second target antigen binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for the first or the second target antigen binding domain, wherein at least one of the first or the second target antigen binding domain is masked from binding its target by the binding moiety, and wherein the first or the second target antigen binding domain that is masked, is able to bind its target upon cleavage of the cleavable linker. In some embodiments, the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264. In some embodiments, the non-CDR loop comprises a binding site specific for the NECTIN4 binding domain. In some embodiments, at least one of the first or the second target antigen binding domain comprises a CD3 binding domain. In some embodiments, the non-CDR loop comprises a binding site specific for the CD3 binding domain. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 613. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ IDWSGR Docket No.47517-766.601 NOS: 1-66. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

[0028] Provided herein is a method of increasing a therapeutic index of an NECTIN4 binding protein comprising an NECTIN4 binding domain and a CD3 binding domain, the method comprising conjugating CD3 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for CD3 binding domain. In some embodiments, the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 613. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific proteinWSGR Docket No.47517-766.601 comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

[0029] Provided herein is an NECTIN4 targeting conditionally active multispecific protein, comprising: an NECTIN4 binding domain, a CD3 binding domain, an albumin binding domain, wherein the albumin binding domain comprises a non-CDR loops that comprises a binding site specific for the CD3 binding domain and a cleavable linker, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264. In some embodiments, the albumin binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

[0030] Provided herein is a pharmaceutical composition comprising an NECTIN4 targeting conditionally active multispecific protein of descriptions herein. In some embodiments, further comprising a pharmaceutically acceptable carrier.WSGR Docket No.47517-766.601

[0031] Provided herein is a process for producing NECTIN4 targeting conditionally active multispecific protein of descriptions herein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the NECTIN4 targeting conditionally active multispecific protein of descriptions herein, under conditions allowing the expression of the NECTIN4 targeting conditionally active multispecific protein and recovering and purifying the produced protein from the culture.

[0032] Provided herein is a method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering an of NECTIN4 targeting conditionally active multispecific protein of descriptions herein, or a pharmaceutical composition according to descriptions herein, to the subject. In some embodiments, the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the tumorous disease comprises at least one of: a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a lung cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a bladder cancer, a gall bladder cancer, a head and neck squamous cell cancer, a squamous cell carcinoma of head and neck, an esophageal cancer, a hepatocellular carcinoma, and a skin cancer, or a combination of any thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0034] FIG.1 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L2, NECTIN4- L10, NECTIN4-L14, NECTIN4-L17, and NECTIN4-L26.

[0035] FIG.2 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing lama anti-NECTIN4 sequences NECTIN4-L28, NECTIN4- L31, NECTIN4-L34, NECTIN4-L36, and NECTIN4-L42.

[0036] FIG.3 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L46, NECTIN4- L48, NECTIN4-L51, NECTIN4-L54, and NECTIN4-L57.

[0037] FIG.4 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L65, NECTIN4- L68, and NECTIN4-L70.WSGR Docket No.47517-766.601

[0038] FIG.5 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L71, NECTIN4- L79, NECTIN4-L81, NECTIN4-L82, and NECTIN4-L85.

[0039] FIG.6 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L86, NECTIN4- L87, NECTIN4-L89, NECTIN4-L90, and NECTIN4-L91.

[0040] FIG.7 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L93, NECTIN4- L95, NECTIN4-L98, NECTIN4-L101, and NECTIN4-L102.

[0041] FIG.8 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L107, NECTIN4- L110, NECTIN4-L112, NECTIN4-L114, and NECTIN4-L115.

[0042] FIG.9 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L118, NECTIN4- L126, NECTIN4-L131, and NECTIN4-L134.

[0043] FIG.10 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L152, NECTIN4- L157, NECTIN4-L158, NECTIN4-L160, and NECTIN4-L162.

[0044] FIG.11 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L167, NECTIN4- L169, NECTIN4-L177, NECTIN4-L187, and NECTIN4-L462.

[0045] FIG.12 provides results from a TDCC assay with UBLC1 cells and anti-CD3 / anti- NECTIN4 fusion proteins containing llama anti-NECTIN4 sequences NECTIN4-L482, NECTIN4- L892, NECTIN4-L952, and GFP.

[0046] FIG.13 provides results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L107 and NECTIN4-H107.

[0047] FIG.14 provides results from a TDCC assay with UBLC1 cells anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L134 and NECTIN4-H134.

[0048] FIG.15 provides results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L177 and NECTIN4-H177.WSGR Docket No.47517-766.601

[0049] FIG.16 shows results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L46 and NECTIN4-H46.

[0050] FIG.17 shows results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L462 and NECTIN4-H462.

[0051] FIG.18 shows results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L82 and NECTIN4-H82.

[0052] FIG.19 shows results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L91 and NECTIN4-H91.

[0053] FIG.20 shows results from a TDCC assay with UBLC1 cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L952 and NECTIN4-H952.

[0054] FIG.21 shows results from a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L107 and NECTIN4-H107.

[0055] FIG.22 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L134 and NECTIN4-H134.

[0056] FIG.23 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L177 and NECTIN4-H177.

[0057] FIG.24 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L46 and NECTIN4-H46.

[0058] FIG.25 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L462 and NECTIN4-H462.

[0059] FIG.26 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L82 and NECTIN4-H82.WSGR Docket No.47517-766.601

[0060] FIG.27 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L91 and NECTIN4-H91.

[0061] FIG.28 shows results of a TDCC assay with LUDLU1 Cells and anti-ALB / anti- CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized or llama anti-NECTIN4 sequences NECTIN4-L952 and NECTIN4-H952.

[0062] FIG.29 shows an SDS-PAGE Gel of anti-ALB / anti-CD3 / anti-NECTIN4 fusion proteins with humanized anti-NECTIN4 sequences NECTIN4-H46, NECTIN4-H462, NECTIN4-H82, and NECTIN4-H952.

[0063] FIG.30 shows an SDS-PAGE Gel of anti-cd3 / anti-NECTIN4 fusion proteins with humanized anti-NECTIN4 sequences NECTIN4-H46, NECTIN4-H462, NECTIN4-H82, and NECTIN4-H952.

[0064] FIG.31 shows results of a TDCC assay with SW780 cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H46 and NECTIN4-H462.

[0065] FIG.32 shows results of a TDCC assay with SW780 cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H82 and NECTIN4-H952 and GFP.

[0066] FIG.33 shows results of a TDCC assay with SCC9 Cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H46 and NECTIN4-H462.

[0067] FIG.34 shows results of a TDCC assay with SCC9 Cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H82 and NECTIN4-H952 and GFP.

[0068] FIG.35 shows results of a TDCC assay with HT1376 Cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H46 and NECTIN4-H462.

[0069] FIG.36 shows results of a TDCC assay with HT1376 Cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H82 and NECTIN4-H952 and GFP.

[0070] FIG.37 shows results of a TDCC assay with HPAFII Cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H46 and NECTIN4-H462.WSGR Docket No.47517-766.601

[0071] FIG.38 shows results of a TDCC assay with HPAFII Cells and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing humanized anti- NECTIN4 sequences NECTIN4-H82 and NECTIN4-H952 and GFP.

[0072] FIG.39 shows a Non-Reduced SDS-PAGE Gel of Anti-CD3 / Anti-NECTIN4 fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4-H46, NECTIN$-H82, NECTIN4-H952, and NECTIN4-H462 after treatment without (-) or with (+) matriptase.

[0073] FIG.40 shows results of a TDCC assay with SW780 cells and Anti-CD3 / Anti-NECTIN4 (MAT Act), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV), fusion proteins containing humanized anti-NECTIN4 Sequences NECTIN4- H46 and GFP.

[0074] FIG.41 shows results of a TDCC assay with SW780 cells and Anti-CD3 / Anti-NECTIN4 (MAT Act), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV), fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H82 and GFP.

[0075] FIG.42 shows results of a TDCC assay with SW780 cells and Anti-CD3 / Anti-NECTIN4 treated with matriptase (MAT Act), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), anti- ALB / anti-CD3 / anti-NECTIN4 (NCLV), fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4-H952 and GFP.

[0076] FIG.43 shows results of a TDCC assay with SW780 cells and anti-CD3 / Anti-NECTIN4 treated with matriptase (MAT Act), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), anti- ALB / anti-CD3 / anti-NECTIN4 (NCLV), fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4-H462 and GFP.

[0077] FIG.44 shows results of a TDCC assay with HT1376 cells and Anti-CD3 / Anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H46.

[0078] FIG.45 shows results of a TDCC assay with HT1376 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H82.

[0079] FIG.46 shows results of a TDCC assay with HT1376 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H952.WSGR Docket No.47517-766.601

[0080] FIG.47 shows results of a TDCC assay with HT1376 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H462.

[0081] FIG.48 shows results of a TDCC assay with MV411 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H46.

[0082] FIG.49 shows results of a TDCC assay with MV411 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H82.

[0083] FIG.50 shows results of a TDCC assay with MV411 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H952.

[0084] FIG.51 shows results of a TDCC assay with MV411 cells and anti-CD3 / anti-NECTIN4 (CT), anti-ALB / anti-CD3 / anti-NECTIN4 (ProTriTAC L040), and anti-ALB / anti-CD3 / anti- NECTIN4 (NCLV) fusion proteins containing humanized anti-NECTIN4 sequences NECTIN4- H462.

[0085] FIG.52 shows results of a TDCC assay with HCC70 cells and anti-CD3 / anti-NECTIN4 (CT) fusion protein containing humanized NECTIN4-H46 and GFP.

[0086] FIG.53 shows results of a TDCC assay with SW780 cells and anti-CD3 / anti-NECTIN4 (CT) fusion protein containing humanized NECTIN4-H46 and GFP.

[0087] FIG.54 shows results of a TDCC assay with HT29 Cells and anti-CD3 / anti-NECTIN4 (CT) fusion protein containing humanized NECTIN4-H46 and GFP.

[0088] FIG.55 shows results of a TDCC assay with CAL27 Cells and anti-CD3 / anti-NECTIN4 (CT) fusion protein containing humanized NECTIN4-H46 and GFP.

[0089] FIG.56 shows results of Tumor Volume in NSG mice treated with anti-ALB / anti- CD3 / anti-NECTIN4 (ProTriTAC L040) fusion protein containing humanized NECTIN4-H46 in admix therapeutic xenograft HPAF-II model.

[0090] FIG.57 shows results of Tumor Volume in NSG mice treated with anti-ALB / anti- CD3 / anti-NECTIN4 (ProTriTAC L040) fusion protein containing humanized NECTIN4-H46 in admix therapeutic xenograft FaDu model.WSGR Docket No.47517-766.601

[0091] FIG.58 results of Tumor Volume in NSG mice treated with anti-ALB / anti-CD3 / anti- NECTIN4 (ProTriTAC L040) fusion protein containing humanized NECTIN4-H46 in admix prophylactic xenograft SW780 model.

[0092] FIG.59 shows an IHC assay with NECTIN4 rabbit polyclonal antibody staining on HT29, SW780, and Jurkat cell pellets at Day 0 (40X magnification).

[0093] FIG.60 shows an IHC assay with NECTIN4 rabbit polyclonal antibody staining on SW780 tumors at Day 7 and Day 21 (40X magnification).

[0094] FIG.61 shows an IHC assay with NECTIN4 rabbit polyclonal antibody staining on HT29 tumors at Day 7 and Day 21 (40X magnification).

[0095] FIG.62 shows an anti-NECTIN4 proTriTAC construct with a L276 cleavable linker (lane 2) showed substantially reduced pre-cleaved active drug in CHO productions versus an anti- proTriTAC construct with a L040 cleavable linker (lane 1). M indicates molecular weight standards, with the molecular weights listed to the left of this lane in KDa. DETAILED DESCRIPTION OF THE DISCLOSURE

[0096] Described herein are trispecific proteins that target NECTIN4, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such proteins thereof. Also provided are methods of using the disclosed NECTIN4 targeting trispecific proteins in the prevention, and / or treatment of diseases, conditions and disorders. The NECTIN4 targeting trispecific proteins are capable of specifically binding to NECTIN4 as well as CD3 and have a half-life extension domain, such as a domain binding to human albumin (ALB). NECTIN4 binding proteins

[0097] Described herein are proteins that bind NECTIN4, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such proteins thereof. Also provided are methods of using the disclosed NECTIN4 binding proteins in the prevention, and / or treatment of diseases, conditions and disorders. In some embodiments, the NECTIN4 binding proteins are part of multispecific (e.g., trispecific) proteins that comprise a NECTIN4 binding domain as described herein.

[0098] NECTIN4 or poliovirus receptor-like 4 (PVRL4) or PRR4 or LNIR or Poliovirus Receptor-Related 4 or Ig Superfamily Receptor LNIR is a type I transmembrane 66 kDa polypeptide, encoded by NECTIN4. NECTIN4 belongs to the nectin subfamily of immunoglobulin- like adhesion molecules that are involved in Ca(2+)-independent cell to cell adhesion. NECTIN4 comprises two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain.WSGR Docket No.47517-766.601

[0099] NECTIN4 plays an essential role in various cell functions, such as cell polarity, proliferation, differentiation, migration, and invasion. It is involved in cell adhesion through trans- homophilic and -heterophilic interactions. NECTIN4 overexpression is observed in many types of malignant tumors such as gastric cancer, thyroid cancer, papillary thyroid cancer, colorectal cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, bladder cancer, prostate cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, uterine corpus endometrial carcinoma, gallbladder cancer, head and neck cancer (e.g., a squamous cell carcinoma of head and neck, esophageal cancer), hepatocellular carcinoma, and skin cancers, or any combination thereof. NECTIN4 overexpression is linked to a poor prognosis, for survival and drug resistance.

[0100] An exemplary protein sequences for NECTIN4 is provided in UniProtKB ID NOS: Q96NY8 or Ref Seq NP_112178.2 (SEQ ID NO: 619). In some embodiments, the protein sequences for NECTIN4 are provided in Ref Seq XP_005245565.1, Ref Seq XP_047287244.1, Ref Seq XP_011508324.1, or Ref Seq EP_011508323.1. In some embodiments, the NECTIN4 binding proteins of this disclosure binds to a NECTIN4 protein comprising an amino acid sequence as provided in UniProtKB ID NOS: Q96NY8 or Ref Seq NP_112178.2. In some embodiments, the protein sequence for NECTIN4 is provided in Ref Seq NP_001116152.1. In some embodiments, the protein sequences for NECTIN4 are provided in Ref Seq XP_005541277.1, or XP_005541280.1, or XP_045254978.1, or XP_045254993.1. In some embodiments, the NECTIN4 binding proteins of this disclosure binds to a NECTIN4 protein encoded by a nucleic acid as provided in Ref Seq NM_030916.3. In some embodiments, the NECTIN4 binding proteins of this disclosure binds to a NECTIN4 protein encoded by a nucleic acid as provided in Ref Seq XM_005245508.4, or XM_011510021.3, XM_011510022.3, or XM_047431288.1. In some embodiments, the Nectin4 protein lacks N terminal methionine (e.g., SEQ ID NO: 620).

[0101] In some embodiments, the NECTIN4 binding proteins of this disclosure binds to a NECTIN4 protein comprising an amino acid sequence as set forth in SEQ ID NO: 619 or 620.

[0102] MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFY RGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVL LRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAE GSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQR ITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVD GDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIA ALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVG LRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIK QAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 619).WSGR Docket No.47517-766.601

[0103] GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHS KYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLR VLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTS EFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAM LKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVT VDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYE EELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLT TVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 620).

[0104] In some embodiments, the NECTIN4 binding domain binds to an extracellular domain of the mature NECTIN4 protein. In some embodiments, the NECTIN4 binding domain binds to a transmembrane domain of the mature NECTIN4 protein. In some embodiments, the NECTIN4 binding domain binds to an intracellular tail of the mature NECTIN4 protein.

[0105] In some embodiments, the NECTIN4 binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 619 or 620. In some embodiments, the NECTIN4 binding domain binds to a protein comprising the sequence of SEQ ID NO: 619 or 620. In some embodiments, the NECTIN4 binding domains disclosed herein recognize full-length NECTIN4. In certain instances, the NECTIN4 binding domains disclosed herein recognize an epitope within NECTIN4, such as, in some cases the NECTIN4 binding proteins interact with one or more amino acids found within a domain of human NECTIN4. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of NECTIN4. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within a domain of NECTIN4.

[0106] In some embodiments, the NECTIN4 binding domains disclosed herein recognize full- length NECTIN4. In certain instances, the NECTIN4 binding domains disclosed herein recognize an epitope within NECTIN4, such as, in some cases the NECTIN4 binding proteins interact with one or more amino acids found within a domain of human NECTIN4. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of NECTIN4. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within a domain of NECTIN4.

[0107] In some embodiments, the NECTIN4 binding proteins of this disclosure binds to the full length NECTIN4 protein or to a fragment thereof, such as epitope containing fragments within theWSGR Docket No.47517-766.601 full length NECTIN4 protein, as described above. In some cases, the epitope containing fragment comprises antigenic or immunogenic fragments and derivatives thereof of the NECTIN4 protein. Epitope containing fragments, including antigenic or immunogenic fragments, are, in some embodiments, 12 amino acids or more, e.g., 20 amino acids or more, 50 or 100 amino acids or more. The NECTIN4 fragments, in some embodiments, comprises 95% or more of the length of the full protein, 90% or more, 75% or 50% or 25% or 10% or more of the length of the full protein. In some embodiments, the epitope-containing fragments of NECTIN4 including antigenic or immunogenic fragments are capable of eliciting a relevant immune response in a patient. Derivatives of NECTIN4 include, in some embodiments, variants on the sequence in which one or more (e.g., 1-20 such as 15 amino acids, or up to 20% such as up to 10% or 5% or 1% by number of amino acids based on the total length of the protein) deletions, insertions or substitutions have been made to the NECTIN4 sequence provided in SEQ ID NO: 619 or 620.

[0108] In some embodiments, substitutions comprise conservative substitutions. Derivatives and variants of, in some examples, have essentially the same biological function as the protein from which they are derived. For instance, derivatives and variants of NECTIN4 are, in some cases, comparably antigenic or immunogenic to the protein from which they are derived, have either the ligand-binding activity, or the active receptor-complex forming ability, or preferably both, of the protein from which they are derived, and have the same tissue distribution as NECTIN4.

[0109] In some embodiments, the NECTIN4 binding protein specifically binds NECTIN4 with equivalent or better affinity as that of a reference NECTIN4 binding protein, and the NECTIN4 binding protein in such embodiments comprises an affinity matured NECTIN4 binding molecule, and is derived from the NECTIN4 binding parental molecule, comprising one or more amino acid mutations (e.g., a stabilizing mutation, a destabilizing mutation) with respect to the NECTIN4 binding parental molecule. In some embodiments, the affinity matured NECTIN4 binding molecule has superior stability with respect to selected destabilizing agents, as that of a reference NECTIN4 binding parental molecule. In some embodiments, the affinity matured NECTIN4 binding molecule is identified in a process comprising panning of one or more pre-candidate NECTIN4 binding molecules derived from one or more NECTIN4 binding parental molecule, expressed in a phage display library, against a NECTIN4 protein, such as a human NECTIN4 protein. The pre-candidate NECTIN4 binding molecule comprises, in some embodiments, amino acid substitutions in the variable regions, CDRs, or framework residues, relative to a parental molecule.

[0110] As used herein, “Phage display” refers to a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of phage, e.g., filamentous phage, particles. A utility of phage display lies in the fact that large libraries ofWSGR Docket No.47517-766.601 randomized protein variants can be rapidly and efficiently selected for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins See e.g., Wells and Lowman, Curr. Opin. Struct. Biol, 3:355-362 (1992), and references cited therein. In monovalent phage display, a protein or peptide library is fused to a gene III, or a portion thereof, and expressed at low levels in the presence of wild type gene III protein so that phage particles display one copy or none of the fusion proteins. Avidity effects are reduced relative to polyvalent phage so that selection is on the basis of intrinsic ligand affinity, and phagemid vectors are used, which simplify DNA manipulations. See e.g., Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0111] In some embodiments, the panning comprises using varying binding times and concentrations to identify NECTIN4 binding molecules with increased or decreased on-rates, from pre-candidate NECTIN4 binding molecules. In some embodiments, the panning comprises using varying wash times to identify NECTIN4 binding molecules with increased or decreased off-rates, from pre-candidate NECTIN4 molecules. In some embodiments, the panning comprises using both varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of the affinity matured NECTIN4. binding molecule, for example, by shuffling to create a second-stage combinatorial library from such mutants and conducting a second round of panning followed by a binding selection.

[0112] In some embodiments, the affinity matured NECTIN4 binding molecule comprises an equivalent or better affinity to a NECTIN4 protein (such as human NECTIN4 protein) as that of a v binding parental molecule, but that has reduced cross reactivity, or in some embodiments, increased cross reactivity, with selected substances, such as ligands, proteins, antigens, or the like, other than the NECTIN4 epitope for which the NECTIN4 binding parental molecule is specific, or is designed to be specific for. In regard to the latter, an affinity matured NECTIN4 binding molecule, in some embodiments, is more successfully tested in animal models if the affinity matured v binding molecule is reacted with both human NECTIN4 and the corresponding target of the animal model, e.g., mouse NECTIN4 or cynomolgus monkey (cyno) NECTIN4. In some embodiments, the parental NECTIN4 binding molecule binds to human NECTIN4 with an affinity of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, and to cynomolgus NECTIN4 with an affinity of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 15 nM or less, or 10 nM or less. In someWSGR Docket No.47517-766.601 embodiments, the affinity matured NECTIN4 binding molecule, identified after one round of panning, binds to human NECTIN4 with an affinity of about 5 nM or less, such as 1 nM or less, and to cynomolgus NECTIN4 with an affinity of about 7.5 nM or less, such as 1 nM or less. In some embodiments, the affinity matured NECTIN4 binding molecule, identified after two rounds of panning, binds to human NECTIN4 with an affinity of about 2.5 nM or less, and to cynomolgus NECTIN4 with an affinity of about 3.5 nM or less.

[0113] In some embodiments, the NECTIN4 binding protein comprises an antigen-specific binding domain polypeptide that specifically bind to targets, such as targets on diseased cells, or targets on other cells that support the diseased state, such as targets on stromal cells that support tumor growth or targets on immune cells that support disease-mediated immunosuppression. In some examples, the antigen-specific binding domain includes antibodies, single chain antibodies, Fabs, Fv, T-cell receptor binding domains, ligand binding domains, receptor binding domains, domain antibodies, single domain antibodies, minibodies, nanobodies, peptibodies, or various other antibody mimics (such as AFFIMERS®, affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, DARPINS®, affibodies, affilins, monobodies and armadillo repeat protein-based proteins).

[0114] In some embodiments, the NECTIN4 binding domain is an anti- NECTIN4 antibody or an antigen binding fragment thereof, or an antibody variant of the NECTIN4 binding domain or an antigen binding fragment thereof. As used herein, the term “antibody variant” refers to variants and derivatives of an antibody or an antigen binding fragment as described herein. In certain embodiments, amino acid sequence variants of the anti- NECTIN4 antibodies or antigen binding fragments thereof, as described herein, are contemplated. For example, in certain embodiments amino acid sequence variants of anti- NECTIN4 antibodies or antigen binding fragments thereof, as described herein, are contemplated to improve the binding affinity and / or other biological properties of the same. Exemplary method for preparing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody or antigen binding fragments thereof.

[0115] Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen- binding. In certain embodiments, variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introducedWSGR Docket No.47517-766.601 into an antibody or antigen binding fragments thereof of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, altered Antibody dependent cellular cytotoxicity (ADCC), or improved T-cell mediated cytotoxicity (TDCC). Both conservative and non-conservative amino acid substitutions are contemplated for preparing the antibody variants.

[0116] In another example of a substitution to create a variant anti- NECTIN4 antibody or antigen binding fragments thereof, one or more hypervariable region residues of a parent antibody are substituted. In general, variants are then selected based on improvements in desired properties compared to a parent antibody or antigen binding fragments thereof, for example, increased affinity, reduced affinity, reduced immunogenicity, increased pH dependence of binding.

[0117] In some embodiments, the NECTIN4 binding domain is a single domain antibody (sdAb) such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama derived sdAb, a peptide, a ligand or a small molecule entity specific for NECTIN4. In some embodiments, the NECTIN4 binding domain described herein is any domain that binds to NECTIN4 including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In certain embodiments, the NECTIN4 binding domain is a single-domain antibody. In other embodiments, the NECTIN4 binding domain is a peptide. In further embodiments, the NECTIN4 binding domain is a small molecule.

[0118] Generally, it should be noted that the term single domain antibody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4- chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. For example, in some embodiments, the single domain antibodies of the disclosure are obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, and in particular from a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization” of a “domain antibody” or “Dab,” or by expression of a nucleic acidWSGR Docket No.47517-766.601 encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the field, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0119] In one embodiment, a single domain antibody corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against NECTIN4. As further described herein, such VHH sequences can generally be generated or obtained by suitably immunizing a species of Llama with NECTIN4, (i.e., so as to raise an immune response and / or heavy chain antibodies directed against NECTIN4), by obtaining a suitable biological sample from said Llama (such as a blood sample, serum sample or sample of B-cells), and by generating VHH sequences directed against NECTIN4, starting from said sample, using any suitable technique known in the field.

[0120] In another embodiment, such naturally occurring VHH domains against NECTIN4, are obtained from naïve libraries of Camelid VHH sequences, for example by screening such a library using NECTIN4, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the field. Such libraries and techniques are for example described in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naïve VHH libraries are used, such as VHH libraries obtained from naïve VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as for example described in WO 00 / 43507.

[0121] In a further embodiment, yet another technique for obtaining VHH sequences directed against NECTIN4, involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e., so as to raise an immune response and / or heavy chain antibodies directed against NECTIN4), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample or sample of B-cells), and then generating VHH sequences directed against NECTIN4, starting from said sample, using any suitable technique known in the field. For example, for this purpose, the heavy chain antibody-expressing rats or mice and the further methods and techniques described in WO 02 / 085945 and in WO 04 / 049794 can be used.

[0122] In some embodiments, an anti- NECTIN4 single domain antibody of this disclosure comprises a single domain antibody with an amino acid sequence that corresponds to the amino acid sequence of a non-human antibody and / or a naturally occurring VHH domain, e.g., a llama anti- NECTIN4 antibody, but that has been “humanized,” i.e., by replacing one or more amino acid residues in the amino acid sequence of said non-human anti- NECTIN4 and / or the naturallyWSGR Docket No.47517-766.601 occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g., as indicated above). This can be performed in a manner known in the field, which will be clear to the skilled person, for example on the basis of the further description herein. Again, it should be noted that such humanized anti- NECTIN4 single domain antibodies of the disclosure are obtained in any suitable manner known per se (i.e., as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. In some additional embodiments, a single domain anti- NECTIN4 antibody, as described herein, comprises a single domain antibody with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized” i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae hallmark residues. See, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence that is used as a starting material or starting point for generating or designing the camelized single domain is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized anti- NECTIN4 single domain antibodies of the disclosure, in certain embodiments, are obtained in any suitable manner known in the field (i.e., as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a non-human anti- NECTIN4 antibody and / or the naturally occurring VH domain as a starting material. For example, as further described herein, both “humanization” and “camelization” is performed by providing a nucleotide sequence that encodes a naturally occurring VHH domain or VH domain, respectively, and then changing, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” single domain antibody, respectively. This nucleic acid can then be expressed, so as to provide a desired anti- NECTIN4 single domain antibody of the disclosure. Alternatively, in other embodiments, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized anti- NECTIN4 single domain antibody of the disclosure, respectively, are designed and then synthesized de novo using known techniques for peptide synthesis. In some embodiments, based on the amino acid sequence or nucleotide sequenceWSGR Docket No.47517-766.601 of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized anti- NECTIN4 single domain antibody of the disclosure, respectively, is designed and then synthesized de novo using known techniques for nucleic acid synthesis, after which the nucleic acid thus obtained is expressed in using known expression techniques, so as to provide the desired anti- NECTIN4 single domain antibody of the disclosure.

[0123] Other suitable methods and techniques for obtaining the anti- NECTIN4 single domain antibody of the disclosure and / or nucleic acids encoding the same, starting from naturally occurring VH sequences or VHH sequences for example comprises combining one or more parts of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide an anti- NECTIN4 single domain antibody of the disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0124] In some embodiments, the NECTIN4 binding domain is an anti NECTIN4 specific antibody comprising a heavy chain variable complementarity determining region CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the NECTIN4 binding domain comprises any domain that binds to NECTIN4 including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or antigen binding fragments such as single domain antibodies (sdAb), Fab, (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some embodiments, the NECTIN4 binding domain is a single domain antibody. In some embodiments, the anti- NECTIN4 single domain antibody comprises heavy chain variable complementarity determining regions (CDR), CDR1, CDR2, and CDR3.

[0125] In some embodiments, the NECTIN4 binding domain is a polypeptide comprising an amino acid sequence that is comprised of four framework regions / sequences (f1-f4) interrupted by three complementarity determining regions / sequences, as represented by the formula: f1-r1-f2-r2- f3-r3-f4, wherein r1, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues. The framework residues of the NECTIN4 binding protein of the present disclosure comprise, for example, 75, 76, 77, 78, 79, 80,WSGR Docket No.47517-766.601 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity determining regions comprise, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the NECTIN4 binding domain comprises an amino acid sequence selected from SEQ ID NOs: 1-66.

[0126] In some embodiments, the binding proteins described herein comprise a polypeptide having an amino acid sequence selected from SEQ ID NOS: 1-66, subsequences thereof, and variants thereof. In some embodiments, the NECTIN4 binding protein comprises at least 70%-95% or more homology to an amino acid sequence selected from SEQ ID NOS: 1-66, subsequences thereof, and variants thereof. In some embodiments, the NECTIN4 binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology to an amino acid sequence selected from SEQ ID NOS: 1-66, subsequences thereof, and variants thereof. In some embodiments, the NECTIN4 binding protein comprises at least 70%-95% or more identity to an amino acid sequence selected from SEQ ID NOS: 1-66, subsequences thereof, and variants thereof. In some embodiments, the NECTIN4 binding protein comprises at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from SEQ ID NOS: 1-66, subsequences thereof, and variants thereof.

[0127] In some embodiments, the CDR1 comprises the amino acid sequence as set forth in any one of SEQ ID NOS: 67-132 or an amino acid sequence comprising one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOS: 67-132. In some embodiments, the CDR2 comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 133-198 or an amino acid sequence comprising one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOS: 133-198. In some embodiments, the CDR3 comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 199-264 or an amino acid sequence comprising one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOS: 199-264.

[0128] In various embodiments, the NECTIN4 binding domain of the present disclosure is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOS: 67- 132, 133-198, and 199-264.WSGR Docket No.47517-766.601

[0129] In various embodiments, a complementarity determining region of the NECTIN4 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOS: 67-132.

[0130] In various embodiments, a complementarity determining region of the NECTIN4 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOS: 132-198.

[0131] In various embodiments, a complementarity determining region of the NECTIN4 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NOS: 199-264.

[0132] In some embodiments, the NECTIN4 binding domain is cross-reactive with human cynomolgus and mouse NECTIN4. In some embodiments, the NECTIN4 binding domain is specific for human NECTIN4. In certain embodiments, the NECTIN4 binding domains disclosed herein bind to human NECTIN4 with a human KD (h KD). In certain embodiments, the NECTIN4 binding domains disclosed herein bind to cynomolgus NECTIN4 with a cyno Kd (c KD). In certain embodiments, the NECTIN4 binding domains disclosed herein bind to cynomolgus NECTIN4 with a mouse KD (m KD). In certain embodiments, the NECTIN4 binding domains disclosed herein bind to both cynomolgus NECTIN4 and a human NECTIN4, with a cyno Kd (c KD) and a human Kd (h KD), respectively. In certain embodiments, the NECTIN4 binding domains disclosed herein bind to cynomolgus NECTIN4, mouse NECTIN4, and a human NECTIN4, with a cyno KD (c KD), mouse KD (m KD), and a human KD (h KD), respectively. In some embodiments, the NECTIN4 binding protein binds to human, mouse and cynomolgus NECTIN4 with comparable binding affinities (i.e., h KD, the m KDand the c KDvalues do not differ by more than ± 10%). In some embodiments, the h KD, the m KD and the c KD range from about 0.001 nM to about 500 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.001 nM to about 450 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.001 nM to about 400 nM. InWSGR Docket No.47517-766.601 some embodiments, the h KD, the m KDand the c KDrange from about 0.001 nM to about 350 nM. In some embodiments, the h KD, the m KD and the c KD range from about 0.001 nM to about 300 nM. In some embodiments, the h KD, the m KD and the c KD range from about 0.001 nM to about 250 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.001 nM to about 200 nM. In some embodiments, the h KD, the m KD and the c KD range from about 0.001 nM to about 150 nM. In some embodiments, the h KD, the m KD and the c KD range from about 0.001 nM to about 100 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.1 nM to about 90 nM. In some embodiments, h KD, the m KD and the c KD range from about 0.2 nM to about 80 nM. In some embodiments, h KD, the m KD and the c KD range from about 0.3 nM to about 70 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.4 nM to about 50 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.5 nM to about 30 nM. In some embodiments, the h KD, the m KD and the c KD range from about 0.6 nM to about 10 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.7 nM to about 8 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 0.8 nM to about 6 nM. In some embodiments, the h KD, the m KD and the c KD range from about 0.9 nM to about 4 nM. In some embodiments, the h KD, the m KDand the c KDrange from about 1 nM to about 2 nM.

[0133] In some embodiments, any of the foregoing NECTIN4 binding domains (e.g., anti- NECTIN4 single domain antibodies of SEQ ID NOS: 1-66) are affinity peptide tagged for ease of purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also referred to as 6X-His (SEQ ID NO: 615).

[0134] In certain embodiments, the NECTIN4 binding domains of the present disclosure preferentially bind membrane bound NECTIN4 over soluble NECTIN4 Membrane bound NECTIN4 refers to the presence of NECTIN4 in or on the cell membrane surface of a cell that expresses NECTIN4. Soluble NECTIN4 refers to NECTIN4 that is no longer on in or on the cell membrane surface of a cell that expresses or expressed NECTIN4. In certain instances, the soluble NECTIN4 is present in the blood and / or lymphatic circulation in a subject. In one embodiment, the NECTIN4 binding domains bind membrane-bound NECTIN4 at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold greater than soluble NECTIN4. In one embodiment, the NECTIN4 binding proteins of the present disclosure preferentially bind membrane-bound NECTIN430-fold greater than soluble NECTIN4. Determining the preferential binding of an antigen binding protein to membrane bound NECTIN4 over soluble NECTIN4 can be readily determined using binding assays.WSGR Docket No.47517-766.601

[0135] It is contemplated that in some embodiments the NECTIN4 binding protein is fairly small and no more than 40 kDa, no more than 30 kDa, no more than 25 kDa, no more than 20 kDa, no more than 15 kDa, or no more than 10 kDa in some embodiments. In certain instances, the NECTIN4 binding protein is 5 kDa or less if it is a peptide or small molecule entity.

[0136] In other embodiments, the NECTIN4 binding proteins described herein comprise small molecule entity (SME) binders for NECTIN4. SME binders are small molecules averaging about 500 to 2000 Da in size and are attached to the NECTIN4 binding proteins by known methods, such as sortase ligation or conjugation. In these instances, the NECTIN4 binding protein comprises a domain comprising a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 621). To attach a SME binder to NECTIN4 binding protein comprising a sortase recognition sequence, the protein is incubated with a sortase and a SME binder whereby the sortase attaches the SME binder to the recognition sequence. In yet other embodiments, the NECTIN4 binding proteins described herein comprise a knottin peptide for binding NECTIN4. Knottins are disulfide-stabilized peptides with a cysteine knot scaffold and have average sizes about 3.5 kDa. Knottins have been contemplated for binding to certain tumor molecules such as NECTIN4. In further embodiments, the NECTIN4 binding proteins described herein comprise a natural NECTIN4 ligand.

[0137] In some embodiments, the NECTIN4 binding protein comprises more than one domain and are of a single-polypeptide design with flexible linkage of the domains. This allows for facile production and manufacturing of the NECTIN4 binding proteins as they can be encoded by single cDNA molecule to be easily incorporated into a vector. Further, in some embodiments where the NECTIN4 binding proteins described herein are a monomeric single polypeptide chain, there are no chain pairing issues or a requirement for dimerization. It is contemplated that, in such embodiments, the NECTIN4 binding proteins described herein have a reduced tendency to aggregate.

[0138] In the NECTIN4 binding proteins comprising more than one domain, the domains are linked by one or more internal linker. In certain embodiments, the internal linkers are “short,” i.e., consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the internal linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linkers are “long,” i.e., consist of 15, 20 or 25 amino acid residues. In some embodiments, the internal linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the internal linkers, peptides are selected with properties that confer flexibility to the NECTIN4 binding proteins, do not interfere with the binding domains as well as resist cleavage from proteases. For example, glycine and serine residues generally provide proteaseWSGR Docket No.47517-766.601 resistance. Examples of internal linkers suitable for linking the domains in the NECTIN4 binding proteins include but are not limited to (GS)n (SEQ ID NO: 622), (GGS)n (SEQ ID NO: 623), (GGGS)n (SEQ ID NO: 624), (GGSG)n (SEQ ID NO: 625), (GGSGG)n (SEQ ID NO: 626), (GGGGS)n(SEQ ID NO: 627), (GGGGG)n(SEQ ID NO: 628), or (GGG)n(SEQ ID NO: 629), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 630), (GGGGSGGGGSGGGGS) (SEQ ID NO: 631), or (GGGGSGGGS) (SEQ ID NO: 616).

[0139] In some cases, where the NECTIN4 binding protein comprises more than one domain, the domains within the NECTIN4 binding proteins are conjugated using an enzymatic site-specific conjugation method which involves the use of a mammalian or bacterial transglutaminase enzyme. Microbial transglutaminases (mTGs) are versatile tools in modern research and biotechnology. The availability of large quantities of relatively pure enzymes, ease of use, and lack of regulation by calcium and guanosine-5’-triphosphate (GTP) has propelled mTG to be the main cross-linking enzyme used in both the food industry and biotechnology. Currently, mTGs are used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, as well as to other proteins. See, e.g., Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem.25, 5, 855-862.

[0140] In some examples are provided NECTIN4 binding proteins comprising more than one domain, wherein one of the domains comprises an acceptor glutamine in a constant region, which can then be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain which is a substrate for TGase, e.g. comprising an alkylamine, oxoamine) wherein the conjugation occurs exclusively on one or more acceptor glutamine residues present in the targeting moiety outside of the antigen combining site (e.g., outside a variable region, in a constant region). Conjugation thus does not occur on a glutamine, e.g., an at least partly surface exposed glutamine, within the variable region. The NECTIN4 binding protein, in some examples, is formed by reacting one of the domains with a lysine-based linker in the presence of a TGase.

[0141] In some embodiments, where one or more domains within the NECTIN4 binding proteins are directly joined, a hybrid vector is made where the DNA encoding the directly joined domains are themselves directly ligated to each other. In some embodiments, where linkers are used, a hybrid vector is made where the DNA encoding one domain is ligated to the DNA encoding one end of a linker moiety and the DNA encoding another domain is ligated to the other end of the linker moiety.

[0142] In some embodiments, the NECTIN4 binding protein is a single chain variable fragments (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variableWSGR Docket No.47517-766.601 domain (VL) and a variable domain (VHH) of camelid derived single domain antibody. In other embodiments, the NECTIN4 binding protein is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, AFFIMERS®, affitins, alphabodies, avimers, DARPINS®, fynomers, kunitz domain peptides, and monobodies. In further embodiments, the NECTIN4 binding protein is a ligand or peptide that binds to or associates with NECTIN4. In yet further embodiments, the NECTIN4 binding protein is a knottin. In yet further embodiments, the binding domain to NECTIN4 is a small molecular entity.

[0143] In certain embodiments, the NECTIN4 binding proteins according to the present disclosure may be incorporated into NECTIN4 targeting trispecific proteins. In some embodiments, the trispecific proteins comprise a CD3 binding domain, a half-life extension domain, and a NECTIN4 binding domain according to this disclosure. In some embodiments, the NECTIN4 binding trispecific protein comprises a trispecific antibody. Multispecific NECTIN4 targeting proteins, such as NECTIN4 targeting trispecific proteins (also referred to herein as NECTIN4 targeting TriTAC proteins or molecules)

[0144] In one aspect is described herein a multispecific or a multivalent protein comprising a NECTIN4 binding protein according to the present disclosure. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3. In some embodiments, the multispecific protein further comprises a domain which specifically binds to human CD3. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3-gamma. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3-delta. In some embodiments, the multispecific protein further comprises a domain which specifically binds to CD3-epsilon.

[0145] In additional embodiments, the multispecific protein further comprises a domain which specifically binds to the T cell receptor (TCR). In some embodiments, the multispecific protein further comprises a domain which specifically binds the alpha chain of the TCR. In some chain of the TCR.

[0146] In certain embodiments, the CD3 binding domain of the multispecific protein exhibits not only potent CD3 binding affinities with human CD3, but also shows excellent cross-reactivity with the respective cynomolgus monkey CD3 proteins. In some instances, the CD3 binding domain of the multispecific proteins are cross-reactive with CD3 from cynomolgus monkey. In certain instances, human:cynomolgous KD (h KD : c KD) ratios for CD3 binding are between 20:1 and 1:2.

[0147] In some embodiments, the CD3 binding domain of the multispecific protein is any domain that binds to CD3 including but not limited to domains from a monoclonal antibody, a polyclonalWSGR Docket No.47517-766.601 antibody, a recombinant antibody, a human antibody, a humanized antibody, or antigen binding fragments of the CD3 binding antibodies, such as single domain antibodies (sdAb), Fab, F(ab')2, and Fv fragments, fragments comprised of one or more CDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some instances, it is beneficial for the CD3 binding domain to be derived from the same species in which the multispecific protein comprising a single domain serum albumin binding protein described herein will ultimately be used in. For example, for use in humans, it may be beneficial for the CD3 binding domain of the multispecific protein comprising a NECTIN4 binding protein described herein to comprise human or humanized residues from the antigen binding domain of an antibody or antibody fragment. Exemplary amino acid sequence for the CD3 binding domain of a multispecific (e.g., trispecific) NECTIN4 targeting TriTAC protein of this disclosure is provided as SEQ ID NO: 613, or an amino acid sequence that is at least about 75% to 100% identical to SEQ ID NO: 613, such as at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 613.

[0148] In some embodiments, the serum albumin binding domain (also referred to herein as the half-life extension domain) of a multispecific protein comprising a NECTIN4 binding protein as described herein can be any domain that binds to serum albumin including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the serum albumin binding domain is a single chain variable fragments (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived sdAb, or antigen binding fragments of the HSA binding antibodies, such as Fab, F(ab')2, and Fv fragments, fragments comprised of one or more CDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain, peptide, ligand or small molecule entity specific for serum albumin. In certain embodiments, the HSA binding domain is a single-domain antibody. In other embodiments, the serum albumin binding domain is a peptide. In further embodiments, the serum albumin binding domain is a small molecule. It is contemplated that the serum albumin binding domain of the multispecific binding protein comprising a single chain variable fragment CD3 binding protein is fairly small and no more than 25 kD, no more thanWSGR Docket No.47517-766.601 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the serum albumin binding is 5 kD or less if it is a peptide or small molecule entity. Exemplary amino acid sequence for a serum albumin binding domain of a multispecific (e.g., trispecific) NECTIN4 targeting TriTAC protein of this disclosure is provided as SEQ ID NO: 612, or an amino acid sequence that is at least about 75% to 100% identical to SEQ ID NO: 612, such as at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 612.

[0149] The half-life extension domain of a multispecific binding protein, as described herein, comprising a single chain variable fragment CD3 binding protein provides for altered pharmacodynamics and pharmacokinetics of the single chain variable fragment CD3 binding protein itself. As above, the half-life extension domain extends the elimination half-time. The half- life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the single chain variable fragment CD3 binding protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the multispecific binding protein comprising a single chain variable fragment CD3 binding protein, resulting in reduced side effects, such as reduced off-target, such as non-tumor cell cytotoxicity.

[0150] Further, the binding affinity of the half-life extension domain, in some embodiments, is selected so as to target a specific elimination half-time in a particular multispecific binding protein comprising a NECTIN4 binding protein as described herein. Thus, in some embodiments, the half- life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a medium binding affinity. In yet other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include KD of 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to serum albumin are determined by known methods such as Surface Plasmon Resonance (SPR).

[0151] A NECTIN4 targeting multispecific protein of this disclosure, in certain embodiments, comprises (A) a first domain which binds to a CD3; (B) a second domain which is a half-life extension domain; and (C) a third domain which is a NECTIN4 binding protein as described herein. In certain embodiments, the first domain comprises an scFv that specifically binds the CD3. The CD3, for instance, is a human CD3 protein. In certain embodiments, the second domain comprises an sdAb that specifically binds a bulk serum protein. In some instances, the bulk serum protein isWSGR Docket No.47517-766.601 albumin, such as, a serum albumin, such as, a human serum albumin. The domains (A), (B), and (C), are, in some embodiments, linked via linkers L1 and L2, in any one of the following orientations: H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(C)-L1-(B)- L2-(A)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(B)-(C)-L2-COOH, or H2N-(B)-(C)- (A)-COOH.

[0152] A NECTIN4 targeting multispecific protein of this disclosure, in some embodiments, comprises an amino acid sequence that is at least about 70% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-66 and 265-347. In some embodiments, a NECTIN4 targeting multispecific protein of this disclosure comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 76%, at least about 77%, about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-66 and 265-347. Conditionally active multispecific NECTIN4 targeting proteins, such as conditionally active NECTIN4 targeting trispecific proteins (also referred to herein as NECTIN4 targeting ProTriTAC or protrispecific proteins or molecules)

[0153] One embodiment of this disclosure provides a conditionally active multispecific protein comprising a NECTIN4 binding domain as disclosed herein (for example, in some embodiment this disclosure provides a NECTIN4 targeting protrispecific / ProTriTAC protein comprising a NECTIN4 binding domain of this disclosure).

[0154] In some embodiments, the conditionally active multispecific protein further comprises a domain which specifically binds to a CD3 and a binding moiety which specifically binds to a bulk serum protein, such as a human serum albumin. In some embodiments, the binding moiety masks the interaction of the NECTIN4 binding domain or the CD3 binding domain, to their targets. In some embodiments, a binding moiety of this disclosure comprises a masking moiety and a cleavable linker, such as a protease cleavable linker. Exemplary sequences for masking moiety within a binding moiety are provided in SEQ ID NO: 612, or an amino acid sequence comprising one or more substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 612. In some embodiments, the binding moiety comprises a modified non-CDR loop sequence and a cleavable linker. In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 618 and 632-647, or an aminoWSGR Docket No.47517-766.601 acid sequence that comprises one or substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NOS: 618 and 632-647. In some embodiments, the masking moiety comprises a modified non-CDR loop sequence and a non-cleavable linker. In some embodiments the non-cleavable linker comprises an amino acid sequence as set forth in SEQ ID NO: 617, or an amino acid sequence comprising one or more substitutions relative to SEQ ID NO: 617. In some embodiments, a binding moiety comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 612. In some embodiments, a CD3 binding domain of a NECTIN4 ProTriTAC of this disclosure comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 613.

[0155] In some embodiments, a NECTIN4 targeting ProTriTAC of this disclosure, comprises, from N-terminal to C-terminal, comprise a binding moiety that is an anti-ALB domain comprising a non-CDR loop with a binding site for a CD3 binding domain (e.g., a CD3 binding domain having the sequence of SEQ ID NO:612, or at least about 75% identity to the same), a cleavable linker, the CD3 binding domain, and on the C-terminal end the anti- NECTIN4 binding domain. The NECTIN4 binding domain of the ProTriTAC, in some embodiments, is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOS: 1-66.

[0156] In some embodiments, a NECTIN4 targeting ProTriTAC of this disclosure comprises an amino acid sequence that is at least about at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NOS: 1-66 and 265-347. In some embodiments, a NECTIN4 targeting ProTriTAC of this discloses comprises an amino acid sequence as set forth in SEQ ID NO: 1-66 and 265-347, a pharmaceutical composition comprising the same, and method of using the same for treating a disease, such as a tumorous disease as described herein.WSGR Docket No.47517-766.601

[0157] In some embodiments, a NECTIN4 targeting ProTriTAC of this disclosure, in a non- cleavable prodrug format, comprises an amino acid sequence that is at least about at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO: 1-66.

[0158] An exemplary sequence for an active NECTIN4 targeting drug (CT), as described herein, is an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 1-66 and 265-347.

[0159] The binding moiety is capable of synergistically expanding a therapeutic window of a conditionally active NECTIN4 targeting protrispecific protein, by both steric masking and specific masking. In some embodiments, the binding moiety combines both steric masking (for example, via binding to a bulk serum albumin) and specific masking (for example, via non-CDR loops binding to the CDRs of an anti- NECTIN4 domain or an anti-CD3 scFv domain). For example, the binding moiety masks or is capable of masking binding of the NECTIN4 binding domain (e.g., hides the NECTIN4 binding domain and / or prevents premature binding), until activation and the ability of a sequence to “mask” can be tested utilizing an assay where activity with and without the masking sequence (e.g., a sequence selected from the group consisting of SEQ ID NOS: 721-765, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 721-765) is compared. Briefly, to test masking, ProCAR constructs with and without masking moieties in, for example, CC' loops are made. T cells are infected with lentivirus made from the constructs to generate CAR-T cells, which are subsequently stained with anti-FLAG antibodies and NECTIN4-Fc along with fluorescently labeled secondary antibodies and analyzed by flow cytometry. Dot plots of staining are generated and compared. In some instances, the masking moiety is masking peptide / moiety, inserted into one or more non-CDR loops, such that the binding moiety binds to and inhibits the NECTIN4 antigen binding domain until such time that the construct is delivered to a tumor microenvironment. In some cases, modifying the non-CDR loops within the binding moiety does not affect albumin binding. The protease cleavable linker, in some cases, enables activation of a NECTIN4 targeting protrispecific protein in a single proteolytic event, thereby allowing more efficient conversion of the protrispecific molecule in tumor microenvironment. Further, tumor-associated proteolytic activation, in some cases, reveals active T cell engager with minimal off-tumor activity after activation. The present disclosure, in some embodiments, provides a half-life extended T cell engager format (ProTriTAC) comprising a NECTIN4 binding moiety as described herein, which in some cases represents a new and improved approach to engineer conditionally active T cell engagers.WSGR Docket No.47517-766.601

[0160] The half-life of the NECTIN4 binding domain in a conditionally active protrispecific format is, in some embodiments, extended in systemic circulation by using the binding moiety as described above which acts as a safety switch that keeps the multispecific protein in the pro format in an inert state until it reaches the tumor microenvironment where it is conditionally activated by cleavage of the linker and is able to bind its target antigen(s). The safety switch, in certain instances, provides several advantages: some examples including (i) expanding the therapeutic window of the conditionally active NECTIN4 targeting protein; (ii) reducing target-mediated drug disposition by maintaining the conditionally active NECTIN4 targeting protein in systemic circulation; (iii) reducing the concentration of undesirable activated protein in systemic circulation, thereby minimizing the spread of chemistry, manufacturing, and controls related impurities, e.g., pre-activated drug product, endogenous viruses, host-cell proteins, DNA, leachables, anti-foam, antibiotics, toxins, solvents, heavy metals; (iv) reducing the concentration of undesirable activated proteins in systemic circulation, thereby minimizing the spread of product related impurities, aggregates, breakdown products, product variants due to: oxidation, deamidation, denaturation, loss of C-term Lys in MAbs; (v) preventing aberrant activation in circulation; (vi) reducing the toxicities associated with the leakage of activated species from diseased tissue or other pathophysiological conditions, e.g., tumors, autoimmune diseases, inflammations, viral infections, tissue remodeling events (such as myocardial infarction, skin wound healing), or external injury (such as X-ray, CT scan, UV exposure); and (vii) reducing non-specific binding of the conditionally active NECTIN4 targeting protein. Furthermore, post-activation, or in other words post breaking of the safety switch, the conditionally active NECTIN4 targeting protein is separated from the safety switch which provided extended half-life, and thus is cleared from circulation. For instance, if the drug is inadvertently activated outside of a tumor environment or if it leaks out of a tumor environment after activation, it is likely to be cleared rapidly and is less likely to cause damage to normal tissues, thus reducing toxicity.

[0161] In some embodiments, a conditionally active multispecific NECTIN4 binding protein as described herein has an improved therapeutic index as compared to that of a NECTIN4 binding protein that is not conditionally active but is, rather, constitutively active. For instance, a NECTIN4 ProTriTAC, in some embodiments, has an increased therapeutic index than a NECTIN4 TriTAC. The increase in therapeutic index, in some embodiments, is from at least about 2-fold to about 1000-fold, such as about 4-fold to about 800-fold, about 6-fold to about 800-fold, about 6-fold to about 600-fold, about 10-fold to about 400-fold, about 20-fold to about 200-fold, about 30-fold to about 150-fold, about 50-fold to about 100-fold. The increase in therapeutic index, in someWSGR Docket No.47517-766.601 embodiments, is attributed to the conjugation of the NECTIN4 binding domain to a binding moiety as described above, with the non-CDR loop and the cleavable linker.

[0162] A “therapeutic index” (TI) (also referred to as “therapeutic window”) is, in some embodiments, a comparison of the minimum amount of a therapeutic agent (e.g., a NECTIN4 TriTAC, a NECTIN4 ProTriTAC, a NECTIN4 CAR, a NECTIN4 ProCAR) that causes the therapeutic effect (e.g., improved survival of a patient with a NECTIN4 expressing cancer, treated with a therapeutic agent as mentioned above) to minimum tolerated dose. In some instances, the therapeutic index improvement is manifested in terms of an improved EC50 of a NECTIN4 TriTAC compared to a NECTIN4 ProTriTAC, in T cell mediated killing of cancer cells.

[0163] In some embodiments, the conditionally active NECTIN4 targeting protein format gives the NECTIN4 binding domain a significantly longer serum half-life and reduces the likelihood of its undesirable activation in circulation, thereby producing a ‘‘biobetter’’ version.

[0164] A binding moiety as described herein comprises at least one non-CDR loop. In some embodiments, a non-CDR loop provides a binding site for binding of the binding moiety to a NECTIN4 binding domain of this disclosure. In some cases, the binding moiety masks binding of the NECTIN4 binding domain to its target antigen, e.g., via steric occlusion, via specific intermolecular interactions, or a combination of both.

[0165] In some embodiments, a binding moiety as described herein further comprises complementarity determining regions (CDRs), for instance, specific for binding a bulk serum protein (e.g., a human serum albumin). In some instances, a binding moiety of this disclosure is a domain derived from an immunoglobulin molecule (Ig molecule). The Ig may be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). A polypeptide chain of an Ig molecule folds into a series of parallel beta strands linked by loops. In the variable region, three of the loops constitute the “complementarity determining regions” (CDRs) which determine the antigen binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) with are hypervariable in sequence and / or involved in antigen recognition and / or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRsWSGR Docket No.47517-766.601 and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0166] In some embodiments, a binding moiety of this disclosure is a heavy chain only antibody. In some embodiments, the variable domain of a heavy chain only antibody has several beta strands that are arranged in two sheets. The variable domain of a heavy chain only antibody contains three hypervariable loops, or complementarity-determining regions (CDRs) and framework regions FR1, FR2, FR3, and FR4. The three CDRs of the variable domain (CDR1, CDR2, CDR3) cluster at one end of the beta barrel. The CDRs are the loops that connect beta strands B-C, C'-C", and F-G of the immunoglobulin fold, whereas the bottom loops that connect beta strands AB, CC', C" -D and E-F of the immunoglobulin fold, and the top loop that connects the D-E strands of the immunoglobulin fold are the non-CDR loops.

[0167] In some embodiments of this disclosure at least some, or all, of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the “non-CDR loop” of the binding moieties described herein, such as a binding moiety that is a heavy chain only antibody. In some embodiments of this disclosure, at least some amino acid residues of a constant domain, CH1, CH2, or CH3, are part of the “non-CDR loop” of the binding moieties described herein. Non-CDR loops comprise, in some embodiments, one or more of AB, CD, EF, and DE loops of a C1-set domain of an Ig or an Ig-like molecule; AB, CC’, EF, FG, BC, and EC’ loops of a C2-set domain of an Ig or an Ig-like molecule; DE, BD, GF, A(A1A2)B, and EF loops of I(Intermediate)-set domain of an Ig or Ig-like molecule.

[0168] Within the variable domain, the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are considered a scaffold for the CDRs. However, in certain cases, some of the FR residues play an important role in antigen recognition and binding. Framework region residues that affect Ag binding are divided into two categories. The first are FR residues that contact the antigen, thus are part of the binding-site, and some of these residues are close in sequence to the CDRs. Other residues are those that are far from the CDRs in sequence but are in close proximity to it in the 3-D structure of the molecule, e.g., a loop in heavy chain.

[0169] In some embodiments, the non-CDR loop is modified to generate an antigen binding site specific for a bulk serum protein, such as albumin. In some embodiments, the non-CDR loop is modified to generate an antigen binding site specific for a NECTIN4 binding domain as described herein. In some embodiments, the non-CDR loop is modified to generate an antigen binding site specific for a CD3 binding domain as described herein.

[0170] It is contemplated that various techniques can be used for modifying the non-CDR loop, e.g., site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid that is foreign to the non-CDR loop amino acid sequence, amino acid substitution. An antigen peptide isWSGR Docket No.47517-766.601 inserted into a non-CDR loop, in some examples. In some examples, an antigenic peptide is substituted for the non-CDR loop. The modification, to generate an antigen binding site, is in some cases in only one non-CDR loop. In other instances, more than one non-CDR loop are modified. For instance, the modification is in any one of the non-CDR loops are AB, CC', C" D, EF, and D-E. In some cases, the modification is in the DE loop. In other cases, the modifications are in all four of AB, CC', C"D, E-F loops.

[0171] In certain examples, the binding moieties described herein are bound to the NECTIN4 binding domain via their AB, CC', C" D, or EF loop and are bound to a bulk-serum protein, such as albumin, via their B-C, C'-C", or F-G loop. In certain examples, the binding moiety is bound to the NECTIN4 binding domain via its AB, CC', C" D, and EF loop and is bound to a bulk-serum protein, such as albumin, via its BC, C'C", and FG loop. In certain examples, the binding moiety is bound to the NECTIN4 binding domain via one or more of AB, CC', C" D, and E-F loop and is bound to a bulk-serum protein, such as albumin, via one or more of BC, C'C", and FG loop. In certain examples, the binding moiety is bound to a bulk serum protein, such as albumin, via its AB, CC', C" D, or EF loop and is bound to the NECTIN4 binding domain via its BC, C'C", or FG loop. In certain examples, the binding moiety is bound to a bulk serum protein, such as albumin, via its AB, CC', C" D, and EF loop and is bound to the NECTIN4 binding domain via its BC, C'C", and FG loop. In certain examples, the binding moiety of the first embodiment is bound to a bulk serum protein, such as albumin, via one or more of AB, CC', C" D, and E-F loop and is bound to the NECTIN4 binding protein, via one or more of BC, C'C", and FG loop. In certain examples, the binding moieties described herein are bound to a CD3 binding domain via their AB, CC', C" D, or EF loop and are bound to a bulk-serum protein, such as albumin, via their B-C, C'-C", or F-G loop. In certain examples, the binding moieties described herein are bound to a bulk serum protein, such as albumin, via their AB, CC', C" D, or EF loop and are bound to a CD3 binding domain, via their B-C, C'-C", or F-G loop. In certain examples, the binding moieties described herein are bound to a CD3 binding domain via their AB, CC', C" D, or EF loop and are bound to a NECTIN4 binding domain, via their B-C, C'-C", or F-G loop. In certain examples, the binding moieties described herein are bound to a NECTIN4 binding domain via their AB, CC', C" D, or EF loop and are bound to a CD3 binding domain, via their B-C, C'-C", or F-G loop.

[0172] The bulk serum protein comprises, for example, albumin, fibrinogen, or a globulin. In some embodiments, the binding moieties are engineered scaffolds. Engineered scaffolds comprise, for example, sdAb, a scFv, a Fab, a VHH, a fibronectin type III domain, immunoglobulin-like scaffold (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knotWSGR Docket No.47517-766.601 peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin-binding module, or a DNA or RNA aptamer scaffold.

[0173] In some cases, the binding moieties comprise a binding site for the bulk serum protein. In some embodiments, the CDRs within the binding moieties provide a binding site for the bulk serum protein. The bulk serum protein is, in some examples, a globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, or pentameric IgM. In some embodiments, the binding moieties comprise a binding site for an immunoglobulin light chain. In some embodiments, the CDRs provide a binding site for the immunoglobulin light chain. The

[0174] In additional embodiments, the binding moieties are any kinds of polypeptides. For example, in certain instances the binding moieties are natural peptides, synthetic peptides, or fibronectin scaffolds, or engineered bulk serum proteins. In some examples, the binding moieties comprise any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the binding moiety is a single chain variable fragment (scFv), a soluble TCR fragment, a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody. In other embodiments, the binding moieties are non-Ig binding domains, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, AFFIMERS®, affitins, alphabodies, avimers, DARPINS®, fynomers, kunitz domain peptides, or monobodies.

[0175] It is contemplated herein that the binding moiety described herein comprises at least one cleavable linker. In one aspect, the cleavable linker comprises a polypeptide having an amino acid sequence recognized and cleaved in an amino acid sequence-specific manner. The binding moiety described herein, in some cases, comprises a protease cleavable linker recognized and cleaved in an amino acid sequence-specific manner. In some embodiments, the protease cleavable linker is recognized in an amino acid sequence-specific manner by a matrix metalloprotease (MMP), for example a MMP9. In some cases, the protease cleavable linker recognized by a MMP9 comprises a polypeptide having an amino acid sequence PR(S / T)(L / I)(S / T) (SEQ ID NO: 647). In some cases, the protease cleavable linker recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 648). In some cases, the protease cleavable linker is recognized in an amino acid sequence-specific manner by a MMP11.

[0176] Proteases are proteins that cleave proteins, in some cases, in an amino acid sequence- specific manner. Proteases include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagineWSGR Docket No.47517-766.601 peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin K, Cathepsin L, kallikreins, hK1, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), peptidase IV (DPPIV / CD26). Table 1: Exemplary proteases and protease recognition sequences Protease Cleavage Domain Sequence SEQ ID NO: MMP7 KRALGLPG 649 MMP7 (DE)8RPLALWRS(DR)8650 MMP9 PR(S / T)(L / I)(S / T) 651 MMP9 LEATA 652 MMP11 GGAANLVRGG 653 MMP14 SGRIGFLRTA 654 MMP PLGLAG 655 MMP PLGLAX 656 MMP PLGC(me)AG 657 MMP ESPAYYTA 658 MMP RLQLKL 659 MMP RLQLKAC 660 MMP2, MMP9, MMP14 EP(Cit)G(Hof)YL 661 Urokinase plasminogen activator (uPA) SGRSA 662 Urokinase plasminogen activator (uPA) DAFK 663 Urokinase plasminogen activator (uPA) GGGRR 664 Lysosomal Enzyme GFLG 665 Lysosomal Enzyme ALAL 666 Lysosomal Enzyme FK (SEQ IDENTIFIER A667) Cathepsin B NLL (SEQ IDENTIFIER A668)WSGR Docket No.47517-766.601 Protease Cleavage Domain Sequence SEQ ID NO: Cathepsin D PIC(Et)FF 669 Cathepsin K GGPRGLPG 670 Prostate Specific Antigen HSSKLQ 671 Prostate Specific Antigen HSSKLQL 672 Prostate Specific Antigen HSSKLQEDA 673 Herpes Simplex Virus Protease LVLASSSFGY 674 HIV Protease GVSQNYPIVG 675 CMV Protease GVVQASCRLA 676 Thrombin F(Pip)RS (SEQ IDENTIFIER A677) Thrombin DPRSFL678Thrombin PPRSFL679Caspase-3 DEVD 680 Caspase-3 DEVDP681Caspase-3 KGSGDVEG682GWEHDG683Enterokinase EDDDDKA684FAP KQEQNPGST 685 Kallikrein 2 GKAFRR686Plasmin DAFK687Plasmin DVLK688Plasmin DAFK689TOP ALLLALL 690

[0177] Proteases are known to be secreted by some diseased cells and tissues, for example tumor or cancer cells, creating a microenvironment that is rich in proteases or a protease-rich microenvironment. In some case, the blood of a subject is rich in proteases. In some cases, cells surrounding the tumor secrete proteases into the tumor microenvironment. Cells surrounding the tumor secreting proteases include but are not limited to the tumor stromal cells, myofibroblasts, blood cells, mast cells, B cells, NK cells, regulatory T cells, macrophages, cytotoxic T lymphocytes, dendritic cells, mesenchymal stem cells, polymorphonuclear cells, and other cells. In some cases, proteases are present in the blood of a subject, for example proteases that target aminoWSGR Docket No.47517-766.601 acid sequences found in microbial peptides. This feature allows for targeted therapeutics such as antigen binding proteins to have additional specificity because T cells will not be bound by the antigen binding protein except in the protease rich microenvironment of the targeted cells or tissue. Other non-limiting examples of linkers that may be utilized in constructs described herein are provided in the Sequence Listing below. Integration into chimeric antigen receptors (CAR)

[0178] The NECTIN4 binding proteins of the present disclosure can, in certain examples, be incorporated into a chimeric antigen receptor (CAR), or to a ProCAR. An engineered immune effector cell, e.g., a T cell or NK cell, can be used to express a CAR that includes a NECTIN4 binding protein containing, for example, an anti- NECTIN4 single domain antibody as described herein. In one embodiment, the CAR including the NECTIN4 binding protein as described herein is connected to a transmembrane domain via a hinge region, and further a costimulatory domain, e.g., a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the CAR further comprises an amino acid sequence encoding an intracellular signaling domain, such as 4-1BB and / or CD3 zeta. Exemplary sequences for a ProCAR comprising a NECTIN4 binding domain is provided in any one of SEQ ID NOS:1-66, or sequences that are at least about 75% to 100% identical to an amino acid sequence selected from SEQ ID NOS: 1-66, such as about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0179] The conditionally active receptors described herein comprise at least one binding moiety comprising a non-CDR loop. In one aspect, the binding moiety masks binding of the NECTIN4 binding domain, until activation. The cleavable linker, for example, comprises a protease cleavage site or a pH dependent cleavage site. The cleavable linker, in certain instances, is cleaved only in a tumor microenvironment. Thus, the binding moiety, connected to the cleavable linker, and further bound to the NECTIN4 binding domain, in some examples, maintains the NECTIN4 binding domain in an inert state in circulation until the cleavable linker is cleaved off in a tumor microenvironment. In some embodiments, the binding moiety binds to the NECTIN4 binding domain. In some embodiments, a non-CDR loop provides a binding site for binding of the moiety to the NECTIN4 binding domain. In some embodiments, the binding moiety masks binding of the NECTIN4 binding domain to its target antigen, e.g., via steric occlusion, via specific intramolecular interactions, such as interactions within the different domains of the polypeptide comprising the binding moiety. In some embodiments, the binding moiety further comprises complimentary determining regions (CDRs).WSGR Docket No.47517-766.601

[0180] In some instances, the binding moiety of a CAR or a proCAR as described herein is a domain derived from an immunoglobulin molecule (Ig molecule), as described above in the section corresponding to conditionally active multispecific NECTIN4 targeting proteins of this disclosure.

[0181] In some embodiments, the conditionally active receptor comprises a NECTIN4 binding domain (aTarget1), a cleavable linker, and a binding moiety (aTarget2). The NECTIN4 binding domain has specificity for a first target (NECTIN4), while the binding moiety has specificity for a second target. The binding moiety also has a modified non-CDR loop, which inhibits binding of the NECTIN4 binding domain to its target. Once cleaved at the cleavable linker, the binding moiety can be released, enabling binding of the NECTIN4 binding domain.

[0182] In some embodiments, the inactive receptor (Inactive ProCAR) comprises a NECTIN4 binding domain (Anti-Tumor Target sdAb or scFv) connected to a binding moiety (Anti-Target 2 sdAb) via a linker comprising a protease cleavage site. The binding moiety comprises a masking peptide / moiety, inserted into one or more non-CDR loops, such that the binding moiety binds to and inhibits the NECTIN4 antigen binding domain. In some embodiments, the binding moiety has specificity for a given target, as described further elsewhere herein. The receptor also comprises a transmembrane domain and an intracellular signaling domain. The receptor is provided in a T-cell (CAR-T). Upon exposure to a tumor environment, the protease cleavage site is cleaved by tumor- associated proteases, thereby activating the receptor, generating the active receptor which does not comprise the binding moiety. The receptor now comprises an active antigen-binding domain. As the receptor is internalized by the cell, new receptors are generated which comprise the binding moiety and are inactive.

[0183] The cleavable linker of the binding moiety, in some embodiments, comprises a protease cleavable site similar to what is described above with respect to the conditionally active multispecific proteins comprising a NECTIN4 binding domain of this disclosure. For instance, the cleavable linker in some embodiments comprises an amino acid sequence selected from the linker sequences provided in the sequence table. Transmembrane domain

[0184] The conditionally active chimeric antigen receptors, T-cell receptor fusion proteins, and T-cell receptors of the present disclosure include a transmembrane domain for insertion into a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the NECTIN4 binding domain and the intracellular domain. In some embodiments, the transmembrane domain is interposed between the NECTIN4 binding domain and the costimulatory domain.WSGR Docket No.47517-766.601

[0185] Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. As one non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 691) can be used. Additional non-limiting examples of suitable TM sequences include: a) CD8 beta derived: GLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 692); b) CD4 derived: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 693); c) CD3 zeta derived: LCYLLDGILFIYGVILTALFLRV (SEQ ID NO: 694); d) CD28 derived: WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 695); e) CD134 (OX40) derived: AAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 696); and f) CD7 derived: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 697). Hinge Region

[0186] In some cases, the conditionally active chimeric antigen receptors, T-cell receptor fusion proteins, and T-cell receptors of the present disclosure comprise a hinge region (also referred to herein as a “spacer”), where the hinge region is interposed between the NECTIN4 binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0187] The hinge region can have a length of from about 4 amino acids to about 50 amino acids (aa), e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.

[0188] Suitable spacers can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0189] Exemplary spacers include glycine polymers (G)n, glycine- serine polymers (including, for example, (GS)n (SEQ ID NO: 622), (GSGGS)n (SEQ ID NO: 698) and (GGGS)n (SEQ ID NO: 624), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary spacers comprise amino acid sequences including, but notWSGR Docket No.47517-766.601 limited to, GGSG (SEQ ID NO: 699), GGSGG (SEQ ID NO: 700), GSGSG (SEQ ID NO: 701), GSGGG (SEQ ID NO: 702), GGGSG (SEQ ID NO: 704), GSSSG (SEQ ID NO: 705), and the like.

[0190] Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res.14: 1779. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 705); CPPC (SEQ ID NO: 706); CPEPKSCDTPPPCPR (SEQ ID NO: 707); see, e.g., Glaser et al. (2005) J. Biol. Chem.280:41494); ELKTPLGDTTHT (SEQ ID NO: 708); KSCDKTHTCP (SEQ ID NO: 709); KCCVDCP (SEQ ID NO: 710); KYGPPCP (SEQ ID NO: 711); EPKSCDKTHTCPPCP (SEQ ID NO: 712); human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 713); human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 714); human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 715); human IgG4 hinge); and the like.

[0191] In some embodiments, the hinge region comprises an amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4, hinge region. The hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgG1 hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 716); see, e.g., Yan et al. (2012) J. Biol. Chem.287:5891.

[0192] In some embodiments, the hinge region comprises an amino acid sequence derived from human CD8; e.g., the hinge region comprises the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 717), or a variant thereof. Conditionally Active Chimeric Antigen Receptors

[0193] In one embodiment, the disclosure provides a conditionally active chimeric antigen receptor (CAR). A CAR generally comprises multiple domains, including a target antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The conditionally active CAR of the present disclosure comprises multiple domains, including a binding moiety, a target antigen binding domain which binds NECTIN4, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a signaling domain of a protein including, but not limited to, ZAP70, CD3 zeta, and 4-1BB.

[0194] In some embodiments, the conditionally active chimeric antigen receptor further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein including, but not limited to, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequencesWSGR Docket No.47517-766.601 thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0195] In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

[0196] In one aspect, the disclosure provides a cell (e.g., T-cell) engineered to express a CAR. In one aspect a cell is transformed with the CAR and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., T-cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the CAR. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cell may transiently express the CAR. Conditionally Active T-cell Receptor Fusion Proteins

[0197] In one embodiment, the disclosure provides a conditionally active T-cell receptor fusion protein. As used herein, a “T-cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T-cell.

[0198] The conditionally active TFP comprises a binding moiety, a NECTIN4 binding domain, and a T-cell receptor subunit. In some embodiments, the T-cell receptor subunit further comprises at least a portion of a T-cell receptor extracellular domain, a transmembrane domain, and a T-cell receptor intracellular domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, or amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0199] In some embodiments, the T-cell receptor intracellular domain comprises a stimulatory domain. The stimulatory domain may be from T-cell receptor subunit, including but not limited to the beta subunit, alpha subunit, delta subunit, gamma subunit, epsilon subunit, or a combination thereof. In some embodiments, the stimulatory domain comprises an immunoreceptor tyrosine-WSGR Docket No.47517-766.601 based activation motif (ITAM) or portion thereof including, but not limited to, CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CDS, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0200] In some embodiments, the conditionally active TFP further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein including, but not limited to, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.

[0201] In some embodiments, the NECTIN4 binding domain is connected to the T-cell receptor extracellular domain by a linker sequence. In some instances, the encoded linker sequence comprises (G4S)n, wherein n=1 to 4 (SEQ ID NO: 718). In some instances, the encoded linker sequence comprises a long linker (LL) sequence. In some instances, the encoded long linker sequence comprises (G4S)n, wherein n=2 to 4 (SEQ ID NO: 719). In some instances, the encoded linker sequence comprises a short linker (SL) sequence. In some instances, the encoded short linker sequence comprises (G4S)n, wherein n=1 to 3 (SEQ ID NO: 720).

[0202] In one aspect, the disclosure provides a cell (e.g., T-cell) engineered to express a conditionally active T-cell receptor fusion protein (TFP). In one aspect a cell is transformed with the conditionally active TFP and the conditionally active TFP is expressed on the cell surface. In some embodiments, the cell (e.g., T-cell) is transduced with a viral vector encoding a conditionally active TFP. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the conditionally active TFP. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TFP. In some such embodiments, the cell may transiently express the conditionally active TFP. Conditionally Active T-cell Receptors

[0203] In one embodiment, the disclosure provides a conditionally active T-cell receptor. A T- cell receptor generally comprises multiple subunits, including alpha, beta, delta, gamma, epsilon, and zeta subunits. The conditionally active T-cell receptor of the present disclosure comprises aWSGR Docket No.47517-766.601 binding moiety. In some embodiments, the binding moiety is attached to a T-cell receptor subunit including, but not limited to, the alpha subunit, beta subunit, or a combination thereof.

[0204] In some embodiments, the binding moiety is capable of masking or masks the binding of the T-cell receptor to its target. In some embodiments, the binding moiety binds to T-cell receptor. In some embodiments, a non-CDR loop provides a binding site for binding of the moiety to the T- cell receptor. In some embodiments, the non-CDR loop provides a binding site specific for T-cell receptor alpha, T-cell receptor beta, or a combination thereof. In some embodiments, the binding moiety masks binding of the T-cell receptor to its target, e.g., via steric occlusion, via specific intermolecular interactions.

[0205] In one aspect, the disclosure provides a cell (e.g., T-cell) engineered to express a conditionally active T-cell receptor (TCR). In one aspect a cell is transformed with the conditionally active TCR and the conditionally active TCR is expressed on the cell surface. In some embodiments, the cell (e.g., T-cell) is transduced with a viral vector encoding a conditionally active TCR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the conditionally active TCR. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TCR. In some such embodiments, the cell may transiently express the conditionally active TCR. Cells

[0206] In one embodiment, the present disclosure provides a cell comprising the chimeric antigen receptor or the conditionally active chimeric antigen receptor, conditionally active T-cell receptor fusion protein, or conditionally active T-cell receptor of the present disclosure. The cell may be a mammalian cell.

[0207] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, HuT- 78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0208] In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cellWSGR Docket No.47517-766.601 obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

[0209] In recent studies, CAR constructs have been used to direct natural killer (NK) cell activity, reviewed by Hermanson & Kaufman (2015, Front Immunol 6:195) and Carlsten & Childs (2015, Front Immunol 6:266). Similar to T cells, NK cells can be transfected with CAR expression constructs and used to induce an immune response. Because NK cells do not require HLA matching, they can be used as allogeneic effector cells (Harmanson & Kaufman, 2015). Also, peripheral blood NK cells (PB-NK), of use for therapy, may be isolated from donors by a simple blood draw. The CAR constructs of use may contain similar elements to those used to make CAR- T cells.

[0210] As such, in some embodiments, the present disclosure provides a cell comprising an NK cell comprising the chimeric antigen receptor, the conditionally active chimeric antigen receptor, conditionally active T-cell receptor fusion protein, or conditionally active T-cell receptor of the present disclosure.

[0211] As discussed above in the context of a conditionally active NECTIN4 binding protein (e.g., a NECTIN4 ProTriTAC), in some embodiments, a conditionally active chimeric antigen receptor as described herein has an improved therapeutic index as compared to that of a chimeric antigen receptor comprising the same NECTIN4 binding domain as the conditionally active variant but is constitutively active instead of being conditionally active. For instance, a NECTIN4 ProCAR, in some embodiments, has an increased therapeutic index than a NECTIN4 CAR. The increased, in some embodiments, is from at least about 2-fold to about 1000-fold, such as about 4- fold to about 800-fold, about 6-fold to about 800-fold, about 6-fold to about 600-fold, about 10-fold to about 400-fold, about 20-fold to about 200-fold, about 30-fold to about 150-fold, about 50-fold to about 100-fold. The increase in therapeutic index, in some embodiments, is attributed to the conjugation of the NECTIN4 binding domain to a binding moiety as described above, with the non- CDR loop and the cleavable linker. Methods of Generating a Cell Comprising a Conditionally Active Receptor

[0212] The present disclosure provides a method of generating a cell comprising a conditionally active chimeric antigen receptor, T-cell receptor fusion protein, or T-cell receptor. The method generally involves genetically modifying a mammalian cell with an expression vector, or an RNA (e.g., in vitro transcribed RNA), comprising nucleotide sequences encoding a conditionally active chimeric antigen receptor, T-cell receptor fusion protein, or T-cell receptor of the presentWSGR Docket No.47517-766.601 disclosure. The genetic modification can be carried out in vivo, in vitro, or ex vivo. The cell can be, for example, an immune cell (e.g., a T lymphocyte or NK cell), a stem cell, or a progenitor cell.

[0213] In some cases, the genetic modification is carried out ex vivo. For example, a T lymphocyte, a stem cell, or an NK cell is obtained from an individual; and the cell obtained from the individual is genetically modified to express a conditionally active chimeric antigen receptor, T- cell receptor fusion protein, or T-cell receptor of the present disclosure. Sources of T-Cells

[0214] In some embodiments, a source of T-cells is obtained from a subject. The term “subject,” as used throughout this disclosure, is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T-cells can be obtained from a number of sources, including, but not limited to, allogenic T cells (e.g., allogeneic donor-derived CAR T cells), natural killer cells (e.g., donor derived natural killer cells), peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T-cell lines available in the art, may be used. In certain embodiments of the present disclosure, T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T-cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the disclosure, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.WSGR Docket No.47517-766.601

[0215] In one embodiment, T-cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTMgradient or by counterflow centrifugal elutriation. A specific subpopulation of T- cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T-cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T-cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T-cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours. Longer incubation times may be used to isolate T-cells in any situation where there are few T-cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T-cells. Thus, by simply shortening or lengthening the time T-cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T-cells (as described further herein), subpopulations of T-cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T-cells can be preferentially selected for or against at culture initiation or at other desired time points. Multiple rounds of selection can also be used in the context of this disclosure. In certain embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

[0216] Enrichment of a T-cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T- cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.WSGR Docket No.47517-766.601

[0217] In one embodiment, a T-cell population can be selected that expresses one or more of appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. WO 2013 / 126712.

[0218] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet one embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T-cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T-cells that normally have weaker CD28 expression.

[0219] In another embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T-cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T-cells express higher levels of CD28 and are more efficiently captured than CD8+ T-cells in dilute concentrations. In one embodiment, the concentration of cells used is 5×10e6 / ml. In other embodiments, the concentration used can be from about 1×105 / ml to 1×106 / ml, and any integer value in between.

[0220] In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C. or at room temperature.

[0221] T-cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, oneWSGR Docket No.47517-766.601 method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of liquid nitrogen.

[0222] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.

[0223] Also contemplated in the context of the disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T-cells, isolated and frozen for later use in T-cell therapy for any number of diseases or conditions that would benefit from T-cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T-cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

[0224] In a further embodiment of the present disclosure, T-cells are obtained from a patient directly following treatment that leaves the subject with functional T-cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T-cells obtained may be optimal orWSGR Docket No.47517-766.601 improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T-cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and / or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T- cells, B cells, dendritic cells, and other cells of the immune system. Activation and Expansion of T-Cells

[0225] T-cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

[0226] Generally, the T-cells of the disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T-cells. In particular, T-cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co- stimulation of an accessory molecule on the surface of the T-cells, a ligand that binds the accessory molecule is used. For example, a population of T-cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T- cells. To stimulate proliferation of either CD4+ T-cells or CD8+ T-cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol. Meth.227(1-2):53-63, 1999).

[0227] In certain embodiments, the primary stimulatory signal and the costimulatory signal for the T-cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface and the agentWSGR Docket No.47517-766.601 providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In one embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. US 20040101519 A1 and US 20060034810 A1 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T-cells in the present disclosure.

[0228] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1:1 ratio of each antibody bound to the beads for CD4+ T-cell expansion and T-cell growth is used. In certain embodiments of the present disclosure, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T-cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one particular embodiment an increase of from about 1-fold to about 3-fold is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one embodiment of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the disclosure, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet one embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

[0229] Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T-cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1:100 to 100:1 and any integer values in-between and in further embodiments the ratio comprises 1:9 to 9:1 and any integer valuesWSGR Docket No.47517-766.601 in between, can also be used to stimulate T-cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T-cells that result in T-cell stimulation can vary as noted above, however certain values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1 particles per T-cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In one particular embodiment, a particle: cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In one embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation. In one embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present disclosure. In particular, ratios will vary depending on particle size and on cell size and type.

[0230] In further embodiments of the present disclosure, the cells, such as T-cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0231] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28 beads) to contact the T-cells. In one embodiment the cells (for example, 104to 109T-cells) and beads (for example, DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer, for example PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present disclosure. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensureWSGR Docket No.47517-766.601 maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In one embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet one embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T-cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T-cells that normally have weaker CD28 expression.

[0232] In one embodiment of the present disclosure, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In one embodiment, the mixture may be cultured for 21 days. In one embodiment of the disclosure the beads and the T- cells are cultured together for about eight days. In one embodiment, the beads and T-cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T-cells can be 60 days or more. Conditions appropriate for T-cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T-cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0233] T-cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T-cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T-cell population (TC, CD8+). Ex vivo expansion of T-cells by stimulating CD3 andWSGR Docket No.47517-766.601 CD28 receptors produces a population of T-cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T-cells comprises an increasingly greater population of TC cells. NECTIN4 Binding Protein Modifications

[0234] The NECTIN4 binding proteins described herein, including NECTIN4 binding domains (e.g., a NECTIN4 binding sdAb of this disclosure) and NECTIN4 targeting multispecific proteins (e.g., a NECTIN4 targeting trispecific or protrispecific protein as described herein) encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or an amino acid sequence for purification of the protein.

[0235] Typical modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0236] Modifications are made anywhere in the NECTIN4 binding proteins described herein, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Certain common peptide modifications that are useful for modification of the NECTIN4 binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.

[0237] In some embodiments, derivatives of the NECTIN4 binding proteins as described herein comprise immunoreactive modulator derivatives and antigen binding molecules comprising one or more modifications.

[0238] In some embodiments, the NECTIN4 binding proteins of the disclosure are monovalent or multivalent bivalent, trivalent, etc.). As used herein, the term “valency” refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds one target molecule or specific position or locus on a target molecule. When an antibody is monovalent, each binding site of the molecule will specifically bind to a single antigen position orWSGR Docket No.47517-766.601 epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site may specifically bind the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or positions on the same antigen).

[0239] In some embodiments, the NECTIN4 binding proteins as set forth above are fused to an Fc region from any species, including but not limited to, human immunoglobulin, such as human IgG1, a human IgG2, a human IgG3, human IgG4, to generate Fc-fusion NECTIN4 binding proteins. In some embodiments, the Fc-fusion NECTIN4 binding proteins of this disclosure have extended half-life compared to an otherwise identical NECTIN4 binding protein. In some embodiments, the Fc-fusion NECTIN4 binding proteins of this disclosure contain inter alia one or more additional amino acid residue substitutions, mutations and / or modifications, e.g., in the Fc region. which result in a binding protein with preferred characteristics including, but not limited to: altered pharmacokinetics, extended serum half-life, etc.

[0240] In some embodiments, such Fc-fused NECTIN4 binding proteins provide extended half- lives in a mammal, such as in a human, of greater than 5 days, greater than 10 days, greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life, in some cases, results in a higher serum titer which thus reduces the frequency of the administration of the NECTIN4 binding proteins and / or reduces the concentration of the antibodies to be administered. Binding to human FcRn in vivo and serum half-life of human FcRn high affinity binding polypeptides is assayed, in some examples, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered.

[0241] The NECTIN4 binding proteins, in some cases, are differentially modified during or after production, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications are carried out by techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0242] Various post-translational modifications of the NECTIN4 binding proteins also encompassed by the disclosure include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.WSGR Docket No.47517-766.601 Moreover, the NECTIN4 binding proteins are, in some cases, modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the modulator. Polynucleotides Encoding NECTIN4 Binding Proteins

[0243] Also provided, in some embodiments, are polynucleotide molecules encoding NECTIN4 binding proteins described herein. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

[0244] The polynucleotide molecules are constructed by known methods such as by combining the genes encoding a single domain NECTIN4 binding protein or gene encoding various domains of NECTIN4 binding proteins comprising more than one domain. In some embodiments, the gene encoding the domains are either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.

[0245] In some embodiments, the polynucleotide coding for a NECTIN4 binding protein as described herein is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

[0246] A variety of expression vector / host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described NECTIN4 binding protein. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells. Thus, the NECTIN4 binding proteins as described herein, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.WSGR Docket No.47517-766.601 Pharmaceutical Compositions

[0247] Also provided, in some embodiments, are pharmaceutical compositions comprising an anti- NECTIN4 binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the NECTIN4 binding proteins or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents. A further embodiment provides one or more of the above described NECTIN4 binding proteins packaged in lyophilized form or packaged in an aqueous medium.

[0248] In some embodiments of the pharmaceutical compositions, the NECTIN4 binding proteins described herein are encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the NECTIN4 binding protein is attached to liposomes. In some instances, the NECTIN4 binding proteins are conjugated to the surface of liposomes. In some instances, the NECTIN4 binding proteins are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0249] The NECTIN4 binding proteins described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient’s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An “effective dose” refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.WSGR Docket No.47517-766.601

[0250] In some embodiments, the NECTIN4 binding proteins of this disclosure are administered at a dosage of up to 10 mg / kg at a frequency of once a week. In some cases, the dosage ranges from about 1 ng / kg to about 10 mg / kg, for example about 1 ng / kg to about 70 ng / kg. In some embodiments, the dose is from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about 70 ng / kg, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 µg / kg, about 0.5 µg / kg to about 5 µg / kg, about 2 µg / kg to about 10 µg / kg, about 7 µg / kg to about 15 µg / kg, about 12 µg / kg to about 25 µg / kg, about 20 µg / kg to about 50 µg / kg, about 35 µg / kg to about 70 µg / kg, about 45 µg / kg to about 80 µg / kg, about 65 µg / kg to about 90 µg / kg, about 85 µg / kg to about 0.1 mg / kg, about 0.095 mg / kg to about 10 mg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg; about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg. The frequency of administration, in some embodiments, is about less than daily, every other day, less than once a day, twice a week, weekly, once in 7 days, once in two weeks, once in three weeks, once in four weeks, or once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg / kg. In some cases, duration of administration is from about 1 day to about 4 weeks or longer. Methods of treatments and Tumor growth reduction properties

[0251] Also provided in certain embodiments are methods of treating a condition associated with malignant cells expressing NECTIN4 in a subject comprising administering to a subject in need thereof an effective amount of a NECTIN4 binding domains or multispecific proteins (including conditionally active multispecific proteins) comprising a NECTIN4 binding domain of this disclosure, or a CAR or a ProCAR comprising a NECTIN4 binding protein as described herein, or a pharmaceutical composition comprising the same. In some embodiments, the condition is a cancer.

[0252] In another aspect, the disclosure provides a method of inhibiting tumor growth or progression in a subject who has malignant cells expressing NECTIN4, comprising administering to the subject in need thereof an effective amount of a NECTIN4 binding domains or multispecific proteins comprising a NECTIN4 binding domain of this disclosure, or a CAR comprising a NECTIN4 binding protein as described herein, or a pharmaceutical composition comprising the same. In another aspect, the disclosure provides a method of inhibiting metastasis of malignantWSGR Docket No.47517-766.601 cells expressing NECTIN4 in a subject, comprising administering to the subject in need thereof an effective amount of a NECTIN4 binding domains or multispecific proteins comprising a NECTIN4 binding domain of this disclosure, or a pharmaceutical composition comprising the same. In another aspect, the disclosure provides a method of inducing tumor regression in a subject who has malignant cells expressing NECTIN4, comprising administering to the subject in need thereof an effective amount of a NECTIN4 binding domains or multispecific proteins comprising a NECTIN4 binding domain of this disclosure, or a pharmaceutical composition comprising the same. In some embodiments, the methods as described herein further comprise administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is a biotherapeutic agent, for example, an antibody. In some embodiments, the second therapeutic agent inhibitor, an oncolytic virus, a kinase inhibitor, an IDO (Indoleamine-pyrrole 2,3-dioxygenase) inhibitor, a glutaminase GLS1 inhibitor, a CAR (Chimeric Antigen Receptor)-T cell or T cell therapy, a TLR (Toll-Like Receptor) Agonist (e.g., TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine.

[0253] In certain embodiments, the NECTIN4 binding proteins of the disclosure reduce the growth of tumor cells in vivo when administered to a subject who has tumor cells that express NECTIN4. Measurement of the reduction of the growth of tumor cells can be determined by multiple different methodologies well known in the art. Nonlimiting examples include direct measurement of tumor dimension, measurement of excised tumor mass and comparison to control subjects, measurement via imaging techniques (e.g., CT or MRI) that may or may not use isotopes or luminescent molecules (e.g., luciferase) for enhanced analysis, and the like. In specific embodiments, administration of the NECTIN4 binding proteins of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen binding agent by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, with an about 100% reduction in tumor growth indicating a complete response and disappearance of the tumor. In further embodiments, administration of the NECTIN4 binding proteins of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen binding agent by about 50-100%, about 75-100% or about 90-100%. In further embodiments, administration of the NECTIN4 binding proteins of the disclosure results in a reduction of in vivo growth of tumor cells as compared to a control antigen binding agent by about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100%.

[0254] In some embodiments, the NECTIN4 binding proteins of the present disclosure are administered to treat a neoplastic condition. Neoplastic conditions, in some embodiments, areWSGR Docket No.47517-766.601 benign or malignant; solid tumors or other blood neoplasia; and, in some embodiments, are selected from the group including, but not limited to: adrenal gland tumors, AIDS-associated cancers, alveolar soft part sarcoma, astrocytic tumors, autonomic ganglia tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocoelic disorders, bone cancer (adamantinoma, aneurismal bone cysts, osteochondroma, osteosarcoma), brain and spinal cord cancers, metastatic brain tumors, breast cancer including triple negative breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytomas, desmoplastic small round cell tumors, ependymomas, epithelial disorders, Ewing's tumors, extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder and bile duct cancers, gastric cancer, gastrointestinal, gestational trophoblastic disease, germ cell tumors, glandular disorders, head and neck cancers, hypothalamic, intestinal cancer, islet cell tumors, Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemias, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphomas, lung cancers (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma etc.), macrophagal disorders, medulloblastoma, melanoma, meningiomas, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancers, papillary thyroid carcinomas, parathyroid tumors, pediatric cancers, peripheral nerve sheath tumors, phaeochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare hematologic disorders, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcomas, skin cancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer, stromal disorders, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, and uterine cancers (carcinoma of the cervix, endometrial carcinoma, and leiomyoma).

[0255] In certain embodiments the NECTIN4 binding proteins of the present disclosure are used as a front-line therapy and administered to subjects who have not previously been treated for the cancerous condition. In other embodiments the NECTIN4 binding proteins of the present disclosure are used to treat subjects that have previously been treated (with a NECTIN4 binding protein of this disclosure or with other anti-cancer agent) and have relapsed or determined to be refractory to the previous treatment. In some embodiments the NECTIN4 binding proteins of the present disclosure are used to treat subjects that have recurrent tumors.

[0256] In some embodiments, a NECTIN4 binding protein as described herein, including a multispecific protein, a CAR or a ProCAR as described herein, is administered to treat a cancer with widespread NECTIN4 expression and prevalence, including, but not limited to, colorectal,WSGR Docket No.47517-766.601 prostate, neuroendocrine, thyroid, lung (both non-small cell lung and small cell lung cancers), gastric, ovarian, endometrial, pancreatic, biliary tract and gallbladder cancer, esophageal, breast, an adenocarcinoma, or any combination thereof.

[0257] In some aspects, the NECTIN4 binding proteins of the present disclosure are administered to treat a proliferative disorder comprising a solid tumor including, but not limited to, adrenal, liver, kidney, bladder, breast, gastric, ovarian, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinomas, sarcomas, glioblastomas, various head and neck tumors, or any combination thereof.

[0258] In some embodiments, the NECTIN4 binding proteins of the present disclosure are administered to a subject suffering from melanoma. In some embodiments, the NECTIN4 binding proteins of the present disclosure are used to diagnose, monitor, treat or prevent melanoma. The term “melanoma,” as used herein, includes all types of melanoma including, but not limited to, primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polypoid melanoma, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, nodular malignant melanoma, lentigo maligna melanoma, lentiginous melanoma, lentiginous malignant melanoma, mucosal lentiginous melanoma, mucosal melanoma, acral lentiginous melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical mole and melanoma (FAM-M) syndrome, desmoplastic malignant melanoma, uveal melanoma, or any combination thereof.

[0259] In some embodiments, possible indications for administration of the NECTIN4 binding proteins of this disclosure or pharmaceutical compositions comprising the same are tumorous diseases especially epithelial cancers / carcinomas such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the genito-urinary tract, e.g., ovarian cancer, endometrial cancer, cervix cancer and kidney cancer, lung cancer, gastric cancer, cancer of the small intestine, liver cancer, pancreas cancer, gall bladder cancer, cancers of the bile duct, esophagus cancer, cancer of the salivatory glands and cancer of the thyroid gland. In some embodiments, the administration of the NECTIN4 binding proteins of this disclosure or pharmaceutical compositions comprising the same is indicated for minimal residual disease, such as early solid tumor, advanced solid tumor or metastatic solid tumor, which is characterized by the local and non-local reoccurrence of the tumor caused by the survival of single cells, or any combination thereof.

[0260] In selected aspects a NECTIN4 binding proteins of the disclosure is incorporated into a chimeric antigen receptors (CAR) and the NECTIN4 CAR is administered in a CAR based therapy effective at treating a cancer, such as: epithelial cancers / carcinomas such as breast cancer, colonWSGR Docket No.47517-766.601 cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the genito-urinary tract, e.g., ovarian cancer, endometrial cancer, cervix cancer and kidney cancer, lung cancer, gastric cancer, cancer of the small intestine, liver cancer, pancreas cancer, gall bladder cancer, cancers of the bile duct, esophagus cancer, cancer of the salivatory glands and cancer of the thyroid gland, small cell lung cancer, non-small cell lung cancer (e.g., squamous cell non-small cell lung cancer or squamous cell small cell lung cancer), large cell neuroendocrine carcinoma (LCNEC), or any combination thereof.

[0261] A chimeric antigen receptor is generally an artificially constructed hybrid protein or polypeptide containing or comprising an antigen binding domain of an antibody linked to a signaling domain (e.g., T-cell signaling or T-cell activation domains). In some embodiments, CARs comprising the NECTIN4 binding protein of the present disclosure have the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T-cells) toward NECTIN4 positive target cells in a non-MHC-restricted manner by exploiting the antigen-binding properties of antibodies or antigen binding fragments thereof. The non-MHC-restricted antigen recognition gives T-cells expressing NECTIN4 CARs the ability to recognize tumorigenic NECTIN4independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T- cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0262] In some embodiments the disclosed NECTIN4 binding proteins are administered to refractory patients (i.e., those whose disease recurs during or shortly after completing a course of initial therapy); sensitive patients (i.e., those whose relapse is longer than 2-3 months after primary therapy); or patients exhibiting resistance to a platinum based agent (e.g., carboplatin, cisplatin, oxaliplatin) and / or a taxane (e.g., docetaxel, paclitaxel, larotaxel or cabazitaxel). In another embodiment the disclosed NECTIN4 CAR treatments are effective at treating ovarian cancer, including ovarian-serous carcinoma and ovarian-papillary serous carcinoma.

[0263] In another embodiment the NECTIN4 binding proteins of the disclosure, the NECTIN4 CAR, or the NECTIN4 sensitized lymphocytes, or any combination thereof are used in maintenance therapy to reduce or eliminate the chance of tumor recurrence following the initial presentation of the disease. In some cases, the disorder has been treated and the initial tumor mass eliminated, reduced or otherwise ameliorated so the patient is asymptomatic or in remission. At such time the subject is administered pharmaceutically effective amounts of the disclosed the NECTIN4 binding proteins of the disclosure, the NECTIN4 CAR, or the NECTIN4sensitized lymphocytes, or any combination thereof one or more times regardless of if there is little or no indication of disease using standard diagnostic procedures. In some embodiments, the NECTIN4WSGR Docket No.47517-766.601 binding proteins of the disclosure, the NECTIN4 CAR, or the NECTIN4 sensitized lymphocytes, or any combination thereof is administered on a regular schedule over a period of time, such as weekly, every two weeks, monthly, every six weeks, every two months, every three months every six months or annually, for example, to reduce the potential of disease recurrence. Moreover, such treatments are in some embodiments continued for a period of weeks, months, years or even indefinitely depending on the patient response and clinical and diagnostic parameters.

[0264] In yet another embodiment, the NECTIN4 binding proteins of the disclosure, the NECTIN4CAR, or the NECTIN4 sensitized lymphocytes, or any combination thereof are used to prophylactically or as an adjuvant therapy to prevent or reduce the possibility of tumor metastasis following a debulking procedure. As used in the present disclosure a “debulking procedure,” is means any procedure, technique or method that eliminates, reduces, treats or ameliorates a tumor or tumor proliferation. Exemplary debulking procedures include, but are not limited to, surgery, radiation treatments (i.e., beam radiation), chemotherapy, immunotherapy or ablation. In some embodiments, at appropriate times, the NECTIN4 binding proteins of the disclosure, the NECTIN4 CAR, or the NECTIN4 sensitized lymphocytes, or any combination thereof are administered as suggested by clinical, diagnostic or theranostic procedures to reduce tumor metastasis. In some embodiments, the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow it to be modified.

[0265] Yet other embodiments of the disclosure comprise administering the NECTIN4 binding protein of the disclosure, the NECTIN4 CAR, or the NECTIN4 sensitized lymphocytes, or any combination thereof to subjects that are asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, the NECTIN4 binding protein of the disclosure, the NECTIN4 CAR, or the NECTIN4sensitized lymphocytes, or any combination thereof are used in preventative sense and given to patients that have been examined or tested and have one or more noted risk factors (e.g., genomic indications, family history, in vivo or in vitro test results, etc.) but have not developed neoplasia. In such cases those skilled in the art would be able to determine an effective dosing regimen through empirical observation or through accepted clinical practices.

[0266] In some embodiments of the methods described herein, the NECTIN4 binding proteins, or compositions as described herein are administered in combination with an agent for treatment of the particular disease, disorder or condition, also referred to herein as an additional therapeutic agent. Such agents include but are not limited to, therapies involving antibodies, small molecules (e.g. rays, and / or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies. In someWSGR Docket No.47517-766.601 embodiments, a NECTIN4 binding protein as described herein is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics, opioids and / or non-steroidal anti-inflammatory agents. In some embodiments, a NECTIN4 binding protein as described herein is administered in combination with anti-cancer agents. Nonlimiting examples of anti-cancer agents that can be used in the various embodiments of the disclosure, including pharmaceutical compositions and dosage forms and kits of the disclosure, include: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1 interferon alpha-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantroneWSGR Docket No.47517-766.601 hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other examples of anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis- porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5- spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethyleneWSGR Docket No.47517-766.601 bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-I receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (such as but not limited to, Lovastatin, Pravastatin, Fluvastatin, Statin, Simvastatin, and Atorvastatin); loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide;WSGR Docket No.47517-766.601 rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; Vitaxin®; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Additional anti-cancer drugs are 5- fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and a topoisomerase inhibitor. In some embodiments, the NECTIN4 binding protein of the present disclosure is used in combination with gemcitabine. In some embodiments, the NECTIN4 binding protein as described herein is administered before, during, or after surgery. Methods of detection of NECTIN4 expression and diagnosis of NECTIN4 associated cancer

[0267] According to another embodiment of the disclosure, kits for detecting expression of NECTIN4 in vitro or in vivo are provided. The kits include the foregoing NECTIN4 binding protein (e.g., a NECTIN4 binding protein containing a labeled anti- NECTIN4 single domain antibody or antigen binding fragments thereof), and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0268] In some cases, NECTIN4 expression is detected in a biological sample. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections takenWSGR Docket No.47517-766.601 for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

[0269] In one embodiment, provided is a method of determining if a subject has cancer by contacting a sample from the subject with an anti- NECTIN4 single domain antibody as disclosed herein; and detecting binding of the single domain antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0270] In another embodiment, provided is a method of confirming a diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with an anti- NECTIN4 single domain antibody as disclosed herein; and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample as compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0271] In some examples of the disclosed methods, the NECTIN4 single domain antibody is directly labeled. In some examples, the methods further include contacting a second antibody that specifically binds the anti- NECTIN4 single domain antibody with the sample; and detecting the binding of the second antibody. An increase in binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects cancer in the subject or confirms the diagnosis of cancer in the subject. In some cases, the cancer is a neuroendocrine cancer, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative breast cancer or ovarian cancer (such as epithelial ovarian carcinoma), or any other type of cancer that expresses NECTIN4. In some examples, the control sample is a sample from a subject without cancer. In particular examples, the sample is a blood or tissue sample.

[0272] In some cases, the antibody that binds (for example specifically binds) NECTIN4 is directly labeled with a detectable label. In another embodiment, the antibody that binds (for example, specifically binds) NECTIN4 (the first antibody) is unlabeled and a second antibody or other molecule that can bind the antibody that specifically binds NECTIN4 is labeled. A second antibody is chosen such that it is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a llama IgG, then the secondary antibody may be an anti-llama-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially. Suitable labels for the antibody or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-WSGR Docket No.47517-766.601 limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferon, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include125I,131I,35S or3H.

[0273] In an alternative embodiment, NECTIN4 can be assayed in a biological sample by a competition immunoassay utilizing NECTIN4 standards labeled with a detectable substance and an unlabeled antibody that specifically binds NECTIN4. In this assay, the biological sample, the labeled NECTIN4 standards and the antibody that specifically bind NECTIN4 are combined and the amount of labeled NECTIN4 standard bound to the unlabeled antibody is determined. The amount of NECTIN4 in the biological sample is inversely proportional to the amount of labeled NECTIN4 standard bound to the antibody that specifically binds NECTIN4.

[0274] The immunoassays and method disclosed herein can be used for a number of purposes. In one embodiment, the antibody that specifically binds NECTIN4 may be used to detect the production of NECTIN4 in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of NECTIN4 in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, the NECTIN4 is cell-surface NECTIN4. In other examples, the NECTIN4 is soluble NECTIN4 (e.g., NECTIN4 in a cell culture supernatant or soluble NECTIN4 in a body fluid sample, such as a blood or serum sample).

[0275] In one embodiment, a kit is provided for detecting NECTIN4 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of soluble NECTIN4 protein or fragment. Kits for detecting a polypeptide will typically comprise a single domain antibody, according to the present disclosure, that specifically binds NECTIN4. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab is included in the kit. In a further embodiment, the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).

[0276] In one embodiment, a kit includes instructional materials disclosing means of use of an antibody that binds NECTIN4. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk), may be visual (such as video files), or provided through an electronic network, for example, over the internet, World Wide Web, an intranet, or other network. The kits may also include additional components to facilitate the particularWSGR Docket No.47517-766.601 application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

[0277] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting NECTIN4 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to a NECTIN4 polypeptide. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0278] Methods of determining the presence or absence of a cell surface marker are well known in the art. For example, the antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds or drugs. The antibodies can also be utilized in immunoassays such as but not limited to radioimmunoassays (RIAs), ELISA, or immunohistochemical assays. The antibodies can also be used for fluorescence activated cell sorting (FACS). FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells. See U.S. Patent No.5, 061,620). Any of the single domain antibodies that bind NECTIN4, as disclosed herein, can be used in these assays. Thus, the antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation. Certain definitions

[0279] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0280] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example,WSGR Docket No.47517-766.601 “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

[0281] The terms “individual,” “patient,” or “subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker).

[0282] An “antibody” typically refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Human light chains comprise a variable domain (VL) and a constant domain (CL) wherein the constant domain may be readily classified as kappa or lambda based on amino acid sequence and gene loci. Each heavy chain comprises one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD, comprises three domains termed CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In IgG, IgA, and IgD classes the CH1 and CH2 domains are separated by a flexible hinge region, which is a proline and cysteine rich segment of variable length (generally from about 10 to about 60 amino acids in IgG). The variable domains in both the light and heavy chains are joined to the constant domains by a “J” region of about 12 or more amino acids and the heavy chain also has a “D” region of about 10 additional amino acids. Each class of antibody further comprises inter-chain and intra-chain disulfide bonds formed by paired cysteine residues. There are two types of native disulfide bridges or bonds in immunoglobulin molecules: interchain and intrachain disulfide bonds. The location and number of interchain disulfide bonds vary according to the immunoglobulin class and species. Interchain disulfide bonds are located on the surface of the immunoglobulin, are accessible to solvent and are usually relatively easily reduced. In the human IgG1 isotype there are four interchain disulfide bonds, one from each heavy chain to the light chain and two between the heavy chains. The interchain disulfide bonds are not required for chain association. As is well known the cysteine rich IgG1 hinge region of the heavy chain has generally been held to consist of three parts: an upper hinge, a core hinge, and a lower hinge. Those skilled in the art will appreciate that that the IgG1 hinge region contain the cysteines in the heavy chain that comprise the interchain disulfide bonds (two heavy / heavy, two heavy / light), which provide structural flexibility that facilitates Fab movements. The interchain disulfide bond between the light and heavy chain of IgG1 are formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain. The interchain disulfide bonds between the heavy chains are at positions C226 and C229 (all numbered per the EU index according to Kabat, et al., infra.)WSGR Docket No.47517-766.601

[0283] As used herein the term “antibody” includes polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, deimmunized, humanized and primatized antibodies, CDR grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies (e.g., a monovalent IgG), multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof, immunospecific antibody fragments such as: hcIgG, a V-NAR, Fv, Fd, Fab, F(ab')2, F(ab'), Fab2, Fab3 fragments, single-chain fragments (e.g., di-scFv, scFv, scFvFc, scFv- zipper, scFab), disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as nanobodies or single variable domain antibodies comprising merely one variable domain such as sdAb (VH, VL, or VHH domains), “r IgG” (“half antibody”), diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, “minibodies” are in some instances exemplified by a structure which is as follows: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies; and derivatives thereof including Fc fusions and other modifications, and any other immunoreactive molecule so long as it comprises a domain having a binding site for preferential association or binding with a NECTIN4 protein. Moreover, unless dictated otherwise by contextual constraints the term further comprises all classes of antibodies (i.e. IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Heavy-chain constant domains that correspond to the different classes of antibodies are typically denoted by the corresponding lower-case Greek letter alpha, delta, epsilon, gamma, and mu, respectively. Light chains of the antibodies from any vertebrate species can be assigned to one of their constant domains. In some embodiments, the NECTIN4 binding proteins comprise a heavy chain only antibody, such as a VH or a VHH domain. In some cases, the NECTIN4 binding proteins comprise a heavy chain only antibody that is an engineered human VH domain. In some examples, the engineered human VH domain is produced by panning of phage display libraries. In some embodiments, the NECTIN4 binding proteins comprise a VHH. The term “VHH,” as used herein, refers to single chain antibody binding domain devoid of light chain. In some cases, a VHH is derived from an antibody of the type that can be found in Camelidae or cartilaginous fish which are naturally devoid of light chains or to a synthetic and non-immunized VHH which can be constructed accordingly. Each heavy chain comprises a variable region encoded by V-, D- and J exons. A VHH, in some cases, is a natural VHH, such as a Camelid-derived VHH, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camels, llamas, vicunas, guanacos, andWSGR Docket No.47517-766.601 cartilaginous fish (such as, but not limited to, sharks). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, a Huacaya Alpaca or a Suri alpaca).

[0284] As used herein, “Variable region” or “variable domain” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable configuration, connected by three CDRs, which form loops connecting, and in some cases forming regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. The assignment of amino acids to each domain, framework region and CDR is, in some embodiments, in accordance with one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no.91- 3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular / MSI Pharmacopeia) unless otherwise noted. In some embodiments of this disclosure, the NECTIN4 binding proteins comprise heavy chain only antibodies, such as VH or VHH domains, and comprise three CDRs. Such heavy chain only antibodies, in some embodiments, bind NECTIN4 as a monomer with no dependency on dimerization with a VL (light chain variable) region for optimal binding affinity.

[0285] “Variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a singleWSGR Docket No.47517-766.601 amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. It is not intended that CDRs of the present disclosure necessarily correspond to the Kabat numbering convention.

[0286] The term “Framework” or “FR” residues (or regions) refer to variable domain residues other than the CDR or hypervariable region residues as herein defined. A “human consensus framework” is a framework which represents the most commonly occurring amino acid residue in a selection of human immunoglobulin VL or VH framework sequences.

[0287] The term “epitope,” as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0288] As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0289] As used herein, “elimination half-time” is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed.1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of mostWSGR Docket No.47517-766.601 drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, t1 / 2the time required for 50% completion of the process. The units of these reaction are simply related (k×t1 / 2=0.693) and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

[0290] As used herein, the term “binding affinity” refers to the affinity of the proteins described in the disclosure to their binding targets and is expressed numerically using “KD” values. If two or more proteins are indicated to have comparable binding affinities towards their binding targets, then the KDvalues for binding of the respective proteins towards their binding targets, are within ±2-fold of each other. If two or more proteins are indicated to have comparable binding affinities towards single binding target, then the KDvalues for binding of the respective proteins towards said single binding target, are within ±2-fold of each other. If a protein is indicated to bind two or more targets with comparable binding affinities, then the KD values for binding of said protein to the two or more targets are within ±2-fold of each other. In general, a higher KD value corresponds to a weaker binding. In some embodiments, the “KD” is measured by a radiolabeled antigen binding assay (RIA) or surface plasmon resonance assays using a BIACORE -2000 or a BIACORE - 3000 (BIAcore, Inc., Piscataway, N.J.). In certain embodiments, an “on-rate” or “rate of association” or “association rate” or “kon” and an “off-rate” or “rate of dissociation” or “dissociation rate” or “koff” are also determined with the surface plasmon resonance technique using a BIAcore -2000 or a BIAcore -3000 (BIAcore, Inc., Piscataway, N.J.). In additional embodiments, the “KD”, “kon”, and “koff” are measured using the OCTET® Systems (Pall Life Sciences). In an exemplary method for measuring binding affinity using the OCTET® Systems, the ligand, e.g., biotinylated human or cynomolgus NECTIN4, is immobilized on the OCTET® streptavidin capillary sensor tip surface which streptavidin tips are then activated according to manufacturer's instructions using about 20-50 µg / ml human or cynomolgus NECTIN4 protein. A solution of PBS / Casein is also introduced as a blocking agent. For association kinetic measurements, NECTIN4 binding protein variants are introduced at a concentration ranging from about 10 ng / mL to about 100 µg / mL, about 50 ng / mL to about 5 µg / mL, or about 2 ng / mL to about 20 µg / mL. In some embodiments, the NECTIN4 binding single domain proteins are used at aWSGR Docket No.47517-766.601 concentration ranging from about 2 ng / mL to about 20 µg / mL. Complete dissociation is observed in case of the negative control, assay buffer without the binding proteins. The kinetic parameters of the binding reactions are then determined using an appropriate tool, e.g., ForteBio software.

[0291] As used herein, in some embodiments, “treatment” or “treating” or “treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, “treatment” or “treating” or “treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease (e.g., an individual who carries a genetic marker for a disease such as breast cancer).

[0292] A “TriTAC,” a “NECTIN4 targeting TriTAC,” or a “NECTIN4 targeting trispecific protein,” as used herein refers to a trispecific binding protein that is not conditionally activated, and comprises a binding moiety that is specific for a bulk serum protein, a first target antigen binding domain, and a second target antigen binding domain, wherein at least one of the first target antigen binding domain and the second target antigen binding domain comprises a NECTIN4 binding protein as described herein, and at least one of the first target antigen binding domain and the second target antigen binding domain comprises a domain that binds a CD3, such as a human CD3.

[0293] A “ProTriTAC,” or a “NECTIN4 targeting protrispecific protein,” as used herein refers to a trispecific binding protein that is conditionally activated, and comprises (i) a cleavable linker (e.g., comprising an amino acid sequence as set forth in SEQ ID NOS: 618, and 632-646)), (ii) a binding moiety that is specific for a bulk serum protein and also comprises a masking moiety (e.g., comprising an amino acid sequence as set forth in SEQ ID NO: 612) which prohibits the binding of a first target antigen binding domain or a second target antigen binding domain to its target, wherein at least one of the first target antigen binding domain and the second target antigen binding domain comprises a NECTIN4 binding protein as described herein. The ProTriTAC proteins of this disclosure are, in some cases, activated from a masked state to an active state by cleavage of theWSGR Docket No.47517-766.601 cleavable linker, for example, in a protease rich environment, such as in a tumor microenvironment, to form an active drug. An active drug, as provided herein, in some instances, comprises a NECTIN4 binding domain of the disclosure and a CD3 binding domain of the disclosure. An example of an active drug is provided in SEQ ID NOS: 265-329, or an amino acid sequence that is at least about 75% to 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 265-329such as about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 265-329.

[0294] A “non-cleavable prodrug,” as used herein refers to a ProTriTAC as described above where the cleavable linker is replaced by a non-cleavable linker (e.g., a linker as in SEQ ID NO: 696). An example of an active drug is provided in SEQ ID NOS: 265-329, or an amino acid sequence that is at least about 75% to 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 265-329, such as about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 265-329.

[0295] In a non-beta-sandwich scaffold (e.g., a DARPIN®, an AFFIMER®, an affibody), the “non-CDR loops” refer to an area that is (1) amenable for sequence randomization to allow engineered specificities to a second antigen, and (2) distal to the primary specificity determining region(s) typically used on the scaffold to allow simultaneous engagement of the scaffold to both antigens without steric interference. For this purpose, the primary specificity determining region(s) can be defined using the framework established in the Skrlec 2015 publication (Trends in Biotechnol, 33:408-418). An excerpt of the framework is listed below in Table 2: Table 2 Scaffold Primary specificity determining region(s) Affibody 13 residues in two helices AFFIMER® 12-36 residues Anticalin Four loops (up to 24 aa) Avimer 11 residues Centyrin 13 residues DARPIN® 7 residues in each n-repeat, or 8 residues in each n-repeat Fynomer 6 residues in the RT- and n-Src-loop Kunitz domain 1-2 loopsWSGR Docket No.47517-766.601

[0296] “Chimeric antigen receptor” or “CAR” or “CARs”, as used herein, refers to engineered receptors which provide antigen specificity to cells (for example T cells). CARs comprise multiple domains, for example, at least one target antigen binding domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. Each domain may be connected by a linker. A “ProCAR,” as used herein, refers to a conditionally activatable CAR, comprising a NECTIN4 binding domain of this disclosure. EXAMPLES

[0297] The application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented in order to more fully illustrate embodiments and should in no way be construed, however, as limiting the broad scope of the application. Example 1: Screening of Phage Display Library for Identification of NECTIN4 Binding Domains

[0298] Llamas were immunized with purified NECTIN4 dimer protein expressed in 293 cells. A phage display library for expression of heavy variable antibody domains was constructed from circulating B cells isolated from the immunized llamas (van der Linden, et al., 2000. J Immunol Methods 240:185–195). Phage clones were screened for binding to NECTIN4 dimer by expressing llama anti-NECTIN4 proteins in E coli, preparing periplasmic extracts, and performing colorimetric ELISAs.58 unique heavy chain only sequences were identified (SEQ ID NOS 1 to 58) that produced a signal in the ELISA screening relative to the control with human or cynomolgus monkey NECTIN4 protein (Table 3). The CDR1, CDR2, and CDR3 sequences for these heavy variable domains are, respectively, SEQ ID NOS 67 to 132, 133 to 198, and 199 to 264.

[0299] Table 3: Binding of Llama Anti-NECTIN4 Antibodies to Human or Cynomolgus Monkey NECTIN4 in an ELISA Assay Sequence ELISA ELISA cyNECTIN4 / contro name huNECTIN4cyNECTIN4huNECTIN4 / controll NECTIN4-L51 3.8 3.8 12.5 12.5 NECTIN4-L68 3.9 4.0 22.6 22.9 NECTIN4-L82 3.6 3.7 29.9 30.5 NECTIN4-L98 3.1 3.5 40.2 46.3 NECTIN4- L1073.7 3.6 45.0 44.3NECTIN4- L1584.0 4.0 18.7 18.7NECTIN4- L1773.8 3.7 39.7 39.0NECTIN4-L28 3.8 3.7 35.7 35.0 NECTIN4-L81 3.8 3.8 42.6 42.5WSGR Docket No.47517-766.601 Sequence ELISA ELISA cyNECTIN4 / contro name huNECTIN4cyNECTIN4huNECTIN4 / controll NECTIN4-L70 3.8 4.0 34.8 36.9 NECTIN4-L86 3.8 4.0 43.2 45.3 NECTIN4-L89 4.0 4.0 26.9 26.9 NECTIN4-L93 3.8 4.0 41.7 44.4 NECTIN4- L1123.5 3.9 48.9 54.4NECTIN4- L1513.7 3.7 52.8 52.9NECTIN4- L1523.8 3.6 50.1 48.1NECTIN4-L54 3.7 3.8 40.3 40.4 NECTIN4- L1103.7 4.0 51.0 55.2NECTIN4- L1602.3 3.9 30.0 51.2NECTIN4-L46 3.8 4.0 31.5 33.1 NECTIN4- L1673.8 4.0 21.3 22.2NECTIN4-L71 4.0 4.0 44.8 45.4 NECTIN4-L85 4.0 4.0 41.8 41.8 NECTIN4-L95 3.0 4.0 31.2 41.5 NECTIN4- L1623.3 4.0 24.8 29.9NECTIN4-L48 4.0 4.0 24.7 24.7 NECTIN4-L91 4.0 4.0 45.9 45.9 NECTIN4- L1010.9 2.0 9.5 20.9NECTIN4- L1693.7 3.6 38.8 37.2NECTIN4-L31 3.8 3.8 11.9 12.1 NECTIN4- L1183.8 4.0 47.4 49.7NECTIN4-L57 3.7 3.8 20.6 20.7 2NEC164 3.7 4.0 5.8 6.3 NECTIN4-L65 3.8 4.0 29.8 31.5 NECTIN4- L9524.0 4.0 53.4 53.4NECTIN4-L2 3.8 3.8 7.2 7.3 NECTIN4-L10 3.7 3.7 29.3 29.4 NECTIN4-L14 0.5 1.6 4.9 16.0 NECTIN4-L17 4.0 4.0 33.6 33.6 NECTIN4-L26 2.4 2.6 8.9 9.6 NECTIN4-L34 2.4 3.6 24.0 36.2 NECTIN4-L42 4.0 4.0 29.2 29.2WSGR Docket No.47517-766.601 Sequence ELISA ELISA cyNECTIN4 / contro name huNECTIN4cyNECTIN4huNECTIN4 / controll NECTIN4- L4623.8 4.0 27.2 28.8NECTIN4-L47 4.0 4.0 25.0 25.0 NECTIN4-L79 3.8 3.7 31.5 31.4 NECTIN4-L90 1.4 2.7 8.9 17.1 NECTIN4- L1023.7 3.7 30.7 30.5NECTIN4- L1143.8 4.0 46.7 49.7NECTIN4- L1154.0 4.0 34.2 34.2NECTIN4- L1263.5 3.6 42.5 43.6NECTIN4- L1313.8 3.7 29.5 28.8NECTIN4- L1343.4 3.9 16.8 19.2NECTIN4- L1572.1 3.7 27.3 48.6NECTIN4- L1874.0 4.0 48.7 48.7NECTIN4-L36 2.4 3.5 25.3 37.0 NECTIN4- L4823.6 4.0 36.9 40.6NECTIN4-L87 3.8 4.0 46.3 48.5 NECTIN4- L8924.0 4.0 46.1 46.1

[0300] The numerical values in Table 1 represent the absorbance readings for the colorimetric ELISA. Example 2: Incorporation of NECTIN4 Binding Heavy Chain Only Single Domain Antibodies Into Fusion Proteins and T Cell Dependent Cellular Cytotoxicity Assays

[0301] The anti-NECTIN4 antibody sequences were cloned into DNA constructs for expression of recombinant fusion proteins (SEQ ID NOS: 265 to 321). The coding sequences of the fusion proteins contained a signal peptide for secreted cell expression, a stub to mimic the cleaved version of a conditionally-active T cell engager (SEQ ID NO: 614), humanized anti-CD3 antibody scFv fragment (SEQ ID NO: 613), one of the anti-NECTIN4 antibody variable domains (SEQ ID NOS: 1 to 58), and a repeat of six histidine sequences (SEQ ID NO: 615). A linker sequence was inserted at the junctions between antibody domains (SEQ ID NO: 616). These anti-CD3 / anti-NECTIN4 (CT) fusion protein constructs were transfected into Expi293 cells (Life Technologies). The amount fusion protein in the conditioned media from the transfected Expi293 cells was quantitated using byWSGR Docket No.47517-766.601 using an Octet instrument with either Protein A or anti-6× His tips using a fusion protein of similar molecular weight to the anti-CD3 / anti-NECTIN4 proteins as a standard.

[0302] The conditioned media were tested in a T-cell dependent cellular cytotoxicity assay (TDCC) (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA.2015. J Biomol Screen.20:519-27). In this assay, luciferase labelled UBLC1 cells that express NECTIN4, were combined with purified human T cells and a titration of anti-CD3 / anti-NECTIN4 fusion protein. If a fusion protein directs T cells to kill the target cells, the signal in a luciferase assay performed at 48 hours after starting the experiment should decrease. FIGS.1 to 12 have graphs of TDCC viability results. EC50 values from the TDCC assay are listed in Table 4. The most potent molecule had an EC50value of 2.3 pM with UBLC1 cells. A negative control for the TDCC assays was an anti-GFP / anti-albumin / anti-CD3 protein, and this protein did not direct the T cells to kill the target cells except for slight activity at the highest concentration tested (e.g., FIG.12). Table 4: EC50 Values for Redirected T Cell Killing of H292 Cells by anti-CD3 / anti-NECTIN4 fusion proteins containing Llama Anti-NECTIN4 Sequences. NECTIN4 Binder UBLC1 EC50 (pM) NECTIN4-L2 n / a NECTIN4-L10 n / a NECTIN4-L14 570 NECTIN4-L17 1500 NECTIN4-L26 12 NECTIN4-L28 6.8 NECTIN4-L31 250 NECTIN4-L34 1900 NECTIN4-L36 5.3 NECTIN4-L42 4.5 NECTIN4-L46 8.7 NECTIN4-L47 n.d. NECTIN4-L48 34 NECTIN4-L51 16 NECTIN4-L54 4.1 NECTIN4-L57 13 NECTIN4-L65 71 NECTIN4-L68 63 NECTIN4-L70 13 NECTIN4-L71 n / a NECTIN4-L79 11 NECTIN4-L81 47 NECTIN4-L82 n / a NECTIN4-L85 41 NECTIN4-L86 2.4 NECTIN4-L87 5.2WSGR Docket No.47517-766.601 NECTIN4 Binder UBLC1 EC50 (pM) NECTIN4-L89 8500 NECTIN4-L90 9.1 NECTIN4-L91 8.4 NECTIN4-L93 260 NECTIN4-L95 44 NECTIN4-L98 n / a NECTIN4-L101 2.5 NECTIN4-L102 25 NECTIN4-L107 2.3 NECTIN4-L110 47 NECTIN4-L112 14 NECTIN4-L114 550 NECTIN4-L115 n / a NECTIN4-L118 200 NECTIN4-L126 20 NECTIN4-L131 470 NECTIN4-L134 3.6 NECTIN4-L151 n.d. NECTIN4-L152 n / a NECTIN4-L157 18 NECTIN4-L158 44 NECTIN4-L160 n / a NECTIN4-L162 4.88 NECTIN4-L167 63.8 NECTIN4-L169 10.8 NECTIN4-L177 5.17 NECTIN4-L187 10.1 NECTIN4-L462 13.4 NECTIN4-L482 718 NECTIN4-L892 32.2 NECTIN4-L952 <1

[0303] n / a insufficient activity to calculate an EC50 using the protein concentrations tested. n.d. no data with this binder.

[0304] Binding affinity of ten anti-CD3 / anti-NECTIN4 (CT) fusion proteins for human or cynomolgus monkey NECTIN4 extracellular domain in conditioned medium was determined using an Octet instrument with streptavidin tips loaded at a single concentration with the appropriate biotinylated NECTIN4 protein. The affinities of these binders for the for human or cynomolgus monkey NECTIN4 are reported in Table 5. Table 5: Affinities of Anti-CD3 / Anti-NECTIN4 fusion proteins containing Llama Anti- NECTIN4 Sequences for Human NECTIN4 NECTIN4 Binder huNECTIN4 KD(nM) cyNECTIN4 KD(nM) NECTIN4-L46 1.6 1.6WSGR Docket No.47517-766.601 NECTIN4 Binder huNECTIN4 KD (nM) cyNECTIN4 KD (nM) NECTIN4-L54 7.6 7.6 NECTIN4-L79 105.0 6.7 NECTIN4-L82 35.5 35.6 NECTIN4-L86 10.9 11.0 NECTIN4-L107 18.4 19.6 NECTIN4-L134 30.5 22.2 NECTIN4-L177 9.2 11.5 NECTIN4-L462 5.1 4.6 NECTIN4-L952 8.8 8.3 Example 3: Humanization of NECTIN4 Binding Antibodies and T Cell Dependent Cellular Cytotoxicity Assays

[0305] Eight of the llama anti-NECTIN4 antibody sequences were humanized by grafting their CDR sequences onto human germline sequences, while retaining some llama framework sequences to ensure the antibodies did not lose activity (SEQ ID NOS 59 to 66). These sequences were cloned into expression constructs for expression as anti-CD3 / anti-NECTIN4 fusion proteins (SEQ IDS 322 to 329) and fusion proteins containing an anti-ALB domain with a non-CDR loop mask connected to an anti-CD3 domain by a non-cleavable linker (NCLV), and an anti-NECTIN4 domain (SEQ IDS 330 to 347) in Expi293 cells, along with their parental constructs, as before. The fusion proteins were quantitated and used in a TDCC assay as before using luciferase labeled UBLC1 cells and luciferase labeled LUDLU1 cells. The results of the TDCC assays are plotted in FIGS.13 to 28, and the EC50 values for directed T cell killing are listed in Table 6. Potent directed T cell killing was observed with humanized NECTIN4 antibodies. Table 6: EC50 Values for Redirected T Cell Killing of UBLC1 and LUDLU1 Cells by anti- CD3 / anti-NECTIN4 fusion proteins containing Llama or Humanized Anti-NECTIN4 Sequences. UBLC1 EC50 (pM) LUDLU1 EC50 (pM) NECTIN4 BinderNCLV CT NCLV CTNECTIN4- L107n.d. 19.7 n.d. 138NECTIN4- L134n.d. 729.6 n.d. 5610NECTIN4- L177n.d. 8.6 n.d. 131.3NECTIN4-L46 n.d. 2.1 n.d. 19.9 NECTIN4- L462n.d. 5.0 n.d. 45.4NECTIN4-L82 n.d. 33.0 n.d. 237WSGR Docket No.47517-766.601 UBLC1 EC50 (pM) LUDLU1 EC50 (pM) NECTIN4 BinderNCLV CT NCLV CTNECTIN4-L91 n.d. 5.4 n.d. 49.4 NECTIN4- L952n.d. 11.7 n.d. 79.6NECTIN4- H1075741 85.0 12500 517.6NECTIN4- H1344484 794.8 n / a 3524NECTIN4- H177172.8 6.8 8818 97.3NECTIN4-H46 52.5 1.8 6017 21.9 NECTIN4- H46274.6 33.3 48350 140.3NECTIN4-H82 1112 30.7 17700 241.8 NECTIN4-H91 117.3 12.8 9523 72.7 NECTIN4- H95277.7 17.4 15700 70.1

[0306] n.d. no data with this binder.

[0307] Non-cleavable anti-ALB / anti-CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing four different humanized anti-NECTIN4 antibodies were expressed in 293 cells as before and purified by using Protein A affinity chromatography following by desalting and cation exchange chromatography. FIGS.29 and 30 show Coomassie stained SDS- PAGE gels of the purified NCLV (FIG.29) and CT (FIG.30) proteins.

[0308] The purified fusion proteins were tested as before in TDCC assays using luciferase labeled SW780, SCC9, HT1376, and HPAFII cells. The results of the TDCC assays are plotted in FIGS.31 to 38, and the EC50 values for directed T cell killing are listed in Table 7. Potent directed T cell killing was observed with purified humanized NECTIN4 CT fusion protein, whereas the potency of the NCLV fusion proteins was greatly reduced compared to the CT proteins. A negative control for the TDCC assays was an anti-GFP / anti-albumin / anti-CD3 protein, and this protein did not direct the T cells to kill the target cells except for slight activity at the highest concentration tested (e.g., FIGS.32, 34, 36, and 38). Table 7: EC50 Values for Redirected T Cell Killing of SW780, SCC9, HT1376, and HPAFII Cells by anti-ALB / anti-CD3 / anti-NECTIN4 (NCLV) and anti-CD3 / anti-NECTIN4 (CT) fusion proteins containing Humanized Anti-NECTIN4 Sequences SCC9 EC50 HT1376 EC50 HPAFII EC50 SW780 EC50 (pM) (pM) (pM) (pM) NECTIN4 Binder NCLV CT NCLV CT NCLV CT NCLV CTWSGR Docket No.47517-766.601 NECTIN4-H46 5200 10.9 5448 8.2 8110 20.6 3378 5.9 NECTIN4-H462 n / a 77.2 n / a 106.8 n / a 705.7 14500 74.9 NECTIN4-H82 n / a 101.5 n / a 111.1 n / a 220.8 55300 65.5 NECTIN4-H952 13400 32.0 n / a 51.2 n / a 165.2 11700 42.8

[0309] n / a insufficient activity to calculate an EC50 using the protein concentrations tested. Example 4: Redirected T Cell Killing of SW780, HT1376, and MV411 Cells by anti-ALB / anti- CD3 / anti-NECTIN4 and Anti-CD3 / Anti-NECTIN4 fusion proteins containing humanized anti-NECTIN4 sequences

[0310] Four purified anti-CD3 / anti-NECTIN4 fusion proteins were proteolytically activated using Matriptase, a serine protease.100 nM of Matriptase was added to 100 µg of each fusion protein containing humanized anti-NECTIN4 sequences H46, H82, H952, and H462. Each individual mixture was incubated at 37°C for 2 hours. Upon protease activation, 2ug of inactivated and activated sample set of fusion proteins were loaded onto a 10-20% Tris-Glycine SDS-PAGE gel. The results of the protease activation are shown in FIG.39. The matriptase treated protein migrates as two fragments, indicating the protein was cleaved by the matriptase. TDCC assays were done using luciferase labeled SW780, HT1376, and MV411 cells. The results of the TDCC assays are plotted in FIGS.40 to 51, and the EC50values for directed T cell killing are listed in Table 8 and Table 9. Directed T cell killing was observed with humanized NECTIN4 antibodies. Table 8: EC50Values for Redirected T Cell Killing of SW780 cells by fusion proteins containing Humanized Anti-NECTIN4 Sequences: anti-ALB / anti-CD3 / anti-NECTIN4 With a Non- Cleavable Linker (NCLV), anti-ALB / anti-CD3 / anti-NECTIN4 With a Cleavable Linker (ProTriTAC L040), and anti-ALB / anti-CD3 / anti-NECTIN4 With a Cleavable Linker Treated with Matriptase SW780 EC50 (pM) NECTIN4 Binder NCLV MAT-Act ProTriTAC L040 NECTIN4-H46 3047 18.5 659 NECTIN4-H82 36600 130.7 8358 NECTIN4-H952 13740 112.9 1504 NECTIN4-H462 37090 49.9 1122 Table 9: EC50Values for Redirected T Cell Killing of HT1376 and MV411 Cells by fusion proteins containing Humanized Anti-NECTIN4 Sequences: anti-ALB / anti-CD3 / anti-NECTIN4 With a Non-Cleavable Linker (NCLV), anti-ALB / anti-CD3 / anti-NECTIN4 With a CleavableWSGR Docket No.47517-766.601 Linker (ProTriTAC L040), and anti-ALB / anti-CD3 / anti-NECTIN4 With a Cleavable Linker Treated with Matriptase HT1376 MV411 Binder CT NCLV ProTriTAC L040 CT H46 6.68E-12 2.40E-09 1.03E-10 H82 6.40E-11 1.33E-08 1.11E-09 1.30E- 09 H952 2.97E-11 1.51E-08 1.74E-09 9.20E- 09 H462 7.35E-11 4.48E-08 7.67E-09 5.90E- 09 Example 5: Redirected T Cell Killing of HCC70, SW780, HT29 and CAL27 Cells by Anti- CD3 / Anti-NECTIN4 fusion protein containing humanized Anti-NECTIN4 Sequence H46

[0311] Four Binders were narrowed down to a single binder, NECTIN4-H46. Same as before, TDCC assays were conducted using luciferase labeled HCC70 SW780, HT29, and CAL27 cells. The results of the TDCC assays are plotted in FIGS.52 to 55, and the EC50 values for directed T cell killing are listed in Table 10. Potent directed T cell killing was observed with humanized NECTIN4 antibody H46 in all the cell lines. Table 10: EC50 Values for Redirected T Cell Killing of HCC70, SW780, HT29 and CAL27 Cells by Anti-CD3 / Anti-NECTIN4 (CT) fusion protein containing humanized Anti-NECTIN4 Sequence H46 H46-CT HCC70 2.16E-12 SW780 1.15E-11 HT29 5.99E-12 CAL27 4.57E-12 Example 6: Demonstration of improved tolerability in mouse, conferred by exemplary anti- CD3 / anti-NECTIN4 Fusion Proteins

[0312] All animal experiments were conducted according to the protocol approved by Institutional Animal Care and Use Committee of Harpoon Therapeutics (protocol number HAR- 001-2019). Animals were purchased from The Jackson Laboratory then housed in a pathogen free animal facility located at Harpoon Therapeutics in accordance with IACUC guidelines. All studies were performed in NSG (NOD-SCID IL2Rgammanull) female mice 6, 7, or 11 weeks of age with n = 5-10 mice per group. For each experiment, mice were age matched.

[0313] An admixture of Nectin4-expressing human tumor cells, either HPAF-II, FaDu, or SW780 (5E6) and activated and expanded human T cells (5E6) at an E:T ratio of 1:2 was implanted subcutaneously on the right flank of NSG mice (Day 0). For FaDu and SW780 xenografts,WSGR Docket No.47517-766.601 treatment followed 1 day post implant (Day 1). For HPAF-II xenograft, treatment began once tumors were established (average of 127 mm3) on Day 5. Mice were administered a repeat intraperitoneal dose (qdx10 (FaDu) or qdx14 (SW780)) of the negative control, non-Nectin4 targeting anti-GFP TriTAC, or anti-Nectin4 H46 (human binder) ProTriTAC Linker 040. Tumor growth was monitored at least twice weekly as indicated. Average tumor volume shown was calculated from measurements taken on the final day of each xenograft model study. Statistics represent RM one-way ANOVA with Dunnett post-hoc test, all groups compared to the negative control, anti-GFP TriTAC. The statistics for each xenograft model study are reported in Table 11, and results are shown in FIG.56 to 58. Table 11: P Values from Multiple Rodent Xenograft Studies with anti-ALB / anti-CD3 / anti- NECTIN4 H46 ProTriTAC Linker 040 Fusion Protein Xenograft Test Article Dose (mg / kg) Average P Value Tumor Volume (mm3) HPAF-II 3 0 **** < 0.0001 (pancreas) H46 ProTriTAC 1 0 **** < 0.0001 0.3 0 **** < 0.0001 0.3 874 n / a n / a TriTAC FaDu 0.3 1024 ns ns (head and neck) H46 ProTriTAC 0.3 1132 n / a n / a TriTAC SW780 3 1070 ns ns (bladder) H46 ProTriTAC 0.3 1040 ns ns 0.3 1493 n / a n / a TriTAC

[0314] (P > 0.05); P: probability value. Example 7: NECTIN 4 Expression in HT29, SW730, and Jurkat cell pellets

[0315] Nectin4 expression was evaluated by IHC using cell pellets and rodent tumor samples due to the lack of tumor regression observed in vivo. Cell pellets of HT29, SW780, and Jurkat (negative control) parental cells were embedded on a slide for Day 0. For the rodent tumor samples, HT29 and SW780 tumor cells were implanted with T cells and harvested for IHC at Day 7 and 21WSGR Docket No.47517-766.601 followed by paraffin embedding. For IHC staining, a heat-induced epitope retrieval method was applied. Next, a primary commercial Nectin4 rabbit polyclonal antibody was used, followed by a goat-anti-rabbit-HRP as the secondary antibody. Lastly, a deparaffinization step was done, and a chromogenic detection (DAB) was added to observe staining, which was reflected as a brown color. Low level of Nectin4 staining in HT29 and SW780 cell pellets and is absent in Jurkat cells (Nectin4 negative). Nectin4 is highly expressed in SW780 tumors, which increases with time, however this was minimal in HT29 tumors. The results are shown in FIGS.59 to 61. Example 8: ProTriTACs with L276 have less pre-cleaved active drug and are more manufacturable than proTriTACs with L040 when produced in CHO Cells

[0316] In this example, linker variants were evaluated in reducing the presence of pre-cleaved active drug and improve manufacturability were assessed.2e6 CHOSource CHO-K1 GS null cells (Horizon) were nucleofected with 5µg linearized expression vector plasmid DNA harboring ProTriTAC sequences comprising L040 or L276 protease cleavable linkers. Cells were passaged in medium containing glutamine for 2 days then switched to selection medium lacking glutamine and supplemented with 50µM methionine sulfoximine (MSX) for up to three weeks such that stable pools were allowed to recover until they reached > 95% viability with less than 24hr doubling time and then banked.

[0317] Stable pools were thawed, expanded in glutamine free media supplemented with 50µM MSX for two to three passages, and seeded into production media at 0.5e6 viable cells / ml in Optimum Growth Flasks (Thomson) shaken at 150 rpm, 5% CO2, 60% relative humidity, and viability and cell density determined by ViCell. Conditioned media (CM) was harvested on day 10 by centrifugation and sterile filtration. Titer was determined by biolayer interferometry using an OctetRed96 and Protein A tips with binding rates compared against a standard curve prepared from a ProTriTAC of known concentration as reference. CM samples were heated in non-reducing sample buffer and separated on NuPage TRIS-Glycine gels, stained with SimplyBlue Safestain (Invitrogen), and imaged with an Azure c500 near infrared imaging system.

[0318] Table 12 and FIG.62 demonstrates a ProTriTAC has substantially reduced pre-cleaved active drug in CHO productions versus an anti-proTriTAC construct with a L040 cleavable linker. Table 12: Comparison of 10d harvest % viability and ProA titer of ProTriTAC sequences comprising L040 or L276 protease cleavable linkers. 10d Harvest % 10d ProA Titer SDS-PAGE Lane ProTriTAC / linker Viability (mg / L) 1 H46 / L040 78 569WSGR Docket No.47517-766.601 10d Harvest % 10d ProA Titer SDS-PAGE Lane ProTriTAC / linker Viability (mg / L) 2 H46 / L276 82 667 SEQUENCES SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 1 NECTIN4 NECTIN4-L51 QVQLQESGGGLVQAGGSLRLSCTASTRTFSRYAMGWFRQ antibody variable APGKEREVVASISQSGMVTYYVDSVKGRFTISRDNAKNTVY domain LQMNSLKPEDTAVYHCAARNGGLAASTVNDFPHWGQGT QVTVSS 2 NECTIN4 NECTIN4-L68 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWIRQ antibody variable APGKEREVVAAISRSSATTYYVDSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYHCAARNSGLAASALNDFPHWGQGTQ VTVSS 3 NECTIN4 NECTIN4-L82 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWIRQ antibody variable APGKEREVVAAISRSSSTTYYVDSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYHCAARDGGLAASKANDFPHWGQGTQ VTVSS 4 NECTIN4 NECTIN4-L98 QVQLQESGGGLVQAGGSLRLSCAASGGTFSLYGMGWIRA antibody variable APGKEREVVAAISRSSATTYYVDSVKGRFTISRDNAKNTLYL domain QMNSLKPEDTAVYHCAARNNDLAASKANDFPHWGQGTQ VTVSS 5 NECTIN4 NECTIN4-L107 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWFRQ antibody variable APGKEREVVAAISRSGMSTYYVDSVKGRFTISRDNAKNTVY domain LQMNSLKPEDTAVYHCATRNSGLAASAVNDFPHQGQGTQ VTVSS 6 NECTIN4 NECTIN4-L158 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWFRQ antibody variable APGKEREVVAAISRSGMTTYYVDSVKGRFTISRDNAKNTVY domain LRMNSLKPEDTAVYHCAARNSGLAASGVNDFPHWGQGT QVTVSS 7 NECTIN4 NECTIN4-L177 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWLRQ antibody variable APGKEREVVASISQSGMVTYYVDSVKGRFTISRDNAKNTVY domain LQMNSLKPEDTAVYHCAARNGGLAASTVNDFPHWGQGT QVTVSS 8 NECTIN4 NECTIN4-L28 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWFRQ antibody variable APGKEREVVASISQSGMVTYYVDSVKGRFTISRDNAKNTVY domain LQMNSLKPEDTAVYHCAARNGGLAASTVNDFPHWGQGT QVTVSS 9 NECTIN4 NECTIN4-L81 QVQLQESGGGLVQAGGSLTLSCAASGRPFSLYGMGWIRQ antibody variable APGKEREVVAAISSSSTTTYYVDSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYHCAARNNGLAASKANDFPHWGQGTQ VTVSS 10 NECTIN4 NECTIN4-L70 QVQLQESGGGRVQPGGSLRLSCAASGSTFSINVIGWYRQA antibody variable PGKQRELVASISRGGSTNYADSVKGRFTISRDNAKNTAYLQ domain MNSLKPEDTAVYYCNGDGWHSLTDYYTETSWGQGTQVT VSSWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 11 NECTIN4 NECTIN4-L86 QVQLQESGGGLVQAGGSLRLSCAASGSTLNINVIGWYRQA antibody variable PGKQRELVASISRGGSTNYADSVKGRFTLSRDNAKNTAYLQ domain MNSLKPEDTAVYYCNGDGWHGRTGYYGETSWGQGTQVT VSS 12 NECTIN4 NECTIN4-L89 QVQLQESGGGLVQAGGSLRLSCAASGSTLNINVIAWYRQA antibody variable PGKQRELVASISRGGSTRYADSVKGRFTISRDNAKNTAYLQ domain MNSLKPEDTAVYYCNGDGWHSRAGYYSETSWGQGTQVT VSS 13 NECTIN4 NECTIN4-L93 QVQLQESGGGLVQAGGSLRLSCAASGSTFSINVIGWYRQA antibody variable PEKQRELVASISRGGSTRYADSVKGRFTISRDNAKNTANLQ domain MNNLKPEDTAVYYCNADGWHSRTDYYAETSWGQGTQVT VSS 14 NECTIN4 NECTIN4-L112 QVQLQESGGGLVQAGGSLRLSCAASGSTFSINVIGWYRQA antibody variable PGKQRELVASISRGGSTNYADSVKGRFTISRDNAKNTAYLQ domain MNSLKPEDTAVYYCNGDGWHSLTDYYTETSWGQGTQVT VSS 15 NECTIN4 NECTIN4-L151 QVQLQESGGGLVQAGGSLRLSCVASGSTFSINVIGWYRQA antibody variable PGKQRELVASISRGGSTRYADSVKGRFTISRDNAKNTAYLQ domain MNSLASEDTAVYYCNGDGWHSRTDYYTETSWGQGTQVT VSS 16 NECTIN4 NECTIN4-L152 QVQLQESGGGLVQAGGSLSVSCAASGSTFSINVIAWYRQA antibody variable PEKQRELVASISRGGSTRYADSVKGRFTISRDNAKNTATLQ domain MNNLKPEDTAVYYCNADGWHSRTEYYAETSWGQGTQVT VSS 17 NECTIN4 NECTIN4-L54 QVQLQESGGGLVQAGGSLRLSCAASGSTLNINVIGWYRQA antibody variable PGKQRELVASISRGGSVNYADSVKGRFTISRDNAKNTAYLQ domain MNSLKPEDTAVYYCNGDGWHSRTGYYTETSWGQGTQVT VSS 18 NECTIN4 NECTIN4-L110 QVQLQESGGGLVQAGGSLRLSCAASGSTLSIIAIGWYRQAP antibody variable GKQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYLQM domain NSLKPEDTAVYYCNADFSYGLGPPEYDYWGRGTQVTVSS 19 NECTIN4 NECTIN4-L160 QVQLQESGGGLVQAGGSLRLSCAASGGTFTKYAMGWFR antibody variable QAPGKEREFVAAISGYGGSTNYADSVKGRFTISRDNAKNTV domain YLQMNSLKPEDTAVYYCNADFSYGLGPPEYDYWGRGTQV TVSS 20 NECTIN4 NECTIN4-L46 QVQLQESGGGLVQPGGTLRLACAASGSTFSMLAIAWYRQ antibody variable APGKQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYYCNADTSYGLGGREYDYWGQGTQVT VSS 21 NECTIN4 NECTIN4-L167 QVQLQESGGGLGQAGGSLRLSCAASGRTSSTYNMAWFRQ antibody variable GPGKEREFVAHIRWSSGTTYYADSVKGRFTISRDNAKNTM domain FLQMNSLKPEDTAVYYCAADRPPFRYSYNETSRYDYWGQG TQVTVSS 22 NECTIN4 NECTIN4-L71 QVQLQESGGGLVQAGGSLRLSCAASGLTSSTYNMAWFRQ antibody variable GPGKEREFVAQIRWSSGTTYYADSVKGRFTISRDNAKNTM domain FLQMNSLKPEDTAVYYCAADRPPFRYSYNEASRYDYWGQ GTQVTVSSWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 23 NECTIN4 NECTIN4-L85 QVQLQESGGGLVQTGGSLGLSCAASGRTSSIYNMAWFRQ antibody variable GPGKEREFVAHIRWSSGTVYYADSVKGRFTISRDNTKDTIFL domain QMNSLKPEDTAVYYCAADLPPFRYSYNEASRYDYWGQGT QVTVSS 24 NECTIN4 NECTIN4-L95 QVQLQESGGGLVQAGGSLRLSCAASGDTFSINVIGWSRQA antibody variable PGSQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYLQ domain MNSLKAEDTAVYYCSVPSLIDYGKDYWGKGTQVTVSS 25 NECTIN4 NECTIN4-L162 QVQLQESGGGLVQAGGSLRLSCLVSGRTFKSYTMGWFRQ antibody variable APGSQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYL domain QMNSLKAEDTAVYYCSAPSLIDYGKDYWGKGTQVTVSS 26 NECTIN4 NECTIN4-L48 QVQLQESGGGLVQAGGSLRLSCAASGITFSSNYIGWFRQA antibody variable PGKQREWVAGISSGGSINYKDSVKGRFTISRDNAKNTVYLQ domain MNSLKPEDTAVYSCNVDGTHYWGQGTQVTVSS 27 NECTIN4 NECTIN4-L91 QVQLQESGGGLVQAGGFLRLSCAASGITFSSNYIGWFRQA antibody variable PGKQREWVAGISSGGSINYGDSVKGRFTISRDNAKNTVDL domain QMNSLKPDDTAVYYCNVDGTPYWGQGTQVTVSS 28 NECTIN4 NECTIN4-L101 QVQLQESGGGLVQAGGSLRLSCAASGRSLSSYAMAWFRQ antibody variable APGKEREFVAAINWSGGSTNYVDSVKGRFTISRDNAENTIY domain LQMNNLKPEDTAVYYCAERWGGNIGTTKDEYVYWGQGT QVTVSS 29 NECTIN4 NECTIN4-L169 QVQLQESGGGLVQAGDSLRLSCAASGRSLSSYAMAWFRQ antibody variable APGKEREFVAAINWSGDSTNVVDSVKGRFTISRDNAKNTIY domain LQMSNLKPEDTAVYYCAEGWGGNIRTTKDHYVYWGQGT QVTVSS 30 NECTIN4 NECTIN4-L31 QVQLQESGGGLVEAGDSLRLSCAASGRTFRSYALGWFRQA antibody variable PGKEREFVAHIIWNTGTTYYADSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYYCATAPYAAVAVKGYDYWGQGTQVT VSS 31 NECTIN4 NECTIN4-L118 QVQLQESGGGLVEAGDSLRLSCAASGRTFRSYALGQFRQA antibody variable PGKEREFVAHIVWSTGTVYYVDSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYYCATAPYAAVAAQGYDYWGQGTQVT VSS 32 NECTIN4 NECTIN4-L57 QVQLQESGGGLVQAGDSLRLSCEVSERTFGTYVMAWFRQ antibody variable APGKEREFVAAINWSGNTNYADSVKGRFTISKDNADETVYL domain QMNSLKPQDTAVYYCAFRYGARLYFGQNEYSYWGQGTQ VTVSS 33 NECTIN4 2NEC164 QVQLQESGGGLVQPGGSLRLSCEVSERTFGTYVMAWFRQ antibody variable APGKEREFVAAINWSGNTNYTDSVKGRFTISKDNADETVYL domain QMNSLKPEDTAVYYCAFRYGARLYFGQNEYSYWGQGTQV TVSS 34 NECTIN4 NECTIN4-L65 QVQLQESGGGLVQAGGSLRLSCAASGRNFDRYIMGWFRQ antibody variable APGKEREFVAAIRKSGITVYTTSVKGRFTISRDNAKNTVALE domain MNSLTPEDTAVYYCASSQNEFLIIDLESTYNYWGQGTQVTV SS 35 NECTIN4 NECTIN4-L952 QVQLQESGGGLVQAGGSLRLSCAASGRTLSSYAMGWFRQ antibody variable APGKEREFVAAIRKSGITVYTTSVKGRFTISRDNAKNTVALE domain MNSLTPEDTAVYYCASSQNEFLIIDLESTYNYWGQGTQVTV SSWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 36 NECTIN4 NECTIN4-L2 QVQLQESGGGSVQAGGSLNLSCKVSGGTFSRYGLGWFRQ antibody variable APGKEREFVAAVDWSTFTTMYSDSDNSRFTISRNSAKRTVS domain LQISSLEPEDTAVYYCAASDLYFGRDGTLYQYWGQGTQVT VSS 37 NECTIN4 NECTIN4-L10 QVQLQESGGGLVQAGGSLGLSCTASGRTASTYDMGWFR antibody variable QAPGKEREFVAYISRSGTITYYADSVKGRFTISRDRAKNTTYL domain QIDNLKPEDTAVYYCAATISSIRYNDYRFYDPWGQGTQVTV SS 38 NECTIN4 NECTIN4-L14 QVQLQESGGGLVQAGGSLSVSCAASGSTFSINVIAWYRQA antibody variable PEKQRELVASISRGGSTRYADSVKGRFTISRDNAKNTVYLQ domain MNSLKPEDTAVYYCASSKFGYTAYETPAEYDYWGQGTQVT VSS 39 NECTIN4 NECTIN4-L17 QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYDMGQFRQ antibody variable AAGKEREFVSGITWSGGVTLYADSVKGRFTISRDNAKNTVF domain LQMNSLKPEDTAVYYCAAVPRSNLNSALVQLKTYDYWGQ GTQVTVSS 40 NECTIN4 NECTIN4-L26 QVQLQESGGGLVQAGGSLRLSCEASGRTSTIYGMGWFRRT antibody variable PGKEREFVAAVRWNDGIEFYPDSVKGRFTISRDKVKNTVYL domain QIDSLEPEDTAVYACAARTSPPLVTELYNYWGQGTQVTVSS 41 NECTIN4 NECTIN4-L34 QVQLQESGGGLVQAGGSLRLSCAASGRTFSAYTMGWFRQ antibody variable APGKEREFVAAMRWSGGGTGYADSVKGRFTISRDNAKNT domain VNLQMNSLKPEDTAIYFCAAGNILLYRSTLYGSTWRMDPD RIDYWGQGTQVTVSS 42 NECTIN4 NECTIN4-L42 QVQLQESGGGLVQAGGSLRLSCAASGSTFSINYIAWHRQA antibody variable PGKQREWVAGISSGGSTGYADSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTARYYCEIETMAGVPYWGQGTQVTVSS 43 NECTIN4 NECTIN4-L462 QVQLQESGGGSVQAGGSLRLSCSASGLTVSKYIMAWFRQ antibody variable APGKEREFVAAIFWNSGGAYEDFVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYYCAARDYIGSSLLEARDRYDHWGQGT QVTVSS 44 NECTIN4 NECTIN4-L47 QVQLQESGGGLVQPGGSLRLSCAASGFTFSTSYMMWVRQ antibody variable APGKGPEWVSGISGGGSTRYSDDVKGRFTISRDNAKNALYL domain QMNSLKPEDTAVYYCAVDPGRFGTRRDWYYYWGQGTQV TVSS 45 NECTIN4 NECTIN4-L79 QVQLQESGGGLVQAGDSLRLSCAASGSTFNNYAMGWFR antibody variable QAPGKEREFVATISSSSRTTGYADSVKGRFTISRDNADNTVF domain LHMDQLKPDDTAVYYCAARERGTAANGVRNYDYWGQGT QVTVSS 46 NECTIN4 NECTIN4-L90 QVQLQESGGGLAQAGGSLRLSCAASGRTFADYAVGWFRQ antibody variable APGKEREFVGTINWNFDRTTYADSVKGRFTISGDNAKNTV domain YLQMNSLKPEDTAVYFCATRPVWATDRSTYYTMASHFSV WGQGTQVTVSS 47 NECTIN4 NECTIN4-L102 QVQLQESGGGLVQAGGSLRVSCAASGRTFSSYTMGWFRQ antibody variable APGKEREFVAHIRWNGGTTYYADSVKGRFTISRDNAKNTV domain YLQMNSLTPDDTAVYYCAAATFNSVSVSEYLYWGQGTQVT VSSWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 48 NECTIN4 NECTIN4-L114 QVQLQESGGGLVQAGGSLRLSCAASGSTFSINVMGWYRQ antibody variable APGKQRELVASISTGGSTNYADSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTAVYYCNADEFGGSSWAEYWGQGTQVTVS S 49 NECTIN4 NECTIN4-L115 QVQLQESGGGLVQAGGSLRLSCAASGRTFSNYIMGWFRQ antibody variable APGKERDFVSSISGNGGRTSYADSVRGRFTISRDNVKNTVF domain LQMNSLKPEDTAVYYCAARSYSSKTKTDDYDYWGQGTQV TVSS 50 NECTIN4 NECTIN4-L126 QVQLQESGGGLVQAGDSLRLSCAASGRTFSSYSMGWFRQ antibody variable APGKERESVGIITRSGSALYPDSVKGRFTISRDNAKNTVYLQ domain MNSLQPEDTAIYYCAAALADFDDYEGYEYNYWGQGTQVT VSS 51 NECTIN4 NECTIN4-L131 QVQLQESGGGLVQAGDSLRLSCAPSERSSSTYAMGWFRQ antibody variable APGQEREPVAAISWSGRATYYADSVKGRFAISIDNAKNTVY domain LQMNSLKPEDTAVYYCAARSTVYVAQRISDYSYWGQGTQ VTVSS 52 NECTIN4 NECTIN4-L134 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQ antibody variable APGKERASVAAISGSGRTTYYADSVKGRFTISRDNAKNTMY domain LQMNGLKPEDTANYYCAARGRPYASDRSDEYDYWGQGT QVTVSS 53 NECTIN4 NECTIN4-L157 QVQLQESGGGLVQAGGSLKLSCTASVRSFRNYPMGWFRK antibody variable APGKERESVGIVTQSGGTFYADSVKGRFTISRDNAMNTVYL domain LMNNLQPEDTAVYYCAADDPIASTDDYDYWGQGTQVTVS S 54 NECTIN4 NECTIN4-L187 QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYVMGWFRQ antibody variable APGKEREFVAHITWTRGTTYYADSVKGRFTISRDNAKSTVY domain LQMNSLKPEDTAVYYCASSRWGAINYAGYDYWGQGTQV TVSS 55 NECTIN4 NECTIN4-L36 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYYMRWVRQ antibody variable APGKGLQWVSTITSDGGNTYYADSVKGRFTISRDNTKNTLY domain LQMNNLEPEDTAVYYCAKLYGNDKGQGTQVTVSS 56 NECTIN4 NECTIN4-L482 QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQ antibody variable APGKEREFVATISGSGRNTAYSNSVKGRFTISRDNAKNTVYL domain QMNSLKPEDTANYVCAQRLRAYVGGYSHDYQYWGQGTQ VTVSS 57 NECTIN4 NECTIN4-L87 QVQLQESGGGLVQPGGSLRLSCAASGSTASINYFAWHRQA antibody variable PGKQREWVAGISSGGSINYVDSVKGRFTISRDNAKNTVYLQ domain MNSLKPEDTAVYSCNAESLRADYWGQGTQVTVSS 58 NECTIN4 NECTIN4-L892 QVQLQESGGGLVQAGGSLRLSCAASGSTLSINVIGWYRQT antibody variable PGKEREYVASITRGGSTKYADSVKGRFTISRDGAKNTVYLQ domain MNSLKPEDTAVYYCYAEDSWYDEALETMSESTEYWGQGT QVTVSS 59 NECTIN4 NECTIN4-H107 EVQLLESGGGLVQPGGSLTLSCAASGRPFSLYGMGWFRQA antibody variable PGKEREVVAAISRSGMSTYYVDSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCATRNSGLAASAVNDFPHQGQGTLVT VSSWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 60 NECTIN4 NECTIN4-H134 EVQLLESGGGLVQPGGSLTLSCAASGRTFSSYAMGWFRQA antibody variable PGKERASVAAISGSGRTTYYADSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCAARGRPYASDRSDEYDYWGQGTLVT VSS 61 NECTIN4 NECTIN4-H177 EVQLLESGGGLVQPGGSLTLSCAASGRPFSLYGMGWLRQA antibody variable PGKEREVVASISQSGMVTYYVDSVKGRFTISRDNSKNTLYL domain QMNSLRAEDTAVYYCAARNGGLAASTVNDFPHWGQGTL VTVSS 62 NECTIN4 NECTIN4-H46 EVQLLESGGGLVQPGGSLTLSCAASGSTFSMLAIAWYRQA antibody variable PGKQRELVASISSGGSTNYADSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCNADTSYGLGGREYDYWGQGTLVTVS S 63 NECTIN4 NECTIN4-H462 EVQLLESGGGLVQPGGSLTLSCAASGLTVSKYIMAWFRQA antibody variable PGKEREFVAAIFWNSGGAYEDSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCAARDYIGSSLLEARDRYDHWGQGTLV TVSS 64 NECTIN4 NECTIN4-H82 EVQLLESGGGLVQPGGSLTLSCAASGRPFSLYGMGWIRQA antibody variable PGKEREVVAAISRSSSTTYYVDSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCAARDGGLAASKANDFPHWGQGTLV TVSS 65 NECTIN4 NECTIN4-H91 EVQLVESGGGLVQPGGSLTLSCAASGITFSSNYIGWFRQAP antibody variable GKQREWVAGISSGGSINYGDSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCNVDGTPYWGQGTLVTVSS 66 NECTIN4 NECTIN4-H952 EVQLLESGGGLVQPGGSLTLSCAASGRTLSSYAMGWFRQA antibody variable PGKEREFVAAIRKSGITVYTTSVKGRFTISRDNSKNTLYLQM domain NSLRAEDTAVYYCASSQNEFLIIDLESTYNYWGQGTLVTVSS 67 CDR1 NECTIN4-L51 TRTFSRYAMG 68 CDR1 NECTIN4-L68 GRPFSLYGMG 69 CDR1 NECTIN4-L82 GRPFSLYGMG 70 CDR1 NECTIN4-L98 GGTFSLYGMG 71 CDR1 NECTIN4-L107 GRPFSLYGMG 72 CDR1 NECTIN4-L158 GRPFSLYGMG 73 CDR1 NECTIN4-L177 GRPFSLYGMG 74 CDR1 NECTIN4-L28 GRPFSLYGMG 75 CDR1 NECTIN4-L81 GRPFSLYGMG 76 CDR1 NECTIN4-L70 GSTFSINVIG 77 CDR1 NECTIN4-L86 GSTLNINVIG 78 CDR1 NECTIN4-L89 GSTLNINVIA 79 CDR1 NECTIN4-L93 GSTFSINVIG 80 CDR1 NECTIN4-L112 GSTFSINVIG 81 CDR1 NECTIN4-L151 GSTFSINVIG 82 CDR1 NECTIN4-L152 GSTFSINVIA 83 CDR1 NECTIN4-L54 GSTLNINVIG 84 CDR1 NECTIN4-L110 GSTLSIIAIG 85 CDR1 NECTIN4-L160 GGTFTKYAMG 86 CDR1 NECTIN4-L46 GSTFSMLAIAWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 87 CDR1 NECTIN4-L167 GRTSSTYNMA 88 CDR1 NECTIN4-L71 GLTSSTYNMA 89 CDR1 NECTIN4-L85 GRTSSIYNMA 90 CDR1 NECTIN4-L95 GDTFSINVIG 91 CDR1 NECTIN4-L162 GRTFKSYTMG 92 CDR1 NECTIN4-L48 GITFSSNYIG 93 CDR1 NECTIN4-L91 GITFSSNYIG 94 CDR1 NECTIN4-L101 GRSLSSYAMA 95 CDR1 NECTIN4-L169 GRSLSSYAMA 96 CDR1 NECTIN4-L31 GRTFRSYALG 97 CDR1 NECTIN4-L118 GRTFRSYALG 98 CDR1 NECTIN4-L57 ERTFGTYVMA 99 CDR1 2NEC164 ERTFGTYVMA 100 CDR1 NECTIN4-L65 GRNFDRYIMG 101 CDR1 NECTIN4-L952 GRTLSSYAMG 102 CDR1 NECTIN4-L2 GGTFSRYGLG 103 CDR1 NECTIN4-L10 GRTASTYDMG 104 CDR1 NECTIN4-L14 GSTFSINVIA 105 CDR1 NECTIN4-L17 GRTFSRYDMG 106 CDR1 NECTIN4-L26 GRTSTIYGMG 107 CDR1 NECTIN4-L34 GRTFSAYTMG 108 CDR1 NECTIN4-L42 GSTFSINYIA 109 CDR1 NECTIN4-L462 GLTVSKYIMA 110 CDR1 NECTIN4-L47 GFTFSTSYMM 111 CDR1 NECTIN4-L79 GSTFNNYAMG 112 CDR1 NECTIN4-L90 GRTFADYAVG 113 CDR1 NECTIN4-L102 GRTFSSYTMG 114 CDR1 NECTIN4-L114 GSTFSINVMG 115 CDR1 NECTIN4-L115 GRTFSNYIMG 116 CDR1 NECTIN4-L126 GRTFSSYSMG 117 CDR1 NECTIN4-L131 ERSSSTYAMG 118 CDR1 NECTIN4-L134 GRTFSSYAMG 119 CDR1 NECTIN4-L157 VRSFRNYPMG 120 CDR1 NECTIN4-L187 GRTFSTYVMG 121 CDR1 NECTIN4-L36 GFTFSSYYMR 122 CDR1 NECTIN4-L482 GRTFSSYAMG 123 CDR1 NECTIN4-L87 GSTASINYFA 124 CDR1 NECTIN4-L892 GSTLSINVIG 125 CDR1 NECTIN4-H107 GRPFSLYGMG 126 CDR1 NECTIN4-H134 GRTFSSYAMG 127 CDR1 NECTIN4-H177 GRPFSLYGMG 128 CDR1 NECTIN4-H46 GSTFSMLAIA 129 CDR1 NECTIN4-H462 GLTVSKYIMA 130 CDR1 NECTIN4-H82 GRPFSLYGMGWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 131 CDR1 NECTIN4-H91 GITFSSNYIG 132 CDR1 NECTIN4-H952 GRTLSSYAMG 133 CDR2 NECTIN4-L51 SISQSGMVTYYVDSVKG 134 CDR2 NECTIN4-L68 AISRSSATTYYVDSVKG 135 CDR2 NECTIN4-L82 AISRSSSTTYYVDSVKG 136 CDR2 NECTIN4-L98 AISRSSATTYYVDSVKG 137 CDR2 NECTIN4-L107 AISRSGMSTYYVDSVKG 138 CDR2 NECTIN4-L158 AISRSGMTTYYVDSVKG 139 CDR2 NECTIN4-L177 SISQSGMVTYYVDSVKG 140 CDR2 NECTIN4-L28 SISQSGMVTYYVDSVKG 141 CDR2 NECTIN4-L81 AISSSSTTTYYVDSVKG 142 CDR2 NECTIN4-L70 SISRGGSTNYADSVKG 143 CDR2 NECTIN4-L86 SISRGGSTNYADSVKG 144 CDR2 NECTIN4-L89 SISRGGSTRYADSVKG 145 CDR2 NECTIN4-L93 SISRGGSTRYADSVKG 146 CDR2 NECTIN4-L112 SISRGGSTNYADSVKG 147 CDR2 NECTIN4-L151 SISRGGSTRYADSVKG 148 CDR2 NECTIN4-L152 SISRGGSTRYADSVKG 149 CDR2 NECTIN4-L54 SISRGGSVNYADSVKG 150 CDR2 NECTIN4-L110 SISSGGSTNYADSVKG 151 CDR2 NECTIN4-L160 AISGYGGSTNYADSVKG 152 CDR2 NECTIN4-L46 SISSGGSTNYADSVKG 153 CDR2 NECTIN4-L167 HIRWSSGTTYYADSVKG 154 CDR2 NECTIN4-L71 QIRWSSGTTYYADSVKG 155 CDR2 NECTIN4-L85 HIRWSSGTVYYADSVKG 156 CDR2 NECTIN4-L95 SISSGGSTNYADSVKG 157 CDR2 NECTIN4-L162 SISSGGSTNYADSVKG 158 CDR2 NECTIN4-L48 GISSGGSINYKDSVKG 159 CDR2 NECTIN4-L91 GISSGGSINYGDSVKG 160 CDR2 NECTIN4-L101 AINWSGGSTNYVDSVKG 161 CDR2 NECTIN4-L169 AINWSGDSTNVVDSVKG 162 CDR2 NECTIN4-L31 HIIWNTGTTYYADSVKG 163 CDR2 NECTIN4-L118 HIVWSTGTVYYVDSVKG 164 CDR2 NECTIN4-L57 AINWSGNTNYADSVKG 165 CDR2 2NEC164 AINWSGNTNYTDSVKG 166 CDR2 NECTIN4-L65 AIRKSGITVYTTSVKG 167 CDR2 NECTIN4-L952 AIRKSGITVYTTSVKG 168 CDR2 NECTIN4-L2 AVDWSTFTTMYSDSDNS 169 CDR2 NECTIN4-L10 YISRSGTITYYADSVKG 170 CDR2 NECTIN4-L14 SISRGGSTRYADSVKG 171 CDR2 NECTIN4-L17 GITWSGGVTLYADSVKG 172 CDR2 NECTIN4-L26 AVRWNDGIEFYPDSVKG 173 CDR2 NECTIN4-L34 AMRWSGGGTGYADSVKG 174 CDR2 NECTIN4-L42 GISSGGSTGYADSVKGWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 175 CDR2 NECTIN4-L462 AIFWNSGGAYEDFVKG 176 CDR2 NECTIN4-L47 GISGGGSTRYSDDVKG 177 CDR2 NECTIN4-L79 TISSSSRTTGYADSVKG 178 CDR2 NECTIN4-L90 TINWNFDRTTYADSVKG 179 CDR2 NECTIN4-L102 HIRWNGGTTYYADSVKG 180 CDR2 NECTIN4-L114 SISTGGSTNYADSVKG 181 CDR2 NECTIN4-L115 SISGNGGRTSYADSVRG 182 CDR2 NECTIN4-L126 IITRSGSALYPDSVKG 183 CDR2 NECTIN4-L131 AISWSGRATYYADSVKG 184 CDR2 NECTIN4-L134 AISGSGRTTYYADSVKG 185 CDR2 NECTIN4-L157 IVTQSGGTFYADSVKG 186 CDR2 NECTIN4-L187 HITWTRGTTYYADSVKG 187 CDR2 NECTIN4-L36 TITSDGGNTYYADSVKG 188 CDR2 NECTIN4-L482 TISGSGRNTAYSNSVKG 189 CDR2 NECTIN4-L87 GISSGGSINYVDSVKG 190 CDR2 NECTIN4-L892 SITRGGSTKYADSVKG 191 CDR2 NECTIN4-H107 AISRSGMSTYYVDSVKG 192 CDR2 NECTIN4-H134 AISGSGRTTYYADSVKG 193 CDR2 NECTIN4-H177 SISQSGMVTYYVDSVKG 194 CDR2 NECTIN4-H46 SISSGGSTNYADSVKG 195 CDR2 NECTIN4-H462 AIFWNSGGAYEDSVKG 196 CDR2 NECTIN4-H82 AISRSSSTTYYVDSVKG 197 CDR2 NECTIN4-H91 GISSGGSINYGDSVKG 198 CDR2 NECTIN4-H952 AIRKSGITVYTTSVKG 199 CDR3 NECTIN4-L51 RNGGLAASTVNDFPH 200 CDR3 NECTIN4-L68 RNSGLAASALNDFPH 201 CDR3 NECTIN4-L82 RDGGLAASKANDFPH 202 CDR3 NECTIN4-L98 RNNDLAASKANDFPH 203 CDR3 NECTIN4-L107 RNSGLAASAVNDFPH 204 CDR3 NECTIN4-L158 RNSGLAASGVNDFPH 205 CDR3 NECTIN4-L177 RNGGLAASTVNDFPH 206 CDR3 NECTIN4-L28 RNGGLAASTVNDFPH 207 CDR3 NECTIN4-L81 RNNGLAASKANDFPH 208 CDR3 NECTIN4-L70 DGWHSLTDYYTETS 209 CDR3 NECTIN4-L86 DGWHGRTGYYGETS 210 CDR3 NECTIN4-L89 DGWHSRAGYYSETS 211 CDR3 NECTIN4-L93 DGWHSRTDYYAETS 212 CDR3 NECTIN4-L112 DGWHSLTDYYTETS 213 CDR3 NECTIN4-L151 DGWHSRTDYYTETS 214 CDR3 NECTIN4-L152 DGWHSRTEYYAETS 215 CDR3 NECTIN4-L54 DGWHSRTGYYTETS 216 CDR3 NECTIN4-L110 DFSYGLGPPEYDY 217 CDR3 NECTIN4-L160 DFSYGLGPPEYDY 218 CDR3 NECTIN4-L46 DTSYGLGGREYDYWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 219 CDR3 NECTIN4-L167 DRPPFRYSYNETSRYDY 220 CDR3 NECTIN4-L71 DRPPFRYSYNEASRYDY 221 CDR3 NECTIN4-L85 DLPPFRYSYNEASRYDY 222 CDR3 NECTIN4-L95 PSLIDYGKDY 223 CDR3 NECTIN4-L162 PSLIDYGKDY 224 CDR3 NECTIN4-L48 DGTHY 225 CDR3 NECTIN4-L91 DGTPY 226 CDR3 NECTIN4-L101 RWGGNIGTTKDEYVY 227 CDR3 NECTIN4-L169 GWGGNIRTTKDHYVY 228 CDR3 NECTIN4-L31 APYAAVAVKGYDY 229 CDR3 NECTIN4-L118 APYAAVAAQGYDY 230 CDR3 NECTIN4-L57 RYGARLYFGQNEYSY 231 CDR3 2NEC164 RYGARLYFGQNEYSY 232 CDR3 NECTIN4-L65 SQNEFLIIDLESTYNY 233 CDR3 NECTIN4-L952 SQNEFLIIDLESTYNY 234 CDR3 NECTIN4-L2 SDLYFGRDGTLYQY 235 CDR3 NECTIN4-L10 TISSIRYNDYRFYDP 236 CDR3 NECTIN4-L14 SKFGYTAYETPAEYDY 237 CDR3 NECTIN4-L17 VPRSNLNSALVQLKTYDY 238 CDR3 NECTIN4-L26 RTSPPLVTELYNY 239 CDR3 NECTIN4-L34 GNILLYRSTLYGSTWRMDPDRIDY 240 CDR3 NECTIN4-L42 ETMAGVPY 241 CDR3 NECTIN4-L462 RDYIGSSLLEARDRYDH 242 CDR3 NECTIN4-L47 DPGRFGTRRDWYYY 243 CDR3 NECTIN4-L79 RERGTAANGVRNYDY 244 CDR3 NECTIN4-L90 RPVWATDRSTYYTMASHFSV 245 CDR3 NECTIN4-L102 ATFNSVSVSEYLY 246 CDR3 NECTIN4-L114 DEFGGSSWAEY 247 CDR3 NECTIN4-L115 RSYSSKTKTDDYDY 248 CDR3 NECTIN4-L126 ALADFDDYEGYEYNY 249 CDR3 NECTIN4-L131 RSTVYVAQRISDYSY 250 CDR3 NECTIN4-L134 RGRPYASDRSDEYDY 251 CDR3 NECTIN4-L157 DDPIASTDDYDY 252 CDR3 NECTIN4-L187 SRWGAINYAGYDY 253 CDR3 NECTIN4-L36 LYGND 254 CDR3 NECTIN4-L482 RLRAYVGGYSHDYQY 255 CDR3 NECTIN4-L87 ESLRADY 256 CDR3 NECTIN4-L892 EDSWYDEALETMSESTEY 257 CDR3 NECTIN4-H107 RNSGLAASAVNDFPH 258 CDR3 NECTIN4-H134 RGRPYASDRSDEYDY 259 CDR3 NECTIN4-H177 RNGGLAASTVNDFPH 260 CDR3 NECTIN4-H46 DTSYGLGGREYDY 261 CDR3 NECTIN4-H462 RDYIGSSLLEARDRYDH 262 CDR3 NECTIN4-H82 RDGGLAASKANDFPHWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 263 CDR3 NECTIN4-H91 DGTPY 264 CDR3 NECTIN4-H952 SQNEFLIIDLESTYNY 265 anti-CD3 / anti- NECTIN4-L2 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGSVQ AGGSLNLSCKVSGGTFSRYGLGWFRQAPGKEREFVAAVD WSTFTTMYSDSDNSRFTISRNSAKRTVSLQISSLEPEDTAVY YCAASDLYFGRDGTLYQYWGQGTQVTVSSHHHHHH 266 anti-CD3 / anti- NECTIN4-L10 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLGLSCTASGRTASTYDMGWFRQAPGKEREFVAYISR SGTITYYADSVKGRFTISRDRAKNTTYLQIDNLKPEDTAVYYC AATISSIRYNDYRFYDPWGQGTQVTVSSHHHHHH 267 anti-CD3 / anti- NECTIN4-L14 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLSVSCAASGSTFSINVIAWYRQAPEKQRELVASISRGG STRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCA SSKFGYTAYETPAEYDYWGQGTQVTVSSHHHHHH 268 anti-CD3 / anti- NECTIN4-L17 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCAASGRTFSRYDMGQFRQAAGKEREFVSGITW SGGVTLYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVY YCAAVPRSNLNSALVQLKTYDYWGQGTQVTVSSHHHHHHWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 269 anti-CD3 / anti- NECTIN4-L26 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCEASGRTSTIYGMGWFRRTPGKEREFVAAVRW NDGIEFYPDSVKGRFTISRDKVKNTVYLQIDSLEPEDTAVYA CAARTSPPLVTELYNYWGQGTQVTVSSHHHHHH 270 anti-CD3 / anti- NECTIN4-L28 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLTLSCAASGRPFSLYGMGWFRQAPGKEREVVASISQ SGMVTYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YHCAARNGGLAASTVNDFPHWGQGTQVTVSSHHHHHH 271 anti-CD3 / anti- NECTIN4-L31 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVE AGDSLRLSCAASGRTFRSYALGWFRQAPGKEREFVAHIIW NTGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCATAPYAAVAVKGYDYWGQGTQVTVSSHHHHHH 272 anti-CD3 / anti- NECTIN4-L34 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCAASGRTFSAYTMGWFRQAPGKEREFVAAMR WSGGGTGYADSVKGRFTISRDNAKNTVNLQMNSLKPEDT AIYFCAAGNILLYRSTLYGSTWRMDPDRIDYWGQGTQVTV SSHHHHHHWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 273 anti-CD3 / anti- NECTIN4-L36 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ PGGSLRLSCAASGFTFSSYYMRWVRQAPGKGLQWVSTITS DGGNTYYADSVKGRFTISRDNTKNTLYLQMNNLEPEDTAV YYCAKLYGNDKGQGTQVTVSSHHHHHH 274 anti-CD3 / anti- NECTIN4-L42 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCAASGSTFSINYIAWHRQAPGKQREWVAGISS GGSTGYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTARY YCEIETMAGVPYWGQGTQVTVSSHHHHHH 275 anti-CD3 / anti- NECTIN4-L46 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ PGGTLRLACAASGSTFSMLAIAWYRQAPGKQRELVASISSG GSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CNADTSYGLGGREYDYWGQGTQVTVSSHHHHHH 276 anti-CD3 / anti- NECTIN4-L47 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ PGGSLRLSCAASGFTFSTSYMMWVRQAPGKGPEWVSGIS GGGSTRYSDDVKGRFTISRDNAKNALYLQMNSLKPEDTAV YYCAVDPGRFGTRRDWYYYWGQGTQVTVSSHHHHHH 277 anti-CD3 / anti- NECTIN4-L48 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCAASGITFSSNYIGWFRQAPGKQREWVAGISSGWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. GSINYKDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSC NVDGTHYWGQGTQVTVSSHHHHHH 278 anti-CD3 / anti- NECTIN4-L51 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCTASTRTFSRYAMGWFRQAPGKEREVVASISQS GMVTYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY HCAARNGGLAASTVNDFPHWGQGTQVTVSSHHHHHH 279 anti-CD3 / anti- NECTIN4-L54 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCAASGSTLNINVIGWYRQAPGKQRELVASISRG GSVNYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYY CNGDGWHSRTGYYTETSWGQGTQVTVSSHHHHHH 280 anti-CD3 / anti- NECTIN4-L57 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGDSLRLSCEVSERTFGTYVMAWFRQAPGKEREFVAAINW SGNTNYADSVKGRFTISKDNADETVYLQMNSLKPQDTAVY YCAFRYGARLYFGQNEYSYWGQGTQVTVSSHHHHHH 281 anti-CD3 / anti- NECTIN4-L65 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLRLSCAASGRNFDRYIMGWFRQAPGKEREFVAAIRK SGITVYTTSVKGRFTISRDNAKNTVALEMNSLTPEDTAVYYC ASSQNEFLIIDLESTYNYWGQGTQVTVSSHHHHHHWSGR Docket No.47517-766.601 SEQ Sequence Seq. name Amino acid sequence ID descriptor NO. 282 anti-CD3 / anti- NECTIN4-L68 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF NKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGSQVQLQESGGGLVQ AGGSLTLSCAASGRPFSLYGMGWIRQAPGKEREVVAAISRS SATTYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYH CAARNSGLAASALNDFPHWGQGTQVTVSSHHHHHH 283 anti-CD3 / anti- NECTIN4-L70 VVGGGGTQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGN NECTIN4 fusion YPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKA protein ALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAA...

Claims

WSGR Docket No.47517-766.601 CLAIMS WHAT IS CLAIMED IS:

1. A NECTIN4 binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264. 2 The NECTIN4 binding domain of claim 1, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264. 3 The NECTIN4 binding domain of claim 1 or 2, comprising an amino acid sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1- 66 4 The NECTIN4 binding domain of any one of claims 1-3, wherein the NECTIN4 binding domain is part of a multispecific protein. 5 The NECTIN4 binding domain of claim 4, wherein the multispecific protein further comprises a CD3 binding domain. 6 The NECTIN4 binding domain of claim 5, wherein the multispecific protein comprises an active drug format. 7 The NECTIN4 binding domain of claim 4 or 5, wherein the multispecific protein further comprises a bulk serum protein binding domain. 8 The NECTIN4 binding domain of claim 7, wherein the bulk serum protein comprises a serum albumin protein. 9 The NECTIN4 binding domain of claim 8, wherein the serum albumin protein comprises a human serum albumin protein. 10 The NECTIN4 binding domain of any one of claims 7-9, wherein the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 612.WSGR Docket No.47517-766.601 11. The NECTIN4 binding domain of any one of claims 4-9, wherein the CD3 binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO:

613.

12. The NECTIN4 binding domain of any one of claims 4-9, wherein the multispecific protein comprises a sequence that is at least about 75% identical to the sequence as set forth in SEQ ID NOS: 330-347.

13. The NECTIN4 binding domain of any one of claims 7-9, wherein the bulk serum protein binding domain is a binding moiety comprising a linker and a masking moiety, wherein the masking moiety masks the binding of the NECTIN4 binding domain or the CD3 binding domain, to their respective targets.

14. The NECTIN4 binding domain of any one of claims 7-9 and 13, wherein the multispecific protein comprises a non-cleavable prodrug format.

15. The NECTIN4 binding domain of claim 13 or 14, wherein the masking moiety comprises a sequence selected from the group consisting of: SEQ ID NOS: 721-765, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 721- 765.

16. The NECTIN4 binding domain of any one of claims 13-15, wherein the linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766- 770.

17. The NECTIN4 binding domain of any one of claims 13-16, wherein the bulk serum protein binding domain comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO:

612.

18. The NECTIN4 binding domain of any one of claims 5-9 and 13-17, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO:

613.

19. The NECTIN4 binding domain of any one of claims 7-9 and 13-18, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

20. The NECTIN4 binding domain of any one of claims 7-9 and 13-19, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.WSGR Docket No.47517-766.601 21. The NECTIN4 binding domain of any one of claims 7-9 and 13-20, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 330-347.

22. The NECTIN4 binding domain of claim 6, wherein the active drug comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

23. The NECTIN4 binding domain of any one of claims 1-3, wherein the NECTIN4 binding domain is part of a chimeric antigen receptor (a CAR) or a conditionally activatable chimeric antigen receptor (a ProCAR), wherein the CAR further comprises at least one of a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

24. The NECTIN4 binding domain of claim 23, wherein the NECTIN4 binding domain is part of the ProCAR and the ProCAR further comprises (a) a binding moiety comprising a non-CDR loop and a cleavable linker; (b) a transmembrane domain; and (c) an intracellular signaling domain; wherein the binding moiety masks the binding of the NECTIN4 binding domain to its target.

25. The NECTIN4 binding domain of claim 24, wherein the binding moiety further comprises one or more complementarity determining regions (CDRs).

26. The NECTIN4 binding domain of claim 25, wherein the CDR loop provides a binding site specific for a bulk serum protein.

27. The NECTIN4 binding domain of claim 26, wherein the bulk serum protein comprises at least one of: a serum albumin, a transferrin, an IgG1, an IgG2, an IgG4, an IgG3, an IgA monomer, a Factor XIII, a fibrinogen, a pentameric IgM.

28. The NECTIN4 binding domain of claim 27, wherein the bulk serum protein comprises the serum albumin.

29. The NECTIN4 binding domain of claim 28, wherein the serum albumin is a human serum albumin.

30. The NECTIN4 binding domain of any one of claims 23-29, wherein the ProCAR further comprising a costimulatory domain, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM- 1 LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto. 31 The NECTIN4 binding domain of claim 30, wherein the at least one but not more than 20 modifications thereto comprises a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the encoded T-cell receptor fusion protein.WSGR Docket No.47517-766.601 32. The NECTIN4 binding domain of any one of claims 23-31, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto.

33. The NECTIN4 binding domain of any of claims 23-32, wherein the intracellular signaling domain is derived from CD3 epsilon, CD3 gamma, CD3 delta, CD3 alpha, CD3 beta, or a combination thereof.

34. The NECTIN4 binding domain of any of claims 23-32, wherein the intracellular signaling domain is derived from CD3 epsilon.

35. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a NECTIN4 binding domain according to any one of claims 1-34, or a pharmaceutical composition comprising the same, to a subject in need thereof.

36. The method of 35, wherein the subject is human.

37. A conditionally active chimeric antigen receptor (ProCAR) that comprises a single polypeptide chain, comprising: (a) a binding moiety comprising a non-CDR loop and a cleavable linker; (b) a NECTIN4 binding domain, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198 ; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264; (c) a transmembrane domain; and (d) an intracellular signaling domain; wherein the binding moiety masks the binding of the NECTIN4 binding domain to its target.WSGR Docket No.47517-766.601 38. The conditionally active chimeric antigen receptor of claim 37, wherein the NECTIN4 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

39. A conditionally active NECTIN4 binding protein comprising a binding moiety (M) which comprises a non-CDR loop, a cleavable linker (L), a first target antigen binding domain (T1), and a second target antigen binding domain (T2), wherein at least one of the first target antigen binding domain (T1) and the second target antigen binding domain (T2) comprises a NECTIN4 binding domain, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133- 198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264, wherein the non-CDR loop is capable of binding to the NECTIN4 binding domain or the second target antigen binding domain, and wherein the binding moiety masks the binding of the NECTIN4 binding domain or the second target antigen binding domain to its target.

40. The conditionally active NECTIN4 binding protein of claim 39, wherein the binding moiety comprises a masking moiety and wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOS: 721-765, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOS: 721-765.

41. The conditionally active NECTIN4 binding protein of claim 39 or 40, wherein the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 698-704, 718-720, and 766-776, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 698-704, 718-720, and 766-776.

42. The conditionally active NECTIN4 binding protein of any one of claims 39-41, wherein the binding moiety comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO:

612.

43. The conditionally active NECTIN4 binding protein of any one of claims 39-42, wherein the second target antigen binding domain (T2) comprises a CD3 binding domain.WSGR Docket No.47517-766.601 44. The conditionally active NECTIN4 binding protein of claim 43, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO:

613.

45. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a conditionally active chimeric antigen receptor according to claim 37 or 38, or a pharmaceutical composition comprising the same, to a subject in need thereof.

46. The method of 45, wherein the subject is human.

47. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a conditionally active NECTIN4 binding protein according to any one of claims 39-44, or a pharmaceutical composition comprising the same, to a subject in need thereof.

48. The method of 47, wherein the subject is human.

49. The NECTIN4 binding domain of any one of claims 1-3, wherein the binding domain is a humanized antibody or an antigen binding fragment thereof.

50. The NECTIN4 binding domain of any one of claims 1-3 and 49, wherein the binding domain is a single domain antibody, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a Fab’, a non-Ig domain, a ligand, a knottin, or a small molecule entity.

51. The NECTIN4 binding domain of claim 50, wherein the binding domain comprises the single domain antibody.

52. The NECTIN4 binding domain of any one of claims 1-3 and 49-51, wherein the binding domain binds to NECTIN4 with a binding affinity (KD) of about 0.001 nM to about 500 nM.

53. The NECTIN4 binding domain of any one of claims 1-3 and 49-52, wherein the binding domain binds to human NECTIN4, mouse NECTIN4, cynomolgus NECTIN4, or a combination thereof.

54. A multispecific protein comprising a NECTIN4 binding domain, wherein the NECTIN4 binding domain is according to any one of claims 1-3 and 49-53.

55. The multispecific protein of claim 54, comprising the NECTIN4 binding domain according to any one of claims 1-3 and 49-53 (anti- NECTIN4 domain), and a CD3 binding domain (anti- CD3 domain).

56. The multispecific protein of claim 55, wherein the anti- NECTIN4 domain and the anti-CD3 domain are in an anti- NECTIN4:anti-CD3 orientation.

57. The multispecific protein of claim 55, wherein the anti- NECTIN4 domain and the anti-CD3 domain are in an anti-CD3: anti- NECTIN4 orientation.WSGR Docket No.47517-766.601 58. The multispecific protein of any one of claims 54-56, comprising the NECTIN4 binding domain according to any one of claims 1-3 and 49-53 (anti- NECTIN4 domain), the CD3 binding domain (anti-CD3 domain), and an albumin binding domain (anti-ALB domain).

59. The multispecific protein of any one of claims 54-58, wherein the anti-CD3 domain comprises an amino acid as set forth in SEQ ID NO:

613.

60. The multispecific protein of any one of claims 58-59, wherein the anti-ALB domain comprises an amino acid sequence as set forth in SEQ ID NO:

612.

61. The multispecific protein of any one of claims 58-60, wherein the anti- NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ALB: anti- NECTIN4 orientation.

62. The multispecific protein of any one of claims 58-60, wherein the anti- NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-NECTIN4: anti-ALB: anti-CD3 orientation.

63. The multispecific protein of any one of claims 58-60, wherein the anti- NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-NECTIN4: anti-CD3 orientation.

64. The multispecific protein of any one of claims 58-60, wherein the anti-NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti- NECTIN4: anti-ALB orientation.

65. The multispecific protein of any one of claims 58-60, wherein the anti- NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB: anti-CD3: anti- NECTIN4 orientation.

66. The multispecific protein of any one of claims 58-60, wherein the anti- NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti- NECTIN4: anti-CD3: anti-ALB orientation.

67. A multivalent protein comprising a sequence as set forth in any one of SEQ ID NOS: 330- 347.

68. An active drug comprising a sequence as set forth in any one of SEQ ID NOS: 265-329.

69. An active drug comprising a sequence as set forth in any one of SEQ ID NOS: 1-66.

70. A pharmaceutical composition comprising (i)(a) a NECTIN4 binding domain according to any one of claims 1-34 and 49-53; (i)(b) a conditionally active chimeric antigen receptor according to any one of claims 37-38; (i)(c) a conditionally active NECTIN4 binding protein according to any one of claims 39-44; (i)(d) a multispecific protein according to any one of claims 54-66; (i)(e) aWSGR Docket No.47517-766.601 multivalent protein according to claim 67 or 69; or (i)(f) an active drug according to claim 68, and (ii) a pharmaceutically acceptable carrier.

71. A process for producing a NECTIN4 binding domain, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the NECTIN4 binding domain according to any one of claims 1-3 and 49-53, under conditions allowing the expression of the NECTIN4 binding domain and recovering and purifying the produced protein from the culture.

72. A process for producing a multispecific protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multispecific NECTIN4 binding protein according to any one of claims 54-66 under conditions allowing the expression of the multispecific protein and recovering and purifying the produced protein from the culture.

73. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a NECTIN4 binding domain according to any one of claims 1-34 and 49-53, or a pharmaceutical composition according to claim 70, to a subject in need thereof.

74. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of the multispecific protein according to any one of claims 54-66, a multivalent protein according to claim 67 or 69, an active drug according to claim 68, or a pharmaceutical composition according to claim 70, to a subject in need thereof.

75. The method of claim 73 or 74, wherein the subject is human.

76. The method of claim 75, wherein the method further comprises administration of an agent in combination with a NECTIN4binding domain according to any one of claims 1-34 and 49-53, a multispecific protein according to any one of claims 54-66, a multivalent protein according to claim 67 or 69, an active drug according to claim 68, or a pharmaceutical composition according to claim 70.

77. The method of any one of claims 73-76, wherein the NECTIN4 binding domain selectively binds to tumor cells expressing NECTIN4.

78. The method of any one of claims 73-77, wherein the tumorous disease comprises a solid tumor disease.

79. The method of claim 78, wherein the solid tumor disease is metastatic.

80. The method of any one of claims 73-79, wherein the tumorous disease comprises at least one of: an oral cancer, a colorectal cancer, a head and neck cancer, a prostate cancer, a liver cancer, a cervical cancer, a nasopharyngeal cancer, a thyroid cancer, a non-small cell lung cancer, a smallWSGR Docket No.47517-766.601 cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, a bladder cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, a nasal NK / T cell lymphoma, a gliomas, a glioblastoma, a osteosarcoma, a pituitary adenoma, or any combination thereof.

81. The method of any one of claims 45-48, wherein the method further comprises administration of an agent in combination with conditionally active chimeric antigen receptor according to claim 37 or 38, conditionally active NECTIN4 binding protein according to any one of claims 39-44, or a pharmaceutical composition comprising the same.

82. The method of claim 81, wherein the NECTIN4 binding domain selectively binds to tumor cells expressing NECTIN4.

83. The method of claim 81 or 82, wherein the tumorous disease comprises a solid tumor disease.

84. The method of claim 83, wherein the solid tumor disease is metastatic.

85. The method of any one of claims 81-84, wherein the tumorous disease is at least one of: a colorectal cancer, an oral cancer, a colorectal cancer, a head and neck cancer, a prostate cancer, a liver cancer, a cervical cancer, a nasopharyngeal cancer, a thyroid cancer, a non-small cell lung cancer, a small cell lung cancer, a gastric cancer, an ovarian cancer, an endometrial cancer, a pancreatic cancer, , a bladder cancer, a gall bladder cancer, an esophageal cancer, a breast cancer, an adenocarcinoma, a nasal NK / T cell lymphoma, a gliomas, a glioblastoma, a osteosarcoma, a pituitary adenoma, or any combination thereof.

86. A process for producing a conditionally active chimeric antigen receptor, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the conditionally active chimeric antigen receptor according to any one of claims 37-38, under conditions allowing the expression of the conditionally active chimeric antigen receptor and recovering and purifying the produced protein from the culture.

87. A process for producing a conditionally active NECTIN4 binding protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the conditionally active NECTIN4 binding protein according to any one of claims 39-44, under conditions allowing the expression of the conditionally active NECTIN4 binding protein and recovering and purifying the produced protein from the culture.

88. A process for producing a multivalent protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the multivalent protein according to claims 67 or 69, under conditions allowing theWSGR Docket No.47517-766.601 expression of the multivalent protein and recovering and purifying the produced protein from the culture.

89. A process for producing an active drug, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the active drug according to claim 68, under conditions allowing the expression of the active drug and recovering and purifying the produced drug from the culture.

90. A cell comprising a CAR according to any one of claims 23, and 30-33.

91. A cell comprising a ProCAR according to any one of claims 23-33 and 37-38.

92. The cell of claim 90 or 91, wherein the cell is a T cell or an NK cell.

93. A method comprising transfecting a cell according to any one of claims 90-92, with a vector or an RNA comprising a nucleotide sequence encoding the CAR or the ProCAR.

94. The conditionally active chimeric antigen receptor of claim 37 or 38, wherein the conditionally active chimeric antigen receptor has a greater therapeutic index compared to a chimeric antigen receptor (CAR) that does not comprise the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor.

95. The conditionally active chimeric antigen receptor of claim 94, wherein the conditionally active chimeric antigen receptor has a therapeutic index that is at least about 5-fold to about 100- fold greater than that of a chimeric antigen receptor (CAR) that does not comprise the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor.

96. The conditionally active NECTIN4 binding protein of any one of claims 39-44, wherein the protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-347.

97. The conditionally active NECTIN4 binding protein of any one of claims 39-44, wherein the protein comprises a sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-347.

98. The conditionally active NECTIN4 binding protein of any one of claims 39-44, wherein the protein comprises a sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-347.

99. The conditionally active NECTIN4 binding protein of any one of claims 39-44 and 96-98, wherein the conditionally active NECTIN4 binding protein has a greater therapeutic index compared to a NECTIN4 binding protein that does not comprise the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active NECTIN4 binding protein.

100. The conditionally active NECTIN4 binding protein of claim 99, wherein the conditionally active NECTIN4 binding protein has a therapeutic index that is at least about 5-fold to about 100-WSGR Docket No.47517-766.601 fold greater than that of a NECTIN4 binding protein that does not comprise the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active NECTIN4 binding protein.

101. A pharmaceutical composition comprising: (i)(a) a conditionally active chimeric antigen receptor according to claim 94 or 95; or (i)(b) a conditionally active NECTIN4 binding protein according to claim 99 or 100, and (ii) a pharmaceutically acceptable carrier.

102. A method for treating or ameliorating a proliferative disease or a tumorous disease, comprising administering of the conditionally active chimeric antigen receptor according to claim 94 or 95, or the pharmaceutical composition according to claim 101, to a subject in need thereof.

103. A method for treating or ameliorating a proliferative disease or a tumorous disease, comprising administering of the conditionally active NECTIN4 binding protein according to claim 99 or 100, or the pharmaceutical composition according to claim 101, to a subject in need thereof.

104. The method of claim 102 or 103, wherein the subject is human.

105. The method of any one of claims 102-104, wherein the tumorous disease comprises at least one of: a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a lung cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a bladder cancer, a gallbladder cancer, a head and neck squamous cell cancer, a squamous cell carcinoma of head and neck, an esophageal cancer, a hepatocellular carcinoma, a skin cancer, or any combination thereof.

106. A method of increasing a therapeutic index of a NECTIN4 binding domain, the method comprising conjugating the NECTIN4 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, - wherein the non-CDR loop comprises a binding site specific for the NECTIN4 binding domain, - wherein the NECTIN4 binding domain is masked from binding its target by the binding moiety, - wherein the NECTIN4 binding domain is able to bind its target upon cleavage of the cleavable linker.

107. The method of claim 106, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected fromWSGR Docket No.47517-766.601 the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264.

108. The method of claim 106 or 107, wherein the NECTIN4 binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, wherein the multispecific protein further comprises a CD3 binding domain.

109. The method of any one of claims 106-108, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO:

612.

110. The method of claim 108 or 109, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO:

613.

111. The method of any one of claims 106-110, wherein the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766- 770.

112. The method of claim any one of claims 106-111, wherein the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

113. The method of any one of claims 106-112, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

114. The method of any one of claims 106-112, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

115. The method of any one of claims 106-112, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

116. The method of claim 106, wherein the NECTIN4 binding domain conjugated to the binding moiety is part of a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor further comprises at least one of: a transmembrane domain, an intracellular signaling domain, and a costimulatory domain.

117. The method of claim 116, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1WSGR Docket No.47517-766.601 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264.

118. The method of claim 116 or 117, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS:

612.

119. The method of any one of claims 116-118, wherein the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

120. The method of any one of claims 116-118, wherein the NECTIN4 binding domain comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

121. A method of increasing a therapeutic index of a NECTIN4 binding protein comprising a first target antigen binding domain and a second target antigen binding domain, wherein at least one of the first and the second target antigen binding domain comprises a NECTIN4 binding domain, the method comprising conjugating the first or the second target antigen binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, - wherein the non-CDR loop comprises a binding site specific for the first or the second target antigen binding domain, - wherein at least one of the first or the second target antigen binding domain is masked from binding its target by the binding moiety, and - wherein the first or the second target antigen binding domain that is masked, is able to bind its target upon cleavage of the cleavable linker.

122. The method of claim 121, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected fromWSGR Docket No.47517-766.601 the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264.

123. The method of claim 121 or 122, wherein the non-CDR loop comprises a binding site specific for the NECTIN4 binding domain.

124. The method of claim 121 or 122, wherein at least one of the first or the second target antigen binding domain comprises a CD3 binding domain.

125. The method of claim 124, wherein the non-CDR loop comprises a binding site specific for the CD3 binding domain.

126. The method of claim 125, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO:

613.

127. The method of any one of claims 121-126, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS:

612.

128. The method of claim any one of claims 121-127, wherein the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

129. The method of any one of claims 121-128, wherein the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766- 770.

130. The method of any one of claims 121-129, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

131. The method of any one of claims 121-129, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

132. The method of any one of claims 121-129, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

133. A method of increasing a therapeutic index of a NECTIN4 binding protein comprising a NECTIN4 binding domain and a CD3 binding domain, the method comprising conjugating CD3 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for CD3 binding domain.WSGR Docket No.47517-766.601 134. The method of claim 133, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264.

135. The method of claim 133 or 134, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO:

613.

136. The method of any one of claims 133-135, wherein the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS:

612.

137. The method of claim any one of claims 133-136, wherein the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

138. The method of any one of claims 133-137, wherein the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766- 770.

139. The method of any one of claims 133-138, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

140. The method of any one of claims 133-138, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

141. The method of any one of claims 133-138, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-329.

142. A NECTIN4 targeting conditionally active multispecific protein, comprising: a NECTIN4 binding domain, a CD3 binding domain, an albumin binding domain, wherein the albumin binding domain comprises a non-CDR loops that comprises a binding site specific for the CD3 bindingWSGR Docket No.47517-766.601 domain and a cleavable linker, wherein the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence selected from the group consisting of SEQ ID NOS: 133-198 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 133-198; and the CDR3 comprising a sequence selected from the group consisting of SEQ ID NOS: 199-264 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOS: 199-264.

143. The NECTIN4 targeting conditionally active multispecific protein of claim 142, wherein the albumin binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOS:

612.

144. The NECTIN4 targeting conditionally active multispecific protein of claim 142 or 143, wherein the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-66.

145. The NECTIN4 targeting conditionally active multispecific protein of any one of claims 142- 144, wherein the cleavable linker comprises a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of: SEQ ID NOS: 616, 617, 618, 622-646, 649-690, 718-720, and 766-770.

146. The NECTIN4 targeting conditionally active multispecific protein of any one of claims 142- 145, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-347.

147. The NECTIN4 targeting conditionally active multispecific protein of any one of claims 142- 145, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-347.

148. The NECTIN4 targeting conditionally active multispecific protein of any one of claims 142- 145, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOS: 265-347.

149. A pharmaceutical composition comprising a NECTIN4 targeting conditionally active multispecific protein of any one of claims 142-148.

150. The pharmaceutical composition of claim 149, further comprising a pharmaceutically acceptable carrier.WSGR Docket No.47517-766.601 151. A process for producing NECTIN4 targeting conditionally active multispecific protein, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding the domains of the NECTIN4 targeting conditionally active multispecific protein of any one of claims 142-148, under conditions allowing the expression of the NECTIN4 targeting conditionally active multispecific protein and recovering and purifying the produced protein from the culture.

152. A method for treating or ameliorating a proliferative disease or a tumorous disease in a subject in need thereof, comprising administering of a NECTIN4 targeting conditionally active multispecific protein of any one of claims 142-148, or a pharmaceutical composition according to claim 149 or 150, to a subject in need thereof.

153. The method of claim 152, wherein the tumorous disease comprises a solid tumor disease.

154. The method of claim 153, wherein the solid tumor disease is metastatic.

155. The method of any one of claims 152-154, wherein the tumorous disease comprises at least one of: a gastric cancer, a thyroid cancer, a papillary thyroid cancer, a colorectal cancer, a lung cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a bladder cancer, a gallbladder cancer, a head and neck squamous cell cancer, a squamous cell carcinoma of head and neck, an esophageal cancer, a hepatocellular carcinoma, a skin cancer, or any combination thereof.

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