EP4646267A1 - Protéine trispécifique ciblant la nectine-4 pour le traitement du cancer - Google Patents

Protéine trispécifique ciblant la nectine-4 pour le traitement du cancer

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Publication number
EP4646267A1
EP4646267A1 EP23915124.4A EP23915124A EP4646267A1 EP 4646267 A1 EP4646267 A1 EP 4646267A1 EP 23915124 A EP23915124 A EP 23915124A EP 4646267 A1 EP4646267 A1 EP 4646267A1
Authority
EP
European Patent Office
Prior art keywords
nectin4
seq
binding
sequence
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23915124.4A
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German (de)
English (en)
Inventor
Holger Wesche
Shuoyen Jack LIN
Kevin J. Wright
Richard J. Austin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harpoon Therapeutics Inc
Original Assignee
Harpoon Therapeutics Inc
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Filing date
Publication date
Application filed by Harpoon Therapeutics Inc filed Critical Harpoon Therapeutics Inc
Publication of EP4646267A1 publication Critical patent/EP4646267A1/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • 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.
  • 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 consist
  • the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-132; the CDR2 comprising a sequence WSGR 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.
  • 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.
  • the NECTIN4 binding domain is part of a multispecific protein.
  • the multispecific protein further comprises a CD3 binding domain.
  • the multispecific protein comprises an active drug format.
  • the multispecific protein further comprises a bulk serum protein binding domain.
  • the bulk serum protein comprises a serum albumin protein.
  • the serum albumin protein comprises a human serum albumin protein.
  • 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.
  • the CD3 binding domain comprises a sequence that is at least 75% identical the sequence as set forth in SEQ ID NO: 613.
  • 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.
  • 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.
  • the multispecific protein comprises a non-cleavable prodrug format.
  • 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.
  • 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.
  • 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.
  • the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 613.
  • 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.
  • 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.
  • 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 consist
  • 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.
  • 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.
  • the binding moiety comprises a sequence that is at least 75% identical the sequence selected from the group consisting of SEQ ID NO: 612.
  • the second target antigen binding domain (T2) comprises a CD3 binding domain.
  • the CD3 binding domain comprises a sequence that is at least 75% identical to the sequence as set forth in SEQ ID NO: 613.
  • 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.
  • the subject is human.
  • WSGR Docket No.47517-766.601 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.
  • the subject is human.
  • the binding domain is a humanized antibody or an antigen binding fragment thereof.
  • 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.
  • binding domain comprises the single domain antibody.
  • the binding domain binds to NECTIN4 with a binding affinity (Kd) of about 0.001 nM to about 500 nM.
  • the binding domain binds to human NECTIN4, mouse NECTIN4, cynomolgus NECTIN4, or a combination thereof.
  • a multispecific protein comprising an NECTIN4 binding domain, wherein the NECTIN4 binding domain is according to descriptions herein.
  • the NECTIN4 binding domain according to descriptions herein anti-NECTIN4 domain
  • a CD3 binding domain anti-CD3 domain
  • the anti-NECTIN4 domain and the anti-CD3 domain are in an anti-NECTIN4:anti-CD3 orientation.
  • the anti-NECTIN4 domain and the anti-CD3 domain are in an anti-CD3: anti-NECTIN4 orientation.
  • 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).
  • the anti-CD3 domain comprises an amino acid as set forth in SEQ ID NO: 613.
  • the anti-ALB domain comprises an amino acid sequence as set forth in SEQ ID NO: 612.
  • the anti-NECTIN4 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3: anti-ALB: anti-NECTIN4 orientation.
  • 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.
  • 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 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.
  • an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 265-329.
  • an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 330-347.
  • an active drug comprising a sequence as set forth in any one of SEQ ID NOS: 1-66.
  • 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.
  • a process for producing a NECTIN4 binding domain 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.
  • a process for producing a multispecific protein 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.
  • 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.
  • 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.
  • the subject is human.
  • 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, a WSGR 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.
  • the NECTIN4 binding domain selectively binds to tumor cells expressing NECTIN4.
  • the tumorous disease comprises a solid tumor disease.
  • the solid tumor disease is metastatic.
  • 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.
  • 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.
  • the NECTIN4 binding domain selectively binds to tumor cells expressing NECTIN4.
  • the tumorous disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic.
  • 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.
  • a gastric 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.
  • a process for producing a conditionally active chimeric antigen receptor 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.
  • a process for producing a conditionally active NECTIN4 binding protein 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 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.
  • a process for producing an active drug according to descriptions herein 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.
  • 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.
  • 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.
  • 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.
  • the subject is human.
  • 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.
  • 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.
  • a method of increasing a therapeutic index of an NECTIN4 binding domain 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.
  • the NECTIN4 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, WSGR Docket No.47517-766.601
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CDR1 comprises a sequence selected from the group consisting of SEQ ID NOS: 67-
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the NECTIN4 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID WSGR Docket No.47517-766.601 NOS: 1-66.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 613.
  • 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.
  • 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.
  • 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.
  • 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.
  • conditionally active multispecific protein WSGR 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the tumorous disease comprises a solid tumor disease.
  • the solid tumor disease is metastatic.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NCLV anti-ALB/anti- CD3/anti-NECTIN4
  • CT anti-CD3/anti-NECTIN4
  • 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.
  • 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.
  • 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.
  • NCLV anti-ALB/anti- CD3/anti-NECTIN4
  • CT anti-CD3/anti-NECTIN4
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NCLV anti-ALB/anti- CD3/anti-NECTIN4
  • CT anti-CD3/anti-NECTIN4
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NCLV anti-ALB/anti-CD3/anti- NECTIN4
  • CT anti-CD3/anti-NECTIN4
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • CT Anti-CD3/Anti-NECTIN4
  • ProTriTAC L040 anti-ALB/anti-CD3/anti-NECTIN4
  • NCLV anti-ALB/anti-CD3/anti- NECTIN4
  • 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.
  • 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.
  • 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.
  • 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.
  • CT anti-CD3/anti-NECTIN4
  • ProTriTAC L040 anti-ALB/anti-CD3/anti-NECTIN4
  • NCLV anti-ALB/anti-CD3/anti- NECTIN4
  • 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.
  • 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.
  • 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.
  • CT anti-CD3/anti-NECTIN4
  • NCLV anti-ALB/anti-CD3/anti- NECTIN4
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG.59 shows an IHC assay with NECTIN4 rabbit polyclonal antibody staining on HT29, SW780, and Jurkat cell pellets at Day 0 (40X magnification).
  • FIG.60 shows an IHC assay with NECTIN4 rabbit polyclonal antibody staining on SW780 tumors at Day 7 and Day 21 (40X magnification).
  • FIG.61 shows an IHC assay with NECTIN4 rabbit polyclonal antibody staining on HT29 tumors at Day 7 and Day 21 (40X magnification).
  • 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.
  • 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.
  • 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.
  • 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.
  • NECTIN4 An exemplary protein sequences for NECTIN4 is provided in UniProtKB ID NOS: Q96NY8 or Ref Seq NP_112178.2 (SEQ ID NO: 619).
  • 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.
  • 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.
  • 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.
  • 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.
  • the Nectin4 protein lacks N terminal methionine (e.g., SEQ ID NO: 620).
  • 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.
  • 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. [00105] 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.
  • the NECTIN4 binding domains disclosed herein recognize full-length NECTIN4.
  • 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.
  • the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within a domain of NECTIN4.
  • the NECTIN4 binding domains disclosed herein recognize full- length NECTIN4.
  • 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.
  • the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within a domain of NECTIN4.
  • 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 the WSGR Docket No.47517-766.601 full length NECTIN4 protein, as described above.
  • 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.
  • 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. [00108] 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.
  • 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.
  • the NECTIN4 binding protein specifically binds NECTIN4 with equivalent or better affinity as that of a reference NECTIN4 binding protein
  • 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.
  • the affinity matured NECTIN4 binding molecule has superior stability with respect to selected destabilizing agents, as that of a reference NECTIN4 binding parental molecule.
  • 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.
  • “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 of WSGR 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.
  • 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.
  • 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.
  • 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.
  • a NECTIN4 protein such as human NECTIN4 protein
  • 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.
  • 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.
  • the affinity matured NECTIN4 binding molecule 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.
  • 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.
  • 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).
  • AFFIMERS® affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, DARPINS®, affibodies, affilins, monobodies and arma
  • 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.
  • antibody variant refers to variants and derivatives of an antibody or an antigen binding fragment as described herein.
  • amino acid sequence variants of the anti- NECTIN4 antibodies or antigen binding fragments thereof, as described herein are contemplated.
  • 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. [00115] 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 introduced WSGR 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. [00116] 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.
  • 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.
  • sdAb single domain antibody
  • VH heavy chain variable domain
  • VHH variable domain
  • peptide a peptide
  • ligand a small molecule entity specific for NECTIN4.
  • 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.
  • the NECTIN4 binding domain is a single-domain antibody.
  • the NECTIN4 binding domain is a peptide.
  • the NECTIN4 binding domain is a small molecule.
  • 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.
  • 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 acid WSGR 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
  • a single domain antibody corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against NECTIN4.
  • 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.
  • VHH domains against NECTIN4 are obtained from na ⁇ ve 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.
  • libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694.
  • improved synthetic or semi-synthetic libraries derived from na ⁇ ve VHH libraries are used, such as VHH libraries obtained from na ⁇ ve VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.
  • 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.
  • a suitable biological sample such as a blood sample, serum sample or sample of B-cells
  • VHH sequences directed against NECTIN4 starting from said sample, using any suitable technique known in the field.
  • 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.
  • 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 naturally WSGR 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).
  • 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.
  • a single domain anti- NECTIN4 antibody 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.
  • 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.
  • camelized anti- NECTIN4 single domain antibodies of the disclosure 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.
  • 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.
  • the amino acid sequence of the desired humanized or camelized anti- NECTIN4 single domain antibody of the disclosure are designed and then synthesized de novo using known techniques for peptide synthesis.
  • 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.
  • 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.
  • naturally occurring VH sequences such as one or more framework (FR) sequences and/or complementarity determining region (CDR) sequences
  • CDR complementarity determining region
  • 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.
  • 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.
  • sdAb single domain antibodies
  • Fab e.g., single chain Fv fragments (scFv)
  • dsFv disulfide stabilized Fv fragments
  • heteroconjugate antibodies e.g., bispecific antibodies
  • pFv fragments heavy chain monomers or dimers, light chain monomers
  • the NECTIN4 binding domain is a single domain antibody.
  • the anti- NECTIN4 single domain antibody comprises heavy chain variable complementarity determining regions (CDR), CDR1, CDR2, and CDR3.
  • CDR heavy chain variable complementarity determining regions
  • 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.
  • the NECTIN4 binding domain comprises an amino acid sequence selected from SEQ ID NOs: 1-66.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the NECTIN4 binding domain is cross-reactive with human cynomolgus and mouse NECTIN4.
  • 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 K D ). In certain embodiments, the NECTIN4 binding domains disclosed herein bind to cynomolgus NECTIN4 with a mouse KD (m KD).
  • the NECTIN4 binding domains disclosed herein bind to both cynomolgus NECTIN4 and a human NECTIN4, with a cyno Kd (c K D ) and a human Kd (h K D ), respectively.
  • 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.
  • the NECTIN4 binding protein binds to human, mouse and cynomolgus NECTIN4 with comparable binding affinities (i.e., h K D , the m K D and the c K D values do not differ by more than ⁇ 10%).
  • the h KD, the m KD and the c KD range from about 0.001 nM to about 500 nM.
  • the h K D , the m K D and the c K D range from about 0.001 nM to about 450 nM.
  • the h K D , the m K D and the c K D range from about 0.001 nM to about 400 nM.
  • the h K D , the m K D and the c K D range 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 K D , the m K D and the c K D range from about 0.001 nM to about 200 nM.
  • 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 K D , the m K D and the c K D range 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.
  • the h K D , the m K D and the c K D range from about 0.4 nM to about 50 nM. In some embodiments, the h K D , the m K D and the c K D range 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 K D , the m K D and the c K D range from about 0.7 nM to about 8 nM. In some embodiments, the h K D , the m K D and the c K D range from about 0.8 nM to about 6 nM.
  • 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 K D , the m K D and the c K D range from about 1 nM to about 2 nM.
  • 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.
  • the affinity peptide tag is six consecutive histidine residues, also referred to as 6X-His (SEQ ID NO: 615).
  • 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.
  • the soluble NECTIN4 is present in the blood and/or lymphatic circulation in a subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • the NECTIN4 binding protein comprises a domain comprising a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 621).
  • the protein is incubated with a sortase and a SME binder whereby the sortase attaches the SME binder to the recognition sequence.
  • 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.
  • the NECTIN4 binding proteins described herein comprise a natural NECTIN4 ligand.
  • 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.
  • 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.
  • the domains are linked by one or more internal linker.
  • 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.
  • 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.
  • 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 protease WSGR 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.
  • the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 630), (GGGGSGGGGSGGGGS) (SEQ ID NO: 631), or (GGGGSGGGS) (SEQ ID NO: 616).
  • 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 are versatile tools in modern research and biotechnology.
  • mTG is 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.
  • 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.
  • a lysine-based linker e.g., any primary amine chain which is a substrate for TGase, e.g. comprising an alkylamine, oxoamine
  • 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.
  • a hybrid vector is made where the DNA encoding the directly joined domains are themselves directly ligated to each other.
  • 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.
  • 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 variable WSGR Docket No.47517-766.601 domain (VL) and a variable domain (VHH) of camelid derived single domain antibody.
  • 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.
  • the NECTIN4 binding protein is a ligand or peptide that binds to or associates with NECTIN4.
  • the NECTIN4 binding protein is a knottin.
  • the binding domain to NECTIN4 is a small molecular entity.
  • the NECTIN4 binding proteins according to the present disclosure may be incorporated into NECTIN4 targeting trispecific proteins.
  • the trispecific proteins comprise a CD3 binding domain, a half-life extension domain, and a NECTIN4 binding domain according to this disclosure.
  • 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)
  • the multispecific protein further comprises a domain which specifically binds to CD3.
  • the multispecific protein further comprises a domain which specifically binds to human CD3.
  • the multispecific protein further comprises a domain which specifically binds to CD3-gamma.
  • the multispecific protein further comprises a domain which specifically binds to CD3-delta.
  • the multispecific protein further comprises a domain which specifically binds to CD3-epsilon.
  • the multispecific protein further comprises a domain which specifically binds to the T cell receptor (TCR).
  • TCR T cell receptor
  • the multispecific protein further comprises a domain which specifically binds the alpha chain of the TCR. In some chain of the TCR.
  • 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.
  • 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 polyclonal WSGR 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 dim
  • the CD3 binding domain 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.
  • the CD3 binding domain of the multispecific protein comprising a NECTIN4 binding protein described herein 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.
  • 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.
  • 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.
  • scFv single chain variable fragments
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain
  • the HSA binding domain is a single-domain antibody.
  • the serum albumin binding domain is a peptide.
  • 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 than WSGR 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.
  • 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.
  • 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.
  • the binding affinity of the half-life extension domain 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.
  • the half- life extension domain has a high binding affinity.
  • the half-life extension domain has a medium binding affinity.
  • the half-life extension domain has a low or marginal binding affinity.
  • a NECTIN4 targeting multispecific protein of this disclosure 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.
  • the first domain comprises an scFv that specifically binds the CD3.
  • the CD3, for instance, is a human CD3 protein.
  • the second domain comprises an sdAb that specifically binds a bulk serum protein.
  • the bulk serum protein is WSGR 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, H 2 N-(C)-L1-(A)-L2-(B)-COOH, H 2 N-(A)-L1-(B)-(C)-L2-COOH, or H 2 N-(B)-(C)- (A)-COOH.
  • a NECTIN4 targeting multispecific protein of this disclosure 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.
  • 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
  • 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)
  • 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).
  • 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.
  • the binding moiety masks the interaction of the NECTIN4 binding domain or the CD3 binding domain, to their targets.
  • 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.
  • the binding moiety comprises a modified non-CDR loop sequence and a cleavable linker.
  • the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 618 and 632-647, or an amino WSGR 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.
  • the masking moiety comprises a modified non-CDR loop sequence and a non-cleavable linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the NECTIN4 binding domain of the ProTriTAC 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ProTriTAC half-life extended T cell engager format
  • 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 pathophysio
  • 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.
  • 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.
  • 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 some WSGR 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.
  • a “therapeutic index” (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.
  • a therapeutic agent e.g., a NECTIN4 TriTAC, a NECTIN4 ProTriTAC, a NECTIN4 CAR, a NECTIN4 ProCAR
  • 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.
  • 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.
  • a binding moiety as described herein comprises at least one non-CDR loop.
  • a non-CDR loop provides a binding site for binding of the binding moiety to a NECTIN4 binding domain of this disclosure.
  • 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.
  • 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).
  • CDRs complementarity determining regions
  • 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.
  • 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.
  • 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).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs WSGR 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.
  • a binding moiety of this disclosure is a heavy chain only antibody.
  • 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.
  • CDRs complementarity-determining regions
  • 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.
  • 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.
  • 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.
  • the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are considered a scaffold for the CDRs.
  • 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.
  • 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. [00170] 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 is WSGR Docket No.47517-766.601 inserted into a non-CDR loop, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the bulk serum protein comprises, for example, albumin, fibrinogen, or a globulin.
  • 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 knot WSGR Docket No.47517-766.601 peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin-binding module, or a DNA or RNA aptamer scaffold.
  • the binding moieties comprise a binding site for the bulk serum protein.
  • 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.
  • the binding moieties comprise a binding site for an immunoglobulin light chain.
  • the CDRs provide a binding site for the immunoglobulin light chain.
  • the binding moieties are any kinds of polypeptides.
  • the binding moieties are natural peptides, synthetic peptides, or fibronectin scaffolds, or engineered bulk serum proteins.
  • 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.
  • 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.
  • 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.
  • the binding moiety described herein comprises at least one cleavable linker.
  • 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 comprises a protease cleavable linker recognized and cleaved in an amino acid sequence-specific manner.
  • the protease cleavable linker is recognized in an amino acid sequence-specific manner by a matrix metalloprotease (MMP), for example a MMP9.
  • MMP matrix metalloprotease
  • 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).
  • the protease cleavable linker recognized by a MMP9 comprises a polypeptide having an amino acid sequence LEATA (SEQ ID NO: 648).
  • protease cleavable linker is recognized in an amino acid sequence-specific manner by a MMP11.
  • 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, asparagine WSGR 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
  • the blood of a subject is rich in proteases.
  • 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.
  • proteases are present in the blood of a subject, for example proteases that target amino WSGR Docket No.47517-766.601 acid sequences found in microbial peptides.
  • An engineered immune effector cell e.g., a T cell or NK cell
  • a CAR that includes a NECTIN4 binding protein containing, for example, an anti- NECTIN4 single domain antibody as described herein.
  • 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.
  • the CAR further comprises an amino acid sequence encoding an intracellular signaling domain, such as 4-1BB and/or CD3 zeta.
  • 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%.
  • the conditionally active receptors described herein comprise at least one binding moiety comprising a non-CDR loop.
  • 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.
  • 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.
  • the binding moiety binds to the NECTIN4 binding domain.
  • a non-CDR loop provides a binding site for binding of the moiety to the NECTIN4 binding domain.
  • 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.
  • the binding moiety further comprises complimentary determining regions (CDRs).
  • 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.
  • 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.
  • the inactive receptor 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.
  • 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.
  • the cleavable linker of the binding moiety 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.
  • the cleavable linker in some embodiments comprises an amino acid sequence selected from the linker sequences provided in the sequence table.
  • Transmembrane domain [00184]
  • 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.
  • 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.
  • TM transmembrane
  • 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.
  • the TM sequence IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 691
  • 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).
  • 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.
  • the hinge region is an immunoglobulin heavy chain hinge region.
  • the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).
  • 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.
  • aa amino acids to about 50 amino acids
  • 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.
  • 1 amino acid e.g., Gly
  • 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.
  • 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 not WSGR 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.
  • 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.
  • 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.
  • 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 IgG2 hinge ELKTPLGDTTHTCPRCP
  • SPNMVPHAHHAQ SEQ ID NO: 715
  • human IgG4 hinge and the like.
  • 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.
  • 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.
  • 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 [00193]
  • 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.
  • the intracellular signaling domain is a signaling domain of a protein including, but not limited to, ZAP70, CD3 zeta, and 4-1BB.
  • the conditionally active chimeric antigen receptor further comprises a costimulatory domain.
  • 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 WSGR Docket No.47517-766.601 thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications thereto.
  • 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.
  • 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,
  • the disclosure provides a cell (e.g., T-cell) engineered to express a CAR.
  • a cell is transformed with the CAR and the CAR is expressed on the cell surface.
  • the cell e.g., T-cell
  • the cell is transduced with a viral vector encoding a CAR.
  • the viral vector is a retroviral vector.
  • the viral vector is a lentiviral vector.
  • the cell may stably express the CAR.
  • the cell e.g., T cell
  • the cell is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR.
  • the cell may transiently express the CAR.
  • 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.
  • the conditionally active TFP comprises a binding moiety, a NECTIN4 binding domain, and a T-cell receptor subunit.
  • 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.
  • 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.
  • 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, CD
  • 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.
  • 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
  • ITAM
  • the conditionally active TFP further comprises a costimulatory domain.
  • 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.
  • the NECTIN4 binding domain is connected to the T-cell receptor extracellular domain by a linker sequence.
  • the disclosure provides a cell (e.g., T-cell) engineered to express a conditionally active T-cell receptor fusion protein (TFP).
  • a cell is transformed with the conditionally active TFP and the conditionally active TFP is expressed on the cell surface.
  • the cell e.g., T-cell
  • the cell is transduced with a viral vector encoding a conditionally active TFP.
  • the viral vector is a retroviral vector.
  • the viral vector is a lentiviral vector.
  • the cell may stably express the conditionally active TFP.
  • the cell e.g., T cell
  • a nucleic acid e.g., mRNA, cDNA, DNA
  • the cell may transiently express the conditionally active TFP.
  • Conditionally Active T-cell Receptors [00203]
  • 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 a WSGR Docket No.47517-766.601 binding moiety.
  • 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.
  • the binding moiety is capable of masking or masks the binding of the T-cell receptor to its target.
  • the binding moiety binds to T-cell receptor.
  • a non-CDR loop provides a binding site for binding of the moiety to the T- cell receptor.
  • the non-CDR loop provides a binding site specific for T-cell receptor alpha, T-cell receptor beta, or a combination thereof.
  • the binding moiety masks binding of the T-cell receptor to its target, e.g., via steric occlusion, via specific intermolecular interactions.
  • the disclosure provides a cell (e.g., T-cell) engineered to express a conditionally active T-cell receptor (TCR).
  • TCR conditionally active T-cell receptor
  • a cell is transformed with the conditionally active TCR and the conditionally active TCR is expressed on the cell surface.
  • the cell e.g., T-cell
  • the cell is transduced with a viral vector encoding a conditionally active TCR.
  • the viral vector is a retroviral vector.
  • the viral vector is a lentiviral vector.
  • the cell may stably express the conditionally active TCR.
  • the cell e.g., T cell
  • the cell is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TCR.
  • the cell may transiently express the conditionally active TCR.
  • 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.
  • 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.
  • 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
  • the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual.
  • the cell is an immune cell WSGR Docket No.47517-766.601 obtained from an individual.
  • the cell is a T lymphocyte obtained from an individual.
  • the cell is a cytotoxic cell obtained from an individual.
  • the cell is a stem cell or progenitor cell obtained from an individual.
  • 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).
  • peripheral blood NK cells 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.
  • 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.
  • 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.
  • 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.
  • 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 present WSGR 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.
  • an immune cell e.g., a T lymphocyte or NK cell
  • a stem cell e.g., a T lymphocyte or NK cell
  • a progenitor cell e.g., a progenitor cell.
  • the genetic modification is carried out ex vivo.
  • 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 [00214] In some embodiments, a source of T-cells is obtained from a subject.
  • 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.
  • 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 e.g., lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • 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.
  • 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.
  • 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.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • T-cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL TM gradient or by counterflow centrifugal elutriation.
  • T-cells can be further isolated by positive or negative selection techniques.
  • 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.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • 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.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T-cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • 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.
  • 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.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • T regulatory T- cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.
  • T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.
  • WSGR Docket No.47517-766.601 [00217]
  • 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.
  • the concentration of cells and surface can be varied.
  • 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.
  • a concentration of 2 billion cells/ml is used.
  • a concentration of 1 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • 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.
  • concentrations can result in increased cell yield, cell activation, and cell expansion.
  • 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.
  • the concentration of cells used is 5 ⁇ 10e6/ml. In other embodiments, the concentration used can be from about 1 ⁇ 10 5 /ml to 1 ⁇ 10 6 /ml, and any integer value in between.
  • 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.
  • T-cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one WSGR 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.
  • 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.
  • 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.
  • a blood sample or an apheresis is taken from a generally healthy subject.
  • 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.
  • the T-cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • 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.
  • 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
  • T-cells are obtained from a patient directly following treatment that leaves the subject with functional T-cells.
  • the quality of T-cells obtained may be optimal or WSGR Docket No.47517-766.601 improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • 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.
  • 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.
  • 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.
  • 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.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co- stimulation of an accessory molecule on the surface of the T-cells.
  • 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.
  • an anti-CD3 antibody and an anti-CD28 antibody are used to stimulate proliferation of either CD4+ T-cells or CD8+ T-cells.
  • an anti-CD28 antibody examples 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).
  • the primary stimulatory signal and the costimulatory signal for the T-cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface.
  • the agents 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 agent WSGR 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.
  • the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.”
  • 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.
  • a 1:1 ratio of each antibody bound to the beads for CD4+ T-cell expansion and T-cell growth is used.
  • 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.
  • the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between.
  • more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one.
  • 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.
  • 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.
  • 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.
  • 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 values WSGR 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.
  • a ratio of particles to cells of 1:1 or less is used.
  • a particle: cell ratio is 1:5.
  • the ratio of particles to cells can be varied depending on the day of stimulation.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • ratios will vary depending on particle size and on cell size and type.
  • 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.
  • a force such as a magnetic force
  • 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.
  • the cells for example, 10 4 to 10 9 T-cells
  • beads for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1:1
  • a buffer for example PBS (without divalent cations such as, calcium and magnesium).
  • any cell concentration may be used.
  • 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.
  • a concentration of about 2 billion cells/ml is used. In one embodiment, greater than 100 million cells/ml is used.
  • 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.
  • 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
  • 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% CO 2 ).
  • an appropriate temperature e.g., 37° C.
  • atmosphere e.g., air plus 5% CO 2
  • T-cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • 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+).
  • 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.
  • 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.
  • NECTIN4 binding proteins 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. [00237] 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.
  • the NECTIN4 binding proteins of the disclosure are monovalent or multivalent bivalent, trivalent, etc.).
  • 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 or WSGR Docket No.47517-766.601 epitope.
  • 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).
  • 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.
  • the Fc-fusion NECTIN4 binding proteins of this disclosure have extended half-life compared to an otherwise identical NECTIN4 binding protein.
  • 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.
  • 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.
  • 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.
  • NECTIN4 binding proteins 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.
  • 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.
  • a detectable label such as an enzymatic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the modulator.
  • polynucleotide molecules encoding NECTIN4 binding proteins described herein are provided.
  • the polynucleotide molecules are provided as a DNA construct.
  • the polynucleotide molecules are provided as a messenger RNA transcript.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NECTIN4 binding proteins as described herein 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.
  • 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.
  • 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.
  • suitable pharmaceutical carriers 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.
  • 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.
  • the NECTIN4 binding proteins described herein are encapsulated in nanoparticles.
  • the nanoparticles are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods.
  • the NECTIN4 binding protein is attached to liposomes.
  • the NECTIN4 binding proteins are conjugated to the surface of liposomes.
  • the NECTIN4 binding proteins are encapsulated within the shell of a liposome.
  • the liposome is a cationic liposome.
  • 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.
  • 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 [00250]
  • 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.
  • 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 ⁇ g/kg, about 0.5 ⁇ g/kg to about 5 ⁇ g/kg, about 2 ⁇ g/kg to about 10 ⁇ g/kg, about 7 ⁇ g/kg to about 15 ⁇ g/kg, about 12 ⁇ g/kg to about 25 ⁇ g/kg, about 20 ⁇ g/kg to about 50 ⁇ g/kg, about 35 ⁇ g/kg to about 70 ⁇ g/kg, about 45 ⁇ g/kg to about 80 ⁇ g/kg, about 65 ⁇ g/kg, about 1
  • 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 are also provided in certain embodiments.
  • 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.
  • the condition is a cancer.
  • 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.
  • the disclosure provides a method of inhibiting metastasis of malignant WSGR 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.
  • 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.
  • the methods as described herein further comprise administering an effective amount of a second therapeutic agent.
  • the second therapeutic agent is a biotherapeutic agent, for example, an antibody.
  • 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.
  • TLR3, TLR4, TLR5, TLR7, TLR9 tumor vaccine.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • the NECTIN4 binding proteins of the present disclosure are administered to treat a neoplastic condition.
  • Neoplastic conditions are WSGR 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,
  • 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.
  • 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.
  • 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.
  • the NECTIN4 binding proteins of the present disclosure are administered to a subject suffering from melanoma.
  • the NECTIN4 binding proteins of the present disclosure are used to diagnose, monitor, treat or prevent melanoma.
  • melanoma 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
  • 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.
  • 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
  • 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.
  • 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, colon WSGR 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
  • a cancer such
  • 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).
  • 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.
  • T-cells expressing NECTIN4 CARs the ability to recognize tumorigenic NECTIN4independent of antigen processing, thus bypassing a major mechanism of tumor escape.
  • CARs when expressed in T- cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • 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).
  • a platinum based agent e.g., carboplatin, cisplatin, oxaliplatin
  • a taxane e.g., docetaxel, paclitaxel, larotaxel or cabazitaxel.
  • the disclosed NECTIN4 CAR treatments are effective at treating ovarian cancer, including ovarian-serous carcinoma and ovarian-papillary serous carcinoma.
  • 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.
  • the disorder has been treated and the initial tumor mass eliminated, reduced or otherwise ameliorated so the patient is asymptomatic or in remission.
  • 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.
  • the NECTIN4 WSGR 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow it to be modified.
  • 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.
  • 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.
  • risk factors e.g., genomic indications, family history, in vivo or in vitro test results, etc.
  • 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.
  • 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.
  • 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; carubic
  • 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-PT
  • kits for detecting expression of NECTIN4 in vitro or in vivo are provided.
  • 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.
  • 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.
  • 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 taken WSGR 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.
  • 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.
  • 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.
  • the NECTIN4 single domain antibody is directly labeled.
  • 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.
  • 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.
  • the control sample is a sample from a subject without cancer.
  • the sample is a blood or tissue sample.
  • the antibody that binds (for example specifically binds) NECTIN4 is directly labeled with a detectable label.
  • 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 include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase.
  • 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 include 125 I, 131 I, 35 S or 3 H.
  • 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.
  • 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.
  • the immunoassays and method disclosed herein can be used for a number of purposes.
  • the antibody that specifically binds NECTIN4 may be used to detect the production of NECTIN4 in cells in cell culture.
  • 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.
  • the NECTIN4 is cell-surface NECTIN4.
  • 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).
  • 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.
  • kits for detecting a polypeptide will typically comprise a single domain antibody, according to the present disclosure, that specifically binds NECTIN4.
  • an antibody fragment such as an scFv fragment, a VH domain, or a Fab is included in the kit.
  • the antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).
  • 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 particular WSGR Docket No.47517-766.601 application for which the kit is designed.
  • 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.
  • the diagnostic kit comprises an immunoassay.
  • 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.
  • Methods of determining the presence or absence of a cell surface marker are well known in the art.
  • 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.
  • the antibodies can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.
  • a conventional immunoassay including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.
  • 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).
  • 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).
  • 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.
  • interchain and intrachain disulfide bonds 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.
  • 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.
  • 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.
  • 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 [00283]
  • 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
  • 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.
  • the NECTIN4 binding proteins comprise a heavy chain only antibody, such as a VH or a VHH domain.
  • the NECTIN4 binding proteins comprise a heavy chain only antibody that is an engineered human VH domain.
  • the engineered human VH domain is produced by panning of phage display libraries.
  • the NECTIN4 binding proteins comprise a VHH.
  • VHH refers to single chain antibody binding domain devoid of light chain.
  • 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.
  • the VHH is derived from a species selected from the group consisting of camels, llamas, vicunas, guanacos, and WSGR Docket No.47517-766.601 cartilaginous fish (such as, but not limited to, sharks).
  • the VHH is derived from an alpaca (such as, but not limited to, a Huacaya Alpaca or a Suri alpaca).
  • 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).
  • CDRs complementarity-determining regions
  • FR framework
  • 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.
  • the NECTIN4 binding proteins comprise heavy chain only antibodies, such as VH or VHH domains, and comprise three CDRs.
  • Such heavy chain only antibodies bind NECTIN4 as a monomer with no dependency on dimerization with a VL (light chain variable) region for optimal binding affinity.
  • VL light chain variable
  • “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).
  • a heavy chain variable domain may include a single WSGR 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.
  • CDRs of the present disclosure necessarily correspond to the Kabat numbering convention.
  • 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.
  • 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.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • 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.
  • “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 most WSGR 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, t 1/2 the time required for 50% completion of the process.
  • 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 K D values 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 K D values 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.
  • the “K D ” 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.).
  • RIA radiolabeled antigen binding assay
  • surface plasmon resonance assays using a BIACORE -2000 or a BIACORE - 3000 (BIAcore, Inc., Piscataway, N.J.).
  • 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.).
  • the “K D ”, “kon”, and “koff” are measured using the OCTET® Systems (Pall Life Sciences).
  • the ligand e.g., biotinylated human or cynomolgus NECTIN4
  • the ligand 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 ⁇ g/ml human or cynomolgus NECTIN4 protein.
  • a solution of PBS/Casein is also introduced as a blocking agent.
  • NECTIN4 binding protein variants are introduced at a concentration ranging from about 10 ng/mL to about 100 ⁇ g/mL, about 50 ng/mL to about 5 ⁇ g/mL, or about 2 ng/mL to about 20 ⁇ g/mL.
  • the NECTIN4 binding single domain proteins are used at a WSGR Docket No.47517-766.601 concentration ranging from about 2 ng/mL to about 20 ⁇ g/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.
  • 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.
  • 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.
  • “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).
  • 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.
  • 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.
  • a cleavable linker e.g., comprising an amino acid sequence as set forth in SEQ ID NOS: 618, and 632-646
  • the ProTriTAC proteins of this disclosure are, in some cases, activated from a masked state to an active state by cleavage of the WSGR 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 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.
  • non-cleavable prodrug 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 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.
  • 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.
  • the primary specificity determining region(s) can be defined using the framework established in the Skrlec 2015 publication (Trends in Biotechnol, 33:408-418).
  • 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 loops WSGR Docket No.47517-766.601 [00296] “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.
  • Example 1 Screening of Phage Display Library for Identification of NECTIN4 Binding Domains [00298] 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.
  • Table 3 Binding of Llama Anti-NECTIN4 Antibodies to Human or Cynomolgus Monkey NECTIN4 in an ELISA Assay Sequence ELISA ELISA cyNECTIN4/contro name huNECTIN4 cyNECTIN4 huNECTIN4/control l 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- L 107 3.7 3.6 45.0 44.3 NECTIN4- L 158 4.0 4.0 18.7 18.7 NECTIN4- L 177 3.8 3.7 39.7 39.0 NECTIN4-L28 3.8 3.7 35.7 35.0 NECTIN4-L81 3.8 3.8 42.6 42.5 WSGR Docket No.47517-766.601 Sequence ELISA ELISA ELISA cy
  • Example 2 Incorporation of NECTIN4 Binding Heavy Chain Only Single Domain Antibodies Into Fusion Proteins and T Cell Dependent Cellular Cytotoxicity Assays [00301]
  • 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).
  • the amount fusion protein in the conditioned media from the transfected Expi293 cells was quantitated using by WSGR 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.
  • 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).
  • TDCC T-cell dependent cellular cytotoxicity assay
  • 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 EC 50 value 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-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 [00303] n/a insufficient activity to calculate an EC50 using the protein concentrations tested. n.d. no data with this binder.
  • 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.
  • 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.
  • TDCC assays 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.
  • 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.
  • 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 CT WSGR 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 3
  • 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
  • Four purified anti-CD3/anti-NECTIN4 fusion proteins were proteolytically activated using Matriptase, a serine protease.100 nM of Matriptase was added to 100 ⁇ g 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.
  • Table 8 EC 50 Values 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: EC 50 Values for Redirected T Cell Killing of HT1376 and MV411 Cells by fusion
  • 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
  • CT Anti-CD3/Anti-NECTIN4
  • mice were age matched.
  • 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).
  • WSGR Docket No.47517-766.601 treatment followed 1 day post implant (Day 1).
  • 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.
  • Example 7 NECTIN 4 Expression in HT29, SW730, and Jurkat cell pellets
  • 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.
  • HT29 and SW780 tumor cells were implanted with T cells and harvested for IHC at Day 7 and 21 WSGR Docket No.47517-766.601 followed by paraffin embedding.
  • a heat-induced epitope retrieval method was applied.
  • 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 [00316]
  • 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 5 ⁇ g linearized expression vector plasmid DNA harboring ProTriTAC sequences comprising L040 or L276 protease cleavable linkers.
  • Stable pools were thawed, expanded in glutamine free media supplemented with 50 ⁇ M 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.
  • MSX methionine sulfoximine
  • CM Conditioned media
  • Table 12 Comparison of 10d harvest % viability and ProA titer of ProTriTAC sequences comprising L040 or L276 protease cleavable linkers.
  • SEQUENCES SEQ Sequence Seq. name Amino acid sequence ID descriptor NO.
  • Cleavage domain Cleavage NLL (SEQ IDENTIFIER A678) sequence domain recognized by sequence Cathepsin B 669 Cleavage domain Cleavage PIC(Et)FF sequence domain recognized by sequence Cathepsin D 670 Cleavage domain Cleavage GGPRGLPG sequence domain recognized by sequence Cathepsin K 671 Cleavage domain Cleavage HSSKLQ sequence domain recognized by sequence Prostate Specific Antigen 672 Cleavage domain Cleavage HSSKLQL sequence domain recognized by sequence Prostate Specific Antigen 673 Cleavage domain Cleavage HSSKLQEDA sequence domain recognized by sequence Prostate Specific Antigen 674 Cleavage domain Cleavage LVLASSSFGY sequence domain recognized by sequence Herpes Simplex Virus Protease 675 Cleavage domain Cleavage GVSQNYPIVG sequence domain recognized by sequence HIV Protease 676 Cleavage domain Cleavage GVVQAS
  • Exemplary AADISDPGG masking sequence 735 Exemplary ALSVDPSG masking sequence 736 Exemplary ARLSVDPG masking sequence 737 Exemplary AVEAADRG masking sequence 738 Exemplary GGPDGNEEMGGG masking sequence 739 Exemplary GGFDGNEEMGGG masking sequence 740 Exemplary GGGDGNEEMGGG masking sequence 741 Exemplary GGEMDGEGQNGG masking sequence 742 Exemplary GGGGGPDGNEEPGG masking sequence 743 Exemplary GGGGSLDGNEEPGG masking sequence 744 Exemplary GGGGALDGNEEPGG masking sequence 745 Exemplary GGGGGLDGNEEPGG masking sequence 746 Exemplary GGGALDGNEEPGG masking sequence 747 Exemplary GGGGGPDGNEEPGGG masking sequence 748 Exemplary GGSGALDGNEEPGG masking sequence 749 Exemplary GGSGSLDGNEEPGG masking sequence WSGR Docket No.47517-766.601 SEQ Sequence Seq.
  • Exemplary GGSGGPDGNEEPGG masking sequence 751 Exemplary GGVRDGPDGNEEPGG masking sequence 752
  • Exemplary GGSGGPDGNEEPGGGG masking sequence 753 Exemplary GGGRGPDGNEEPGG masking sequence 754
  • Exemplary GGSGGLDGNEEPGG masking sequence 755 Exemplary GGGVGPDGNEEPGG masking sequence 756
  • Exemplary GGGRALDGNEEPGG masking sequence 759 Exemplary GGYAGLDGNEEPGG masking sequence 760
  • Exemplary GGGGPDGNEEPGGGG masking sequence 763 Exemplary GGGEALDGNEEPGG masking sequence 764
  • Exemplary GGDASLDGNEEPGG masking sequence 765 Exemplary GGRDAPDGNEEGG masking sequence WSGR Docket No.47517-76

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Abstract

L'invention concerne des protéines de liaison à la NECTINE-4, des compositions pharmaceutiques comprenant de telles protéines ou des fragments de celles-ci, ainsi que des acides nucléiques, des vecteurs d'expression recombinants et des cellules hôtes pour fabriquer de telles protéines de liaison à la NECTINE-4. Sont également divulguées des méthodes d'utilisation des protéines de liaison à la NECTINE-4 divulguées dans la prévention et/ou le traitement de maladies, d'affections et de troubles.
EP23915124.4A 2023-01-03 2023-12-12 Protéine trispécifique ciblant la nectine-4 pour le traitement du cancer Pending EP4646267A1 (fr)

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