EpCAM-binding proteins and methods of use

Abstract

To provide EpCAM binding proteins, pharmaceutical compositions comprising such proteins or fragments thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such EpCAM binding proteins.SOLUTION: The present invention provides an EpCAM binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1, CDR2, and CDR3 each comprise a specific sequence.SELECTED DRAWING: Figure 21B

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 847,778, filed May 14, 2019, PCT Application No. PCT / US2019 / 032307, filed May 14, 2019, PCT Application No. CT / US2019 / 032224, filed May 14, 2019, PCT Application No. PCT / US2019 / US032302, filed May 14, 2019, and PCT Application No. PCT / US2019 / 032306, filed May 14, 2019, each of which is incorporated herein by reference in its entirety.

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

[0003] Studies have shown that epithelial cell adhesion / activation molecule (EpCAM / CD326) is expressed in essentially all human adenocarcinomas, certain squamous cell carcinomas, retinoblastomas, and hepatocellular carcinomas. See, e.g., Baeuerle PA, Gires O. EpCAM (CD326) finding its role in cancer. EpCAM is part of the signature of cancer-proliferating cells in many solid tumors and of normal progenitor and stem cells. See ibid. EpCAM has been implicated in proliferation, migration, and signaling in cancers, including breast cancer. See, e.g., Osta WA, Chen Y, Mikhitarian K, et al. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 2004;64:5818-24.

[0004] The anti-EpCAM antibody edrecolomab showed limited efficacy in a phase III trial. See Eyvazi S, Farajnia S, Dastmalchi S, Kanipour F, Zarredar H, Bandehpour M. Antibody Based EpCAM Targeted Therapy of Cancer, Review and Update. Curr Cancer Drug Targets. 2018;18(9):857-868. Other anti-EpCAM antibodies in use in various clinical trials include adecatumumab (fully human monoclonal antibody), catumaxomab (chimeric antibody), oportuzumab monatox (scFv antibody conjugated with Pseudomonas exotoxin A (ETA)), sitatuzumab bogatox (Fab fragment with bouganin toxin), and the immunoconjugated antibody tucotuzumab (monoclonal antibody with IL-2). Id.

[0005] There is a need for a greater selection of treatment options that will allow physicians to select the treatment with the best side effect profile for each individual patient. The present disclosure provides methods of treatment, particularly novel polypeptide and protein therapeutics useful for treating conditions associated with aberrant expression of EpCAM. Summary of the Invention

[0006] In one embodiment, an EpCAM-binding domain is provided comprising complementarity determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence selected from the group consisting of SEQ ID NOs: 39-76, with one or more substitutions; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or a sequence selected from the group consisting of SEQ ID NOs: 77-114, with one or more substitutions; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, the EpCAM-binding domain comprises an amino acid sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497.

[0007] In some embodiments, the EpCAM-binding domain is part of a multispecific protein. In some embodiments, the multispecific protein further comprises a CD3-binding domain. In some embodiments, the multispecific protein comprises an active drug format. In some embodiments, the multispecific protein further comprises a bulk serum protein-binding domain. In some embodiments, the bulk serum protein comprises a serum albumin protein. In some embodiments, the serum albumin protein comprises a human serum albumin protein. In some embodiments, the bulk serum protein-binding domain comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 378. In some embodiments, the CD3-binding domain comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 379. In some embodiments, the multispecific protein comprises a sequence at least about 75% identical to the sequence set forth in SEQ ID NO: 492. In some embodiments, the bulk serum protein-binding domain comprises a binding moiety comprising a linker and a masking moiety, wherein the masking moiety is capable of masking binding of the EpCAM-binding domain or the CD3-binding domain to its respective target. In some embodiments, the multispecific protein comprises a non-cleavable prodrug format. In some embodiments, the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOs: 380-424, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 380-424. In some embodiments, the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-507, and 581, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-507, and 581. In some embodiments, the bulk serum protein binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the CD3 binding domain comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 474.

[0008] In some embodiments, the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 495, 498-502, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 495 and 502. In some embodiments, the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 498-501, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the active agent comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 153-179, 180-206, 210-212, 494, 571, and 574.

[0009] In some embodiments, the EpCAM-binding domain is part of a chimeric antigen receptor (CAR) or a conditionally activatable chimeric antigen receptor (ProCAR), where the CAR further comprises at least one of a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In some embodiments, the EpCAM-binding domain is part of a ProCAR, where the ProCAR further comprises (a) a binding moiety comprising a non-CDR loop and a cleavable linker, (b) a transmembrane domain, and (c) an intracellular signaling domain, where the binding moiety can mask binding of the EpCAM-binding domain to its target. In some embodiments, the binding moiety further comprises one or more complementarity-determining regions (CDRs). In some embodiments, the CDR loops provide specific binding sites for bulk serum proteins. In some embodiments, the bulk serum proteins include at least one of serum albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, and pentameric IgM. In some embodiments, the bulk serum protein includes serum albumin. In some embodiments, the serum albumin is human serum albumin. In some embodiments, ProCAR further comprises a costimulatory domain, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the at least one but not more than 20 modifications comprise modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to the encoded T cell receptor fusion protein.In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR zeta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, 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 no more than 20 modifications. In some embodiments, the intracellular signaling domain is derived from CD3 epsilon, CD3 gamma, CD3 delta, CD3 alpha, CD3 beta, or a combination thereof. In some embodiments, the EpCAM-binding domain is a portion of ProCAR, and ProCAR comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 485-491.

[0010] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject an EpCAM-binding domain according to the present disclosure, or a pharmaceutical composition comprising the EpCAM-binding domain. In some embodiments, the subject is a human.

[0011] One embodiment provides a conditionally active chimeric antigen receptor (ProCAR) comprising a single polypeptide chain, the chimeric antigen receptor (ProCAR) comprising: (a) a binding moiety comprising a non-CDR loop and a cleavable linker; (b) an EpCAM-binding domain, wherein the EpCAM-binding domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 39-76; (c) an EpCAM-binding domain, (d) an intracellular signaling domain, wherein CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114 or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 77-114, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152 or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 115-152, wherein the binding moiety is capable of masking binding of the EpCAM-binding domain to its target. In some embodiments, the EpCAM-binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497.

[0012] One embodiment provides a conditionally active EpCAM-binding protein comprising a binding moiety (M) comprising 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 EpCAM-binding domain, the EpCAM-binding domain comprising complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence having one or more amino acids selected from the group consisting of SEQ ID NOs: 39-76. In some embodiments, the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114 or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 77-114, and the CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152 or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 115-152, wherein the non-CDR loops are capable of binding to the EpCAM-binding domain or a second target antigen-binding domain, and the binding moiety is capable of masking binding of the EpCAM-binding domain or the second target antigen-binding domain to its target. In some embodiments, the binding moiety comprises a masking moiety, wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOs: 380-424 or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 380-424. In some embodiments, the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581, or a sequence containing one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581. In some embodiments, the binding moiety comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the second target antigen binding domain (T2) comprises a CD3 binding domain. In some embodiments, the CD3 binding domain comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 474.

[0013] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a conditionally active chimeric antigen receptor according to the present disclosure, or a pharmaceutical composition comprising the chimeric antigen receptor. In some embodiments, the subject is a human. One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a conditionally active EpCAM binding protein according to the present disclosure, or a pharmaceutical composition comprising the EpCAM binding protein. In some embodiments, the subject is a human.

[0014] In some embodiments, the binding domain is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the binding domain is a single-domain antibody, a VHH domain, an scFv, a VH domain, a VL domain, a Fab, a Fab', a non-Ig domain, a ligand, a knottin, or a small molecule entity. In some embodiments, the binding domain comprises a single-domain antibody. In some embodiments, the binding domain binds to EpCAM with a binding affinity (Kd) of about 0.001 nM to about 500 nM. In some embodiments, the binding domain binds to human EpCAM, mouse EpCAM, cynomolgus EpCAM, or a combination thereof. One embodiment provides a multispecific protein comprising an EpCAM-binding domain, wherein the EpCAM-binding domain is according to the present disclosure. In some embodiments, the multispecific protein comprises an EpCAM-binding domain (anti-EpCAM domain) and a CD3-binding domain (anti-CD3 domain) according to the present disclosure. In some embodiments, the anti-EpCAM domain and the anti-CD3 domain are in an anti-EpCAM:anti-CD3 orientation. In some embodiments, the anti-EpCAM domain and the anti-CD3 domain are in an anti-CD3:anti-EpCAM orientation. In some embodiments, the EpCAM binding domain (anti-EpCAM domain), the CD3 binding domain (anti-CD3 domain), and the albumin binding domain (anti-ALB domain) according to any one of claims 1-3 and 49-53. In some embodiments, the anti-CD3 domain comprises the amino acid sequence set forth in SEQ ID NO: 379 or SEQ ID NO: 474. In some embodiments, the anti-ALB domain comprises the amino acid sequence set forth in SEQ ID NO: 375, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 482, or SEQ ID NO: 483. In some embodiments, the anti-EpCAM domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3:anti-ALB:anti-EpCAM orientation. In some embodiments, the anti-EpCAM domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-EpCAM:anti-ALB:anti-CD3 orientation. In some embodiments, the anti-EpCAM domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB:anti-EpCAM:anti-CD3 orientation.In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-CD3:anti-EpCAM:anti-ALB orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-ALB:anti-CD3:anti-EpCAM orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-EpCAM:anti-CD3:anti-ALB orientation.

[0015] One embodiment provides a multivalent protein comprising a sequence set forth in any one of SEQ ID NOs: 495, 498-502, 569-570, 572, 573, 575, 576, 577, and 578. One embodiment provides an active agent comprising a sequence set forth in any one of SEQ ID NOs: 494, 571, and 574. One embodiment provides a multivalent protein comprising a sequence set forth in any one of SEQ ID NOs: 485-491.

[0016] One embodiment provides a pharmaceutical composition comprising: (i)(a) an EpCAM-binding domain according to the present disclosure; (i)(b) a conditionally active chimeric antigen receptor according to the present disclosure; (i)(c) a conditionally active EpCAM-binding protein according to the present disclosure; (i)(d) a multispecific protein according to the present disclosure; (i)(e) a multivalent protein according to the present disclosure; or (i)(f) an active agent according to the present disclosure; and (ii) a pharmaceutically acceptable carrier.

[0017] One embodiment provides a process for producing an EpCAM binding domain according to the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding an EpCAM binding domain according to the present disclosure under conditions allowing expression of the EpCAM binding domain, and recovering and purifying the protein produced from the culture.

[0018] One embodiment provides a process for producing a multispecific protein according to the present disclosure, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of a multispecific EpCAM binding protein according to the present disclosure under conditions allowing expression of the multispecific protein, and recovering and purifying the protein produced from the culture.

[0019] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, comprising administering to a subject an EpCAM-binding domain according to the present disclosure or a pharmaceutical composition comprising the EpCAM-binding domain. One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, comprising administering to a subject a multispecific protein according to the present disclosure, a multivalent protein according to the present disclosure, an active agent according to the present disclosure, or a pharmaceutical composition according to the present disclosure. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering an agent in combination with the EpCAM-binding domain according to the present disclosure, a multispecific protein according to the present disclosure, a multivalent protein according to the present disclosure, an active agent according to the present disclosure, or a pharmaceutical composition according to the present disclosure. In some embodiments, the EpCAM-binding domain selectively binds to tumor cells that express EpCAM. In some embodiments, the neoplastic disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the neoplastic disease comprises at least one of colon cancer, prostate cancer, neuroendocrine cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, esophageal cancer, breast cancer, and adenocarcinoma. In some embodiments, the method further comprises administering an agent in combination with a conditionally active chimeric antigen receptor according to the present disclosure, a conditionally active EpCAM binding protein according to the present disclosure, or a pharmaceutical composition comprising the same. In some embodiments, the EpCAM-binding domain selectively binds to tumor cells that express EpCAM. In some embodiments, the neoplastic disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the neoplastic disease is at least one of colon cancer, prostate cancer, neuroendocrine cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, esophageal cancer, breast cancer, and adenocarcinoma.

[0020] One embodiment provides a process for producing a conditionally active chimeric antigen receptor according to the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding a conditionally active chimeric antigen receptor according to the present disclosure under conditions allowing for expression of the conditionally active chimeric antigen receptor according to the present disclosure, and recovering and purifying the protein produced from the culture.One embodiment provides a process for producing a conditionally active EpCAM binding protein according to the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of a conditionally active EpCAM binding protein according to the present disclosure under conditions allowing for expression of the conditionally active EpCAM binding protein according to the present disclosure, and recovering and purifying the protein produced from the culture. One embodiment provides a process for producing a multivalent protein according to the present disclosure, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of a multivalent protein according to the present disclosure under conditions allowing for expression of the multivalent protein according to the present disclosure, and recovering and purifying the protein produced from the culture.One embodiment provides a process for producing an active agent according to the present disclosure, said process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of an active agent according to the present disclosure under conditions allowing for expression of the active agent according to the present disclosure, and recovering and purifying the drug produced from the culture.

[0021] One embodiment provides a cell comprising a CAR according to the present disclosure. One embodiment provides a cell comprising a ProCAR according to the present disclosure. In some embodiments, the cell is a T cell or an NK cell. One embodiment includes a method of transfecting a cell according to the present disclosure with a vector or RNA comprising a nucleotide sequence encoding a CAR or ProCAR.

[0022] One embodiment provides a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor has a greater therapeutic index compared to a chimeric antigen receptor (CAR) that does not include the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor. In some embodiments, the conditionally active chimeric antigen receptor has a therapeutic index that is at least about 5- to about 100-fold greater than the therapeutic index of a chimeric antigen receptor (CAR) that does not include the (a) binding moiety but is otherwise identical to the conditionally active chimeric antigen receptor. In some embodiments, the protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 498-501, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the protein comprises a sequence that is at least about 75% identical to the sequence of SEQ ID NO: 576. In some embodiments, the protein comprises the sequence set forth in SEQ ID NO: 576. In some embodiments, the conditionally active EpCAM binding protein has a greater therapeutic index than an EpCAM binding protein that does not include the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active EpCAM binding protein. In some embodiments, the conditionally active EpCAM binding protein has a therapeutic index that is at least about 5-fold to about 100-fold greater than an EpCAM binding protein that does not include the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active EpCAM binding protein.

[0023] One embodiment provides a pharmaceutical composition comprising: (i)(a) a conditionally active chimeric antigen receptor according to the present disclosure, or (i)(b) a conditionally active EpCAM-binding protein according to the present disclosure, and (ii) a pharmaceutically acceptable carrier. One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a conditionally active chimeric antigen receptor according to the present disclosure, or a pharmaceutical composition comprising the conditionally active chimeric antigen receptor.

[0024] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a conditionally active EpCAM binding protein according to the present disclosure or a pharmaceutical composition comprising the conditionally active EpCAM binding protein. In some embodiments, the subject is a human. In some embodiments, the neoplastic disease comprises at least one of colon cancer, prostate cancer, neuroendocrine cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, esophageal cancer, breast cancer, and adenocarcinoma.

[0025] One embodiment provides a method of increasing the therapeutic index of an EpCAM-binding domain, said method comprising conjugating the EpCAM-binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop; the non-CDR loop comprises a binding site specific for the EpCAM-binding domain, - the EpCAM-binding domain is masked from binding to its target by a binding moiety; -The EpCAM-binding domain is capable of binding to its target once the cleavable linker is cleaved.

[0026] In some embodiments, the EpCAM-binding domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 39-76; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 77-114; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, the EpCAM-binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, wherein the multispecific protein further comprises a CD3-binding domain. In some embodiments, the binding moiety comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 379 and 474. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581.

[0027] In some embodiments, the EpCAM-binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 498-501, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 75% identical to SEQ ID NO: 576. In some embodiments, the conditionally active multispecific protein comprises the sequence set forth in SEQ ID NO: 576. In some embodiments, the EpCAM-binding domain conjugated to the binding moiety is part of a conditionally active chimeric antigen receptor, and the conditionally active chimeric antigen receptor further comprises at least one of a transmembrane domain, an intracellular signaling domain, and a costimulatory domain. In some embodiments, the EpCAM-binding domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 39-76; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 77-114; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, the binding moiety comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the EpCAM-binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497. In some embodiments, the conditionally active chimeric antigen receptor comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 485-491.

[0028] One embodiment provides a method of increasing the therapeutic index of an EpCAM-binding protein comprising 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 an EpCAM-binding domain, the method comprising conjugating the first target antigen-binding domain or the second target antigen-binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop; the non-CDR loop comprises a binding site specific for the first target antigen-binding domain or the second target antigen-binding domain; - at least one of the first target antigen-binding domain or the second target antigen-binding domain is masked from binding of its target by a binding moiety, and the first target antigen-binding domain or the second target antigen-binding domain to be masked is capable of binding to its target upon cleavage of the cleavable linker.

[0029] In some embodiments, the EpCAM-binding domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 39-76; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 77-114; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, the non-CDR loop comprises a binding site specific for the EpCAM-binding domain. In some embodiments, at least one of the first target antigen-binding domain 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 at least about 75% identical to SEQ ID NO: 474. In some embodiments, the binding moiety comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the EpCAM-binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 498-501, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to SEQ ID NO: 576. In some embodiments, the conditionally active multispecific protein comprises the sequence set forth in SEQ ID NO: 576.

[0030] One embodiment provides a method for increasing the therapeutic index of an EpCAM-binding protein comprising an EpCAM-binding domain and a CD3-binding domain, the method comprising conjugating the 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 the CD3-binding domain. In some embodiments, the EpCAM-binding domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 39-76, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 77-114, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, the CD3-binding domain comprises a sequence at least about 75% identical to SEQ ID NO: 474. In some embodiments, the binding moiety comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the EpCAM-binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 498-501, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to SEQ ID NO: 576. In some embodiments, the conditionally active multispecific protein comprises the sequence set forth in SEQ ID NO: 576.

[0031] One embodiment provides a conditionally active multispecific protein that targets EpCAM, comprising an EpCAM-binding domain, a CD3-binding domain, and an albumin-binding domain, wherein the albumin-binding domain comprises a non-CDR loop comprising a binding site specific for the CD3-binding domain and a cleavable linker, and the EpCAM-binding domain comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 39-76, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 77-114, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 115-152. In some embodiments, the albumin binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the EpCAM binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38, 207-209, and 496-497. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581, or a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, and 581. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 498-501, 569-570, 572, 573, 575, 576, 577, and 578. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 75% identical to SEQ ID NO: 576. In some embodiments, the conditionally active multispecific protein comprises the sequence set forth in SEQ ID NO: 576.

[0032] One embodiment provides a pharmaceutical composition comprising a conditionally active multispecific protein that targets EpCAM of the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. One embodiment provides a process for producing a conditionally active multispecific protein that targets EpCAM of the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding domains of a conditionally active multispecific protein that targets EpCAM of the present disclosure under conditions allowing expression of the conditionally active multispecific protein that targets EpCAM of the present disclosure, and recovering and purifying the protein produced from the culture.

[0033] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, comprising administering to a subject a conditionally active multispecific protein targeting EpCAM of the present disclosure or a pharmaceutical composition of claim 149 or 150. In some embodiments, the neoplastic disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. In some embodiments, the neoplastic disease comprises at least one of colon cancer, prostate cancer, neuroendocrine cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, esophageal cancer, breast cancer, and adenocarcinoma.

[0034] One embodiment provides an EpCAM-binding domain comprising a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38. In some embodiments, the EpCAM-binding domain comprises CDR1, CDR2, and CDR3. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 39-76, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 39-76. In some embodiments, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 77-114, or one or more amino acid substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 77-114. In some embodiments, CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-152, or one or more substitutions relative to a sequence selected from SEQ ID NOs: 115-152. In some embodiments, the EpCAM-binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-38. In some embodiments, the EpCAM-binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 1-38. In some embodiments, the EpCAM-binding domain is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the EpCAM-binding domain is a single-domain antibody, a VHH domain, an scFv, a VH domain, a VL domain, a Fab, a Fab', a non-Ig domain, a ligand, a knottin, or a small molecule entity. In some embodiments, the EpCAM-binding domain comprises a single-domain antibody. In some embodiments, the binding domain binds to EpCAM with a binding affinity (Kd) of about 0.001 nM to about 500 nM. In some embodiments, the EpCAM-binding domain binds to human EpCAM, mouse EpCAM, cynomolgus EpCAM, or a combination thereof.

[0035] One embodiment provides a multispecific protein comprising an EpCAM-binding domain, wherein the EpCAM-binding domain is according to the present disclosure. In some embodiments, the multispecific protein comprises an EpCAM-binding domain (anti-EpCAM domain) and a CD3-binding domain (anti-CD3 domain) according to the present disclosure. In some embodiments, the anti-EpCAM domain and the anti-CD3 domain are in an anti-EpCAM:anti-CD3 orientation. In some embodiments, the anti-EpCAM domain and the anti-CD3 domain are in an anti-CD3:anti-EpCAM orientation. In some embodiments, the multispecific protein comprises an EpCAM-binding domain (anti-EpCAM domain), a CD3-binding domain (anti-CD3 domain), and an albumin-binding domain (anti-ALB domain) according to the present disclosure. In some embodiments, the anti-CD3 domain comprises the amino acid sequence set forth in SEQ ID NO:379 or SEQ ID NO:474. In some embodiments, the anti-ALB domain comprises the amino acid sequence set forth in SEQ ID NO:375, SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:482, or SEQ ID NO:483. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-CD3:anti-ALB:anti-EpCAM orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-EpCAM:anti-ALB:anti-CD3 orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-ALB:anti-EpCAM:anti-CD3 orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-ALB:anti-EpCAM:anti-CD3 orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-CD3:anti-EpCAM:anti-ALB orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-ALB:anti-CD3:anti-EpCAM orientation. In some embodiments, the anti-EpCAM domain, anti-CD3 domain, and anti-ALB domain are in an anti-EpCAM:anti-CD3:anti-ALB orientation.

[0036] One embodiment provides a multivalent protein comprising a sequence set forth in any one of SEQ ID NOs: 485- 491. One embodiment provides a pharmaceutical composition comprising: (i)(a) an EpCAM-binding domain according to the present disclosure, (i)(b) a multispecific protein according to the present disclosure, or (i)(c) a multivalent protein according to the present disclosure, and (ii) a pharmaceutically acceptable carrier.

[0037] One embodiment provides a process for producing an EpCAM-binding domain according to the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding an EpCAM-binding domain according to the present disclosure under conditions allowing for expression of the EpCAM-binding domain according to the present disclosure, and recovering and purifying the protein produced from the culture.One embodiment provides a process for producing a multispecific protein according to the present disclosure, the process comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding domains of a multispecific EpCAM-binding protein according to the present disclosure under conditions allowing for expression of the multispecific protein, and recovering and purifying the protein produced from the culture.

[0038] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject an EpCAM binding domain according to the present disclosure, or a pharmaceutical composition according to the present disclosure.

[0039] One embodiment provides a method for treating or ameliorating a proliferative or neoplastic disease, the method comprising administering to a subject a multispecific protein according to this disclosure, a multivalent protein according to this disclosure, or a pharmaceutical composition according to this disclosure. In some embodiments, the subject is human. In some embodiments, the method further comprises administering an agent in combination with an EpCAM binding domain according to this disclosure, a multispecific protein according to this disclosure, a multivalent protein according to this disclosure, or a pharmaceutical composition according to this disclosure. In some embodiments, the EpCAM binding domain selectively binds to tumor cells that express EpCAM. In some embodiments, the neoplastic disease comprises a solid tumor disease. In some embodiments, the solid tumor disease is metastatic. [Brief explanation of the drawings]

[0040] The novel features of the invention are set forth with particularity in the appended claims. For a better understanding of the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Figure 1] 1 provides the results of a representative T cell-dependent cytotoxicity assay with NCI-H508 cells using an exemplary fusion protein of the disclosure comprising an anti-EpCAM domain and an anti-CD3 domain as described herein. [Figure 2] 1 provides the results of a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of the disclosure comprising the anti-EpCAM domain and anti-CD3 domain described herein. [Figure 3] 1 provides the results of a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of the disclosure comprising the anti-EpCAM domain and anti-CD3 domain described herein. [Figure 4] 1 provides the results of a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of the disclosure comprising the anti-EpCAM domain and anti-CD3 domain described herein. [Figure 5]1 provides the results of a representative T cell-dependent cytotoxicity assay using an exemplary fusion protein of the disclosure comprising the humanized anti-EpCAM domain and anti-CD3 domain described herein. [Figure 6] Exemplary ProCAR constructs are illustrated. One exemplary construct comprises an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 485). One exemplary construct comprises an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 486). One exemplary construct comprises an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 487). One exemplary construct comprises an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 488). One exemplary construct comprises an anti-human serum albumin sdAb, protease cleavage site 3, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 489). One exemplary construct comprises an anti-human serum albumin sdAb, protease cleavage site 3, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 490). One exemplary construct comprises an anti-GFP sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 491). [Figure 7] Demonstrates steric blocking of anti-EpCAM sdAb H90 by HSA for the indicated constructs at CAR-T:target cell ratios of 10:1, 5:1, 2.5:1, and 1.25:1. [Figure 8]

[0033] Figure 1 provides histograms of EpCAM-Fc / Alexa Fluor 647 staining of CAR-T cells from Figure 6 categorized as low (A), medium (B), or high (C) CAR expression based on anti-FLAG staining demonstrating the effectiveness of EpCAM mask 1 in blocking ProCAR EpCAM binding activity. Numbers refer to sequence identifiers (e.g., 491 = SEQ ID NO: 491). [Figure 9]

[0033] Figure 1 provides histograms of EpCAM-Fc / Alexa Fluor 647 staining of CAR-T cells from Figure 6 categorized as low (A), medium (B), or high (C) CAR expression based on anti-FLAG staining demonstrating the effectiveness of EpCAM mask 2 in blocking ProCAR EpCAM binding activity. Numbers refer to sequence identifiers (e.g., 488 = SEQ ID NO: 488). [Figure 10] 10:1, 5:1, 2.5:1, and 1.25:1 CAR-T:target cell ratios of 10:1, 5:1, 2.5:1, and 1.25:1 demonstrate masking of anti-EpCAM sdAb H90 by the constructs in FIG. 6 . [Figure 11] Demonstrates protease site-dependent activation of EpCAM ProCAR mask 2 cell killing activity. [Figure 12] Figure 1 illustrates protease activation of EpCAM mask 1 ProCAR antigen binding activity at low (A), medium (B), or high (C) CAR expression levels based on anti-FLAG staining. Numbers refer to sequence identifiers (e.g., 489 = SEQ ID NO: 489). [Figure 13] Figure 1 illustrates protease activation of EpCAM mask 2 ProCAR antigen binding activity at low (Figure A), medium (Figure B), or high (Figure C) CAR expression levels based on anti-FLAG. Numbers refer to sequence identifiers (e.g., 485 = SEQ ID NO: 485). [Figure 14A] 14A-14C show the percent change in body weight in mice following administration of exemplary EpCAM ProTriTAC molecules of the present disclosure (EpCAM ProTriTAC with linker L040 in FIG. 14B; EpCAM ProTriTAC with a non-cleavable linker in FIG. 14C), and EpCAM TriTAC molecules (FIG. 14A). [Figure 14B]14A-14C show the percent change in body weight in mice following administration of exemplary EpCAM ProTriTAC molecules of the present disclosure (EpCAM ProTriTAC with linker L040 in FIG. 14B; EpCAM ProTriTAC with a non-cleavable linker in FIG. 14C), and EpCAM TriTAC molecules (FIG. 14A). [Figure 14C] 14A-14C show the percent change in body weight in mice following administration of exemplary EpCAM ProTriTAC molecules of the present disclosure (EpCAM ProTriTAC with linker L040 in FIG. 14B; EpCAM ProTriTAC with a non-cleavable linker in FIG. 14C), and EpCAM TriTAC molecules (FIG. 14A). [Figure 15A] Binding kinetics are shown for various EpCAM binding domains; H13 (FIG. 15A), H90 (FIG. 15B), and H90.2 (FIG. 15C). [Figure 15B] Binding kinetics are shown for various EpCAM binding domains; H13 (FIG. 15A), H90 (FIG. 15B), and H90.2 (FIG. 15C). [Figure 15C] Binding kinetics are shown for various EpCAM binding domains; H13 (FIG. 15A), H90 (FIG. 15B), and H90.2 (FIG. 15C). [Figure 16A] 16A, 16B, and 16C show the results of a T-cell-dependent cytotoxicity assay (TDCC assay) on HCT116 cells using ProTriTAC, TriTAC proteins containing the EpCAM-binding domains H13 (FIG. 16A), H90.2 (FIG. 16B), and H138.2 (FIG. 16C), or active drug (CT). [Figure 16B] 16A, 16B, and 16C show the results of a T-cell-dependent cytotoxicity assay (TDCC assay) on HCT116 cells using ProTriTAC, TriTAC proteins containing the EpCAM-binding domains H13 (FIG. 16A), H90.2 (FIG. 16B), and H138.2 (FIG. 16C), or active drug (CT). [Figure 16C]16A, 16B, and 16C show the results of a T-cell-dependent cytotoxicity assay (TDCC assay) on HCT116 cells using ProTriTAC, TriTAC proteins containing the EpCAM-binding domains H13 (FIG. 16A), H90.2 (FIG. 16B), and H138.2 (FIG. 16C), or active drug (CT). [Figure 17A] 17A, 17B, and 17C show the results of a T-cell-dependent cytotoxicity assay (TDCC assay) on NCI-H929 cells using ProTriTAC, TriTAC proteins containing the EpCAM-binding domains H13 (FIG. 17A), H90.2 (FIG. 17B), and H138.2 (FIG. 17C), or active drug (CT). [Figure 17B] 17A, 17B, and 17C show the results of a T-cell-dependent cytotoxicity assay (TDCC assay) on NCI-H929 cells using ProTriTAC, TriTAC proteins containing the EpCAM-binding domains H13 (FIG. 17A), H90.2 (FIG. 17B), and H138.2 (FIG. 17C), or active drug (CT). [Figure 17C] 17A, 17B, and 17C show the results of a T-cell-dependent cytotoxicity assay (TDCC assay) on NCI-H929 cells using ProTriTAC, TriTAC proteins containing the EpCAM-binding domains H13 (FIG. 17A), H90.2 (FIG. 17B), and H138.2 (FIG. 17C), or active drug (CT). [Figure 18A] The results of a T cell-dependent cytotoxicity assay (TDCC assay) using TriTAC containing the EpCAM-binding domains H13 and H90.2 against HCT116 cells (FIG. 18A) and HCT116 (EpCAM-knockout; KO) (FIG. 18B) are shown. [Figure 18B] The results of a T cell-dependent cytotoxicity assay (TDCC assay) using TriTAC containing the EpCAM-binding domains H13 and H90.2 against HCT116 cells (FIG. 18A) and HCT116 (EpCAM-knockout; KO) (FIG. 18B) are shown. [Figure 19]FIG. 1 shows the results of a flow cytometry assay to measure binding of EpCAM-binding domains H13 and H90.2 to wild-type (WT) and HCT116 (EpCAM-knockout; KO) cells. [Figure 20] 1 shows the results of a TDCC assay on SKBR3 cells using non-cleavable prodrug or active drug (CT) versions containing the EpCAM binding domain H13 or H90.2. [Figure 21A] Representative plots are illustrated demonstrating the masking effect achieved by non-cleavable prodrug versions containing the EpCAM-binding domain H13 (Figure 21A) or H90.2 (Figure 21B) compared to active drugs containing the same. [Figure 21B] Representative plots are illustrated demonstrating the masking effect achieved by non-cleavable prodrug versions containing the EpCAM-binding domain H13 (Figure 21A) or H90.2 (Figure 21B) compared to active drugs containing the same. [Figure 22A] Figure 22 shows the results of TDCC assays on various cell lines using the non-cleavable prodrug (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22A shows the results for CAPAN2 cells. [Figure 22B] Figure 22B shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22B shows the results for DMS53 cells. [Figure 22C] Figure 22C shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22C shows the results for HepG2 cells. [Figure 22D]Figure 22D shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22C shows the results for KMRC3 cells. [Figure 22E] Figure 22B shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22C shows the results for MDAPCA2b cells. [Figure 22F] Figure 22F shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22F shows the results for OVCAR8 cells. [Figure 22G] Figure 22G shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22G shows the results for PECAPJ41 cells. [Figure 22H] Figure 22 shows the results of TDCC assays on various cell lines using non-cleavable prodrugs (NCLV), ProTriTAC (L040), or active drug (CT) versions containing the EpCAM-binding domains H13 or H90.2. Figure 22H shows the results for SKBR3 cells. [Figure 23A] The efficacy of ProTriTAC (Figure 23B) and TriTAC (Figure 23A) versions containing the EpCAM binding domain H13 in mouse tumor models is shown. [Figure 23B] The efficacy of ProTriTAC (Figure 23B) and TriTAC (Figure 23A) versions containing the EpCAM binding domain H13 in mouse tumor models is shown. [Figure 24] Figure 1 shows cytokine profiles in cynomolgus monkeys after administration of versions of ProTriTAC and TriTAC containing the EpCAM-binding domain H13. A illustrates IFN-γ levels, B illustrates IL-2 levels, C illustrates IL-6 levels, and D illustrates IL-10 levels. [Figure 25] Plasma concentrations after administration of ProTriTAC and TriTAC versions containing the EpCAM binding domain H13 (A) or H90.2 (B) in cynomolgus monkeys are shown. [Figure 26] The variable domain of an immunoglobulin molecule is illustrated, including the complementarity determining regions (CDR1, CDR2, and CDR3) and the non-CDR loops connecting the β strands (AB, CC', C"D, EF, and DE). [Figure 27] 1 illustrates an exemplary arrangement of a target antigen binding domain (aTarget 1), a cleavable linker, and a binding site (aTarget 2) of the present disclosure. [Figure 28] 1 illustrates an example of a conditionally active receptor described herein. [Figure 29A] Figure 29 shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29A shows the results for control TriTAC. [Figure 29B] Figure 29B shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29B shows the results for EpCAM TriTAC at 0.003 mg / kg. [Figure 29C] Figure 29C shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29C shows the results for EpCAM TriTAC at 0.01 mg / kg. [Figure 29D]Figure 29D shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29D shows the results for EpCAM TriTAC at 0.03 mg / kg. [Figure 29E] Figure 29E shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29E shows the results for EpCAM TriTAC at 0.1 mg / kg. [Figure 29F] Figure 29F shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29F shows the results for EpCAM TriTAC at 0.1 mg / kg. [Figure 29G] Figure 29G shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29G shows the results for EpCAM ProTriTAC at 0.03 mg / kg. [Figure 29H] Figure 29H shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29H shows the results for EpCAM ProTriTAC at 0.1 mg / kg. [Figure 29I] Figure 29I shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29I shows the results for EpCAM ProTriTAC at 0.3 mg / kg. [Figure 29J] Figure 29J shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29J shows the results for EpCAM ProTriTAC at 1 mg / kg. [Figure 29K] Figure 29 shows the efficacy of control TriTAC, EpCAM ProTriTAC, and EpCAM TriTAC proteins in an established LoVo (colon cancer) tumor model. Figure 29K shows the results for EpCAM ProTriTAC at 3 mg / kg. [Figure 30] Shown are survival rates (A-B), alanine transaminase (ALT) (C), aspartate transaminase (AST) (D), and total bilirubin (E) levels after administration of ProTriTAC or TriTAC versions containing the EpCAM-binding domain. [Figure 31] Results and summaries of histopathological studies using control GFP TriTACs, EpCAM TriTACs, or EpCAM ProTriTACs are shown. [Figure 32] 1 provides a schematic diagram of a trispecific protein that targets EpCAM. DETAILED DESCRIPTION OF THE INVENTION

[0041] Described herein are EpCAM-targeting trispecific proteins, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the disclosed EpCAM-targeting trispecific proteins in the prevention and / or treatment of diseases, illnesses, and disorders. The EpCAM-targeting trispecific proteins can specifically bind to EpCAM, similar to CD3, and have a half-life-extending domain, such as a domain that binds to human albumin (ALB). Figure 32 depicts one non-limiting example of a trispecific EpCAM-binding protein.

[0042] Specific Definitions The terms used herein are for the purpose of describing particular instances only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof, are used in either the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising."

[0043] The terms "about" or "approximately" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean 1 or more than 1 standard deviation per practice for any value. When particular values ​​are described in this application and in the claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0044] The terms "individual," "patient," or "subject" are used interchangeably. None of the terms require or are limited to a condition characterized by the supervision (e.g., continuous or intermittent) of a health care practitioner (e.g., a physician, registered nurse, nurse practitioner, physician assistant, janitor, or hospice worker).

[0045] The term "antibody" typically refers to a Y-shaped tetrameric protein containing two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and noncovalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), which can be easily classified as kappa or lambda based on their amino acid sequence and genetic locus. Interchain disulfide bonds are located on the surface of the immunoglobulin, are solvent accessible, and are usually relatively easily reduced. In the human IgG1 isotype, there are four interchain disulfide bonds: one from each heavy chain to the light chain and two between heavy chains. Interchain disulfide bonds are not required for chain association. As is well known, the cysteine-rich IgG1 hinge region of the heavy chain is generally maintained as consisting of three parts: the upper hinge, the core hinge, and the lower hinge. Those skilled in the art will appreciate that the IgG1 hinge region contains cysteines in the heavy chain that comprise the interchain disulfide bonds (two heavy / heavy, two heavy / light), providing structural flexibility that facilitates Fab movement. The interchain disulfide bond between the light and heavy chains of IgG1 is formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain. The interchain disulfide bond between the heavy chains is at positions C226 and C229 (all numbered according to the EU index according to Kabat, et al., below).

[0046] As used herein, the term "antibody" includes polyclonal antibodies, multiclonal antibodies, monoclonal antibodies, chimeric antibodies, deimmunized antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies (e.g., monovalent IgG), multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, immunospecific antibody fragments such as hcIgG, V-NAR, Fv, Fd, Fab, F(ab'), F(ab'), Fab2, Fab3 fragments, single chain fragments (e.g., di-scFv, scFv, scFvFc, scFv-zipper, scFab), disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (VH, VL, or VHH domains), "r" antibodies, including mutations and variants thereof. Nanobodies or single variable domain antibodies that contain only one variable domain such as "IgG" ("half antibodies"), diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFvs, tandem tri-scFvs, "minibodies", in some examples exemplified by the structures shown below: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3), or (scFv-CH3-scFv)2, multibodies such as triabodies or tetrabodies; and derivatives thereof, including Fc fusions and other modifications, and any other immunoreactive molecule (as long as it contains a domain with a binding site for preferential association with or binding to the EpCAM protein). Furthermore, unless contextual constraints dictate otherwise, the term further includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy chain constant domains corresponding to the various classes of antibodies are typically designated by the corresponding lowercase Greek letters α, δ, ε, γ, and μ, respectively.The light chains of antibodies of any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. In some embodiments, the EpCAM-binding protein comprises a heavy chain-only antibody, such as a VH domain or a VHH domain. In some cases, the EpCAM-binding protein comprises a heavy chain-only antibody that is an engineered human VH domain. In some examples, the engineered human VH domain is generated by panning a phage display library. In some embodiments, the EpCAM-binding protein comprises a VHH. The term "VHH" as used herein refers to a single-chain antibody binding domain lacking a light chain. In some cases, the VHH is derived from antibodies of the type found in camelids or cartilaginous fish that naturally lack light chains, or from synthetic non-immune VHHs that can be constructed accordingly. Each heavy chain comprises a variable region encoded by V, D, and J exons. The VHH may optionally be a naturally occurring VHH, e.g., a VHH from the Camelidae family, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camel, llama, vicuña, guanaco, and cartilaginous fish (such as, but not limited to, shark). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, Huacaya alpaca and Suri alpaca).

[0047] As used herein, the terms "variable region" or "variable domain" refer to the fact that certain portions of the variable domain differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FR regions adopting a β-sheet structure, connected by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together 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 Institutes 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 amino acid assignments for each domain, framework region, and CDR, in some embodiments, follow one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID:3681981; Chothia et al., 1989, PMID:2687698; MacCallum et al., 1996, PMID:8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co. or AbM (Oxford Molecular / MSI Pharmacopia), unless otherwise specified. In some embodiments of the present disclosure, the EpCAM-binding protein comprises a heavy chain-only antibody, such as a VH domain or a VHH domain, and comprises three CDRs. Such heavy chain-only antibodies, in some embodiments, bind EpCAM as a monomer that does not rely on dimerization with the VL (variable light chain) region for optimal binding affinity.

[0048] "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to omissions from, or insertions into, FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the homologous regions of the antibody's sequence with sequences using "standard" Kabat numbering. The CDRs of this disclosure are not intended to necessarily correspond to the Kabat numbering convention.

[0049] The terms "framework" or "FR" residues (or regions) refer to variable domain residues other than the CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0050] As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. An antigen may have more than one epitope. Thus, different antibodies may bind to different sites on an antigen and have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are formed by spatially juxtaposed amino acids in different segments of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphate, or sulfonyl moieties on an antigen.

[0051] As used herein, the term "percent (%) amino acid sequence identity" for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps as necessary, and without considering any conservative substitutions as part of the sequence identity.Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0052] As used herein, "elimination half-life" is used in its ordinary sense, as described in Goodman and Gillman, 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 drugs is exponential (i.e., follows first-order kinetics) because drug concentrations usually do not reach the concentrations required for saturation of the elimination process. The rate of an exponential process can be expressed by its rate constant, k, which represents the fractional rate of change per unit of time, or by its half-life, t, which is the time required for 50% completion of the process. The units of these two constants are hr and h, respectively. The first-order rate constant and the half-life of a reaction are simply related (k × t 1 / 2 =0.693), which may be exchanged accordingly. First-order elimination kinetics dictates that a constant fraction of the drug is lost per unit of time, so a plot of the logarithm of drug concentration versus time is linear for all times after the initial distribution phase (i.e., after drug absorption and distribution are complete). Drug elimination half-life can be accurately determined from such a graph.

[0053] As used herein, the term "binding affinity" refers to the affinity of a protein described in this disclosure for a binding target and is expressed numerically using a "Kd" value. When two or more proteins are shown to have comparable binding affinities for their binding targets, the Kd values ​​for the binding of each protein to the binding targets are within ±2-fold of each other. When two or more proteins are shown to have comparable binding affinities for a single binding target, the Kd values ​​for the binding of each protein to the single binding target are within ±2-fold of each other. When proteins are shown to bind to two or more targets with comparable binding affinities, the Kd values ​​for the binding of each protein to the two or more targets are within ±2-fold of each other. Generally, a high Kd value corresponds to weak binding. In some embodiments, "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, the "on-rate" or "rate of association or association rate" or "k", and the "off-rate" or "rate of dissociation or dissociation rate" or "k" are also determined with surface plasmon resonance technology using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In further embodiments, "K", "k", and "k" are measured using OCTET® systems (Pall Life Sciences).In an exemplary method for measuring binding affinity using the OCTET® Systems, a ligand, e.g., biotinylated human or cynomolgus EpCAM, is immobilized on the OCTET® streptavidin capillary sensor tip, followed by activation of the streptavidin tip with approximately 20-50 μg / ml of human or cynomolgus EpCAM protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocker. For association kinetic measurements, EpCAM-binding protein variants are introduced at concentrations ranging from about 10 ng / mL to about 100 μg / mL, about 50 ng / mL to about 5 μg / mL, or about 2 ng / mL to about 20 μg / mL. In some embodiments, EpCAM-binding single domain proteins are used at concentrations ranging from about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the negative control assay buffer without binding protein. The kinetic parameters of the binding reaction are then determined using an appropriate tool, for example, ForteBio software.

[0054] As used herein, in some embodiments, "treatment" or "treating" or "treated" refers to therapeutic treatment aimed at delaying an undesirable physiological disease, disorder, or condition, or otherwise obtaining a beneficial or desired clinical result. For purposes described herein, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms; reduction in the extent of the disease, disorder, or condition; stabilization of the disease, disorder, or condition (i.e., not worsening); delaying the onset or progression of the disease, disorder, or condition; improvement of the disease, disorder, or disease state; and improvement, whether detectable or undetectable, whether remission (partial or total) or progression of the disease, disorder, or condition. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment further includes prolonging survival compared to expected survival in the absence of treatment. In other embodiments, "treatment" or "treating" or "treated" refers to prophylactic treatment, the purpose of which is to delay the onset of or reduce the severity of an unwanted physiological disease, disorder, or condition, e.g., in an individual predisposed to the condition (e.g., an individual bearing a genetic marker for a condition such as breast cancer).

[0055] "TriTAC," "TriTAC targeting EpCAM," or "trispecific protein targeting EpCAM," as used herein, refers to a trispecific binding protein that is not conditionally activated and comprises binding moieties 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 an EpCAM binding protein as described herein, and wherein at least one of the first target antigen binding domain and the second target antigen binding domain comprises a domain that binds to CD3, such as human CD3.

[0056] "ProTriTAC" or "protrispecific protein targeting EpCAM," as used herein, refers to a conditionally activated trispecific binding protein that includes (i) a cleavable linker (e.g., comprising the sequences set forth in SEQ ID NOS: 425-471 and 503-506), (ii) a binding moiety specific for a bulk serum protein, and also includes a masking moiety (e.g., comprising the sequences set forth in SEQ ID NOS: 380-424) that prohibits 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 an EpCAM-binding protein as described herein. The ProTriTAC proteins of the present disclosure are activated from the masked state to the active state, optionally in a protease-rich environment, such as a tumor microenvironment, by cleavage of the cleavable linker to form an active drug. The active agent, as provided herein, in some embodiments, comprises an EpCAM-binding domain of the present disclosure and a CD3-binding domain of the present disclosure. Exemplary active agents are provided in SEQ ID NOs: 153-179, 180-206, 210-212, 494, 571, and 574, or a sequence that is at least about 75% to 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 153-179, 180-206, 210-212, 494, 571, and 574, e.g., 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 a sequence selected from the group consisting of SEQ ID NOs: 153-179, 180-206, 210-212, 494, 571, and 574.

[0057] "Non-cleavable prodrug," as used herein, refers to a ProTriTAC as described above, in which the cleavable linker has been replaced with a non-cleavable linker (e.g., a linker such as in SEQ ID NO: 507). Exemplary active agents are provided in SEQ ID NOs: 495 and 502, or sequences that are at least about 75% to 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 495 and 502, e.g., 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 a sequence selected from the group consisting of SEQ ID NOs: 495 and 502.

[0058] In non-beta sandwich scaffolds (e.g., DARPins, affimers, affibodies), the term "non-CDR loop" refers to a region distal to the primary specificity-determining region typically used in the scaffold, which (1) can accommodate sequence randomization to allow for engineered specificity for a second antigen, and (2) allows for simultaneous binding of the scaffold to both antigens without steric interference. For this purpose, the primary specificity-determining region can be defined using the framework established in the Skrlec 2015 publication (Trends in Biotechnol, 33:408-418). An excerpt from the framework is provided below:

[0059] [Table 1]

[0060] "Chimeric antigen receptor" or "CAR" or "CARs," as used herein, refers to an engineered receptor that provides antigen specificity to cells (e.g., T cells). A CAR contains multiple domains, such as at least one target antigen-binding domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. Each domain may be connected by a linker. "ProCAR," as used herein, refers to a conditionally activatable CAR that contains the EpCAM-binding domain of the present disclosure.

[0061] EpCAM-binding protein Described herein are proteins that bind to EpCAM, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the disclosed EpCAM-binding proteins in the prevention and / or treatment of diseases, conditions, and disorders. In some embodiments, the EpCAM-binding protein is part of a multispecific (e.g., trispecific) protein that includes an EpCAM-binding domain as described herein.

[0062] Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed in most normal human epithelia and overexpressed in most carcinomas. This molecule is involved in cell-cell adhesion and also in signal transduction, cell migration, proliferation, and differentiation. Therefore, EpCAM has become an immunotherapeutic target in clinical trials for several solid tumors. It has been shown to play an important role in the detection and isolation of circulating tumor cells (CTCs). Various studies have shown that EpCAM is beneficial for the diagnosis and treatment of various carcinomas. Furthermore, tumor cells often express EpCAM to a much higher extent than their parent epithelium or less invasive forms of the cancers mentioned above. For example, EpCAM expression was shown to be significantly higher in tumor tissue and adenocarcinomas than in normal prostate epithelium (n=76; p<0.0001), suggesting that increased EpCAM expression represents an early event in the development of prostate cancer. See Poczatek, J Urol., 1999, 162, 1462-1644. In addition, in the majority of both squamous cell carcinomas and cervical adenocarcinomas, strong EpCAM expression has been shown to correlate with increased proliferation and loss of markers of terminal differentiation. See Litvinov, Am. J. Pathol. 1996, 148, 865-75. One example is breast cancer, where overexpression of EpCAM on tumor cells is a predictor of survival. See Gastl, Lancet. 2000, 356, 1981-1982. Furthermore, EpCAM has been described as a marker for detecting disseminated tumor cells in patients with squamous cell carcinoma of the head and neck and lung. See Chaubal, Anticancer Res. 1999, 19, 2237-2242; Piyathilake, Hum Pathol. 2000, 31, 482-487. Normal squamous epithelium, such as that found in the epidermis, oral cavity, epiglottis, pharynx, larynx, and esophagus, did not significantly express EpCAM. See Quak, Hybridoma, 1990, 9, 377-387.

[0063] EpCAM is thought to help epithelial cells adhere in an oriented, highly ordered manner. See Litvinov, J Cell Biol. 1997, 139, 1337-1348. It is believed that with malignant transformation of epithelial cells, rapidly growing tumor cells abandon the high cellular order of the epithelium. As a result, the surface distribution of EpCAM is thought to become less restricted, making the molecule more readily exposed to tumor cells. Because of their epithelial cell origin, tumor cells in most cancers are expected to express EpCAM on their surface.

[0064] EpCAM is a 314-amino acid, 40-kDa membrane-bound glycoprotein differentially expressed in certain epithelia and many human cancers. (See Balzar, J. Mol. Med. 1999, 77, 699-712.) EpCAM was discovered and subsequently cloned through recognition by the murine monoclonal antibody 17-1A / edrecolomab. (See Goettlinger, Int J Cancer. 1986;38, 47-53 and Simon, Proc. Natl. Acad. Sci. USA. 1990;87, 2755-2759.) Monoclonal antibody 17-1A was generated by immunization of mice with human colon carcinoma cells. (See Koprowski, Somatic Cell Genet. 1979, 5, 957-971.) The EGF-like repeats of EpCAM have been shown to mediate lateral and reciprocal interactions in homophilic cell adhesion. See, for example, Balzar, Mol. Cell. Biol. 2001, 21, 2570-2580), which is why they are predominantly located among epithelial cells (Litvinov, J Cell Biol. 1997, 139, 1337-1348, Balzar, J Mol Med. 1999, 77, 699-712 and Trebak, J Biol Chem. 2001, 276, 2299-2309).

[0065] EpCAM is also known as epithelial cell adhesion molecule, tumor-associated calcium signaling agent, major gastrointestinal tumor-associated protein GA733-2, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 314, TACSTD1, EGP314, MIC18, TROP1, M4S1, KSA, membrane component chromosome 4 surface marker (35 kD glycoprotein), antigen identified by monoclonal antibody AUA-1, human epithelial glycoprotein-2, epithelial cell surface antigen, epithelial glycoprotein, KS 1 / 4 antigen, CD326 antigen, GA722-2, HEGP314, HNPCC8, EpCAM, DIAR5, EGP-2, EGP40, KS1 / 4, MK-1, M1S2, ESA, and EGP. Exemplary protein sequences for EpCAM are provided in UniProtkB ID Nos. P16422 and B5MCA4. In some embodiments, an EpCAM binding protein of the disclosure binds to the EpCAM sequence provided in UniProtkB ID No. P16422 (SEQ ID NO: 478) or B5MCA4 (SEQ ID NO: 475).

[0066] In some embodiments, the EpCAM-binding domain binds to the extracellular domain of the mature EpCAM protein. The human extracellular domain sequence is provided in SEQ ID NO: 479, the cynomolgus monkey extracellular domain sequence is provided in SEQ ID NO: 480, and the mouse extracellular domain sequence is provided in SEQ ID NO: 481.

[0067] In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 475. In some embodiments, the EPCAM-binding domain binds to a protein comprising the sequence of SEQ ID NO: 475. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 476. In some embodiments, the EPCAM-binding domain binds to a protein comprising the sequence of SEQ ID NO: 476. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 477. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 477. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 478. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 478. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 479. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 479. In some embodiments, the EpCAM-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 480. In some embodiments, the EpCAM-binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 480. In some embodiments, the EpCAM-binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 481. In some embodiments, the EpCAM-binding domain binds to a protein that comprises a truncated sequence compared to SEQ ID NO: 481.

[0068] In some embodiments, the EpCAM-binding domain disclosed herein recognizes full-length EpCAM. In certain examples, the EpCAM-binding domain disclosed herein recognizes an epitope within EpCAM; for example, in some cases, the EpCAM-binding protein interacts with one or more amino acids found within a domain of human EpCAM. The epitope to which the antibody binds may consist of a single contiguous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) located within a domain of EpCAM. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within a domain of EpCAM.

[0069] In some embodiments, the EpCAM-binding proteins of the present disclosure bind to the full-length EpCAM protein or fragments thereof, such as epitope-containing fragments within the full-length EpCAM protein, as described above. In some cases, the epitope-containing fragments include antigenic or immunogenic fragments of the EpCAM protein and derivatives thereof. Epitope-containing fragments, including antigenic or immunogenic fragments, in some embodiments are 12 amino acids or more, e.g., 20 amino acids or more, 50, or 100 amino acids or more. In some embodiments, the EpCAM fragments comprise 95% or more of the full-length protein, 90% or more, 75%, 50%, 25%, or 10% or more of the full-length protein. In some embodiments, epitope-containing fragments of EpCAM, including antigenic or immunogenic fragments, can induce a relevant immune response in patients. Derivatives of EpCAM, in some embodiments, include sequence variants in which one or more (e.g., 1-20, e.g., 15 amino acids, or up to 20%, e.g., 10%, or 5%, or 1% by number of amino acids based on the full length of the protein) deletions, insertions, or substitutions have been made to the EpCAM sequence provided in SEQ ID NOs: 475-481.

[0070] In some embodiments, substitutions include conservative substitutions. In some cases, the derivatives and variants thereof have essentially the same biological function as the protein from which they are derived. For example, in some cases, derivatives and variants of EpCAM have antigenicity or immunogenicity comparable to that of the protein from which they are derived, have either the ligand-binding activity or the ability to form an active receptor-complex of the protein from which they are derived, or preferably both, and have the same tissue distribution as EpCAM.

[0071] In some embodiments, the EpCAM-binding protein specifically binds to EpCAM with an affinity equal to or greater than that of a reference EpCAM-binding protein; in such embodiments, the EpCAM-binding protein comprises an affinity-matured EpCAM-binding molecule, which is derived from an EpCAM-binding parent molecule and contains one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the EpCAM-binding parent molecule. In some embodiments, the affinity-matured EpCAM-binding molecule has superior stability against a selected destabilizing agent compared to the reference EpCAM-binding parent molecule. In some embodiments, the affinity-matured EpCAM-binding molecule is identified by a process comprising panning one or more pre-candidate EpCAM-binding molecules derived from one or more EpCAM-binding parent molecules expressed in a phage display library against an EpCAM protein, such as human EpCAM protein. The pre-candidate EpCAM-binding molecule, in some embodiments, contains amino acid substitutions in variable region, CDR, or framework residues relative to the parent molecule.

[0072] As used herein, "phage display" refers to a technique in which mutant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of a phage, e.g., a filamentous phage, particle. The utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently selected for sequences that bind to target molecules with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins by fusing them to either gene III or gene VIII of filamentous phage. See, e.g., Wells and Lowman, Curr. Opin. Struct. Biol, 3:355-362 (1992) and references cited therein. In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein, resulting in phage particles displaying one copy of the fusion protein or none at all. Compared to polyvalent phage, avidity effects are reduced, selection is based on affinity for endogenous ligands, and phagemid vectors are used, which facilitates DNA manipulation. See, e.g., Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0073] In some embodiments, panning involves using varying binding times and concentrations to identify EpCAM-binding molecules with increased or decreased on-rates from pre-candidate EpCAM-binding molecules. In some embodiments, panning involves using varying wash times to identify EpCAM-binding molecules with increased or decreased on-rates from pre-candidate EpCAM-binding molecules. In some embodiments, panning involves using varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined to increase the stability of affinity-matured EpCAM-binding molecules, for example, by shuffling to generate a second-stage combinatorial library from such mutants and performing a second round of panning and subsequent binding selection.

[0074] In some embodiments, the affinity-matured EpCAM-binding molecule has affinity for EpCAM protein (such as human EpCAM protein) that is equal to or greater than that of the EpCAM-binding parent molecule, but it has reduced, or in some embodiments, increased, cross-reactivity with selected substances, such as ligands, proteins, or antigens, other than the EpCAM epitope for which the EpCAM-binding parent molecule is specific or designed to be specific. Regarding the latter, affinity-matured EpCAM-binding molecules are, in some embodiments, more successfully tested in animal models when the affinity-matured EpCAM-binding molecule is reacted with both human EpCAM and the corresponding target in the animal model, e.g., mouse EpCAM or cynomolgus EpCAM. In some embodiments, the parent EpCAM-binding molecule binds to human EpCAM 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 binds to cynomolgus EpCAM with an affinity of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 15 nM or less, or 10 nM or less. In some embodiments, the affinity-matured EpCAM-binding molecule identified after a single round of panning binds to human EpCAM with an affinity of about 5 nM or less, such as 1 nM or less, and binds to cynomolgus EpCAM with an affinity of about 7.5 nM or less, such as 1 nM or less. In some embodiments, affinity-matured EpCAM-binding molecules identified after two rounds of panning bind to human EpCAM with an affinity of about 2.5 nM or less and bind to cynomolgus EpCAM with an affinity of about 3.5 nM or less.

[0075] In some embodiments, the EpCAM-binding protein comprises an antigen-specific binding domain polypeptide that specifically binds to a target, such as a target on a diseased cell, or to a target on another cell that supports a disease state, e.g., a target on a stromal cell that supports tumor growth, or to a target on an immune cell that supports disease-mediated immunosuppression. In some examples, the antigen-specific binding domain comprises an antibody, single-chain antibody, Fab, Fv, T-cell receptor binding domain, ligand-binding domain, receptor-binding domain, domain antibody, single-domain antibody, minibody, nanobody, peptibody, or various other antibody mimetics (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).

[0076] In some embodiments, the EpCAM-binding domain is an anti-EpCAM antibody or antigen-binding fragment thereof, or an antibody variant of the EpCAM-binding domain or antigen-binding fragment thereof. As used herein, the term "antibody variant" refers to variants and derivatives of the antibodies or antigen-binding fragments thereof described herein. In certain embodiments, amino acid sequence variants of the anti-EpCAM antibodies or antigen-binding fragments thereof described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-EpCAM antibodies or antigen-binding fragments thereof described herein are contemplated to improve their binding affinity and / or other biological properties. Exemplary methods 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 peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequence of the antibody or antigen-binding fragment thereof.

[0077] 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 with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include the CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions may be introduced into an antibody or antigen-binding fragment of interest, and the products may be screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, altered antibody-dependent cellular cytotoxicity (ADCC), or improved T-cell-mediated cytotoxicity (TDCC). Conservative and non-conservative amino acid substitutions are contemplated for preparing antibody variants.

[0078] In another example of substitutions to create a variant anti-EpCAM antibody or antigen-binding fragment thereof, one or more hypervariable region residues of the parent antibody are substituted. Generally, variants are selected based on a desired improved property, e.g., increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependent binding, compared to the parent antibody or antigen-binding fragment thereof.

[0079] In some embodiments, the EpCAM-binding domain is a single-domain antibody (sdAb), e.g., a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide specific for EpCAM, a ligand, or a small molecule entity. In some embodiments, the EpCAM-binding domain described herein is any domain that binds to EpCAM, including, but not limited to, a domain derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the EpCAM-binding domain is a single-domain antibody. In other embodiments, the EpCAM-binding domain is a peptide. In further embodiments, the EpCAM-binding domain is a small molecule.

[0080] In general, it should be noted that the term "single domain antibody," as used herein in its broadest sense, is not limited to a specific biological source or a specific preparation method. A single domain antibody is an antibody whose complementarity-determining region is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be any single domain antibody in the art or future. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, or cow. (2) expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanization" of a naturally occurring VHH domain or expression of a nucleic acid encoding such a humanized VHH domain; (4) "camelization" of a naturally occurring VH domain from any animal species, and in particular from a mammalian species such as human, or expression of a nucleic acid encoding such a camelized VH domain; (5) "camelization" of a "domain antibody" or "Dab" or expression of a nucleic acid encoding such a camelized VH domain; (6) use of synthetic or semi-synthetic techniques to prepare proteins, polypeptides, or other amino acid sequences; (7) preparation of a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the art, followed by expression of the nucleic acid obtained as above; and / or (8) any combination of one or more of the foregoing.

[0081] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody directed against EpCAM. As further described herein, such a VHH sequence can typically be generated or obtained by appropriately immunizing a species of llama with EpCAM (i.e., to generate an immune response and / or heavy chain antibodies directed against EpCAM), by obtaining a suitable biological sample from said llama (such as a blood sample, serum sample, or B cell sample), and generating the VHH sequence directed against EpCAM starting from said sample using any suitable technique known in the art.

[0082] In another embodiment, such naturally occurring VHH domains against EpCAM are obtained from a naive library of camelid VHH sequences, for example, by screening such a library with EpCAM, or at least one part, fragment, antigenic determinant, or epitope thereof, using at least one screening technique known in the art. Such libraries and techniques are described, for example, in WO99 / 37681, WO01 / 90190, WO03 / 025020, and WO03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, such as VHH libraries derived from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in WO00 / 43507.

[0083] In a further embodiment, yet another technique for obtaining VHH sequences directed against EpCAM includes appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to generate an immune response and / or heavy chain antibodies directed against EpCAM), obtaining a suitable biological sample from said transgenic mammal (such as a blood sample, serum sample, or B cell sample), and then generating VHH sequences directed against EpCAM starting from said sample using any suitable technique known in the art. For example, heavy chain antibody-expressing rats or mice and the further methods and techniques described in WO02 / 085945 and WO04 / 049794 can be used for this purpose.

[0084] In some embodiments, anti-EpCAM single domain antibodies of the present disclosure include single domain antibodies having an amino acid sequence corresponding to the amino acid sequence of a non-human antibody and / or naturally occurring VHH domain, e.g., a llama anti-EpCAM antibody, but which has been "humanized," i.e., by replacing one or more amino acid residues in the amino acid sequence (and, in particular, within the framework sequence) of said non-human anti-EpCAM and / or naturally occurring VHH sequence with one or more amino acid residues occurring at the corresponding positions in a VH domain from a conventional four-chain antibody of human origin (e.g., as described above). This can be performed in a manner known in the art that will be apparent to those skilled in the art, for example, based on the further description herein. Furthermore, such humanized anti-EpCAM single domain antibodies of the present disclosure can be obtained in any suitable manner known per se (i.e., as described in items (1) to (8) above) and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material. In some further embodiments, single-domain anti-EpCAM antibodies include single-domain antibodies having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain as described herein, 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 four-chain antibody by one or more amino acid residues that occur at corresponding positions in a VHH domain of a heavy-chain antibody. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or occur at the VH-VL interface and / or at so-called Camelidae hallmark residues (see, e.g., WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Preferably, the VH sequence used as starting material or starting point for generating or designing a camelized single domain is a VH sequence, preferably of mammalian origin, more preferably a human VH sequence, such as a VH3 sequence.However, it should be noted that in certain embodiments, such camelized anti-EpCAM single domain antibodies of the present disclosure can be obtained in any suitable manner known in the art (i.e., as described in items (1) to (8) above) and are therefore not strictly limited to polypeptides obtained using a non-human anti-EpCAM antibody and / or a polypeptide comprising a naturally occurring VH domain as the starting material. For example, as further described herein, both "humanization" and "camelization" are carried out by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then altering one or more codons in the nucleotide sequence so that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. The nucleic acid can then be expressed to yield the desired anti-EpCAM single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of the desired humanized or camelized anti-EpCAM single domain antibody of the present disclosure is designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques of peptide synthesis. In some embodiments, a nucleotide sequence encoding the desired humanized or camelized anti-EpCAM single domain antibody of the present disclosure is designed based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques for nucleic acid synthesis, after which the resulting nucleic acid can be expressed using known expression techniques to provide the desired anti-EpCAM single domain antibody of the present disclosure.

[0085] Other suitable methods and techniques for obtaining an anti-EpCAM single domain antibody of the present disclosure and / or a nucleic acid encoding same, starting from a naturally occurring VH or VHH sequence, include, for example, combining, in a suitable manner, one or more portions 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 portions 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 to provide an anti-EpCAM single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding same.

[0086] In some embodiments, the EpCAM-binding domain is an anti-EpCAM-specific antibody comprising heavy chain variable complementarity-determining region CDR1, heavy chain variable CDR2, heavy chain variable CDR3, light chain variable CDR1, light chain variable CDR2, and light chain variable CDR3. In some embodiments, the EpCAM-binding domain comprises any domain that binds to EpCAM, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment such as a single domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single-chain antibody (e.g., a single-chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the EpCAM-binding domain is a single-domain antibody. In some embodiments, the anti-EpCAM single-domain antibody comprises heavy chain variable complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3.

[0087] In some embodiments, the EpCAM-binding domain is a polypeptide comprising an amino acid sequence composed 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, where 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 EpCAM-binding proteins of the disclosure include, for example, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94 amino acid residues, and the complementarity-determining regions include, for example, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues. In some embodiments, the EpCAM-binding domain comprises an amino acid sequence selected from SEQ ID NOs: 1-38, 207-209, and 496-497.

[0088] In some embodiments, the binding proteins described herein comprise polypeptides having a sequence selected from SEQ ID NOs: 1-38, subsequences thereof, and variants thereof. In some embodiments, the EpCAM binding protein comprises at least 70% to 95% or more homology to a sequence selected from SEQ ID NOs: 1-38, subsequences thereof, and variants thereof. In some embodiments, the EpCAM 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 a sequence selected from SEQ ID NOs: 1-38, 207-209, and 496-497, subsequences thereof, and variants thereof. In some embodiments, the EpCAM binding protein comprises at least 70% to 95% or more identity to a sequence selected from SEQ ID NOs: 1-38, 207-209, and 496-497, subsequences thereof, and variants thereof. In some embodiments, the EpCAM 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 a sequence selected from SEQ ID NOs: 1-38, 207-209, and 496-497, subsequences thereof, and variants thereof.

[0089] In some embodiments, CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 39-76, or a sequence that contains one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 39-76. In some embodiments, CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 77-114, or a sequence that contains one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 77-114. In some embodiments, CDR3 comprises the sequence set forth in any one of SEQ ID NOs: 115-152, or a sequence that contains one or more substitutions compared to a sequence selected from the group consisting of SEQ ID NOs: 115-152.

[0090] In various embodiments, the EpCAM-binding domain of the disclosure has 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%, or about 74% identity with an amino acid sequence selected from SEQ ID NOs: 1-38, 207-209, and 496-497. %, 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.

[0091] In various embodiments, the complementarity determining regions of the EpCAM-binding domain of the present disclosure are 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: 39-76.

[0092] In various embodiments, the complementarity determining regions of the EpCAM binding domain of the present disclosure are 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: 77-114.

[0093] In various embodiments, the complementarity determining regions of the EpCAM-binding domain of the present disclosure are 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: 115-152.

[0094] In some embodiments, the EpCAM-binding domain is cross-reactive with human cynomolgus monkey and mouse EpCAM. In some embodiments, the EpCAM-binding domain is specific for human EpCAM. In certain embodiments, the EpCAM-binding domain disclosed herein binds to human EpCAM with a human Kd (hKd). In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus monkey EpCAM with a cyno Kd (cKd). In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus monkey EpCAM with a mouse Kd (mKd). In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus monkey EpCAM and human EpCAM with a cyno Kd (cKd) and a human Kd (hKd), respectively. In certain embodiments, the EpCAM-binding domain disclosed herein binds to cynomolgus monkey EpCAM, mouse EpCAM, and human EpCAM with a cyno Kd (cKd), a mouse Kd (mKd), and a human Kd (hKd), respectively. In some embodiments, the EpCAM-binding protein binds to human, mouse, and cynomolgus EpCAM with comparable binding affinities (i.e., the hKd, mKd, and cKd values ​​do not differ by more than ±10%). In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 500 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 450 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 400 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 350 nM. In some embodiments, the hKd, mKd, and cKd range from about 0.001 nM to about 300 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 250 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 200 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 150 nM.In some embodiments, the hKd, mKd, and cKd are in the range of about 0.001 nM to about 100 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.1 nM to about 90 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.2 nM to about 80 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.3 nM to about 70 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.4 nM to about 50 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.5 nM to about 30 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.6 nM to about 10 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.7 nM to about 8 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.8 nM to about 6 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 0.9 nM to about 4 nM. In some embodiments, the hKd, mKd, and cKd are in the range of about 1 nM to about 2 nM.

[0095] In some embodiments, any of the aforementioned EpCAM-binding domains (e.g., anti-EpCAM single-domain antibodies of SEQ ID NOs: 1-38) are affinity peptide tagged to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also known as 6X-his (SEQ ID NO: 377).

[0096] In some embodiments, the EpCAM-binding domain of the present disclosure preferentially binds to membrane-bound EpCAM over soluble EpCAM. Membrane-bound EpCAM refers to the presence of EpCAM in or on the cell membrane surface of cells that express EpCAM. Soluble EpCAM refers to EpCAM that is no longer present in or on the cell membrane surface of cells that express or have expressed EpCAM. In some examples, soluble EpCAM is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the EpCAM-binding domain binds to membrane-bound EpCAM at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold more than soluble EpCAM. In one embodiment, the EpCAM-binding protein of the present disclosure preferentially binds to membrane-bound EpCAM 30-fold more than soluble EpCAM. Determining whether an antigen binding protein binds preferentially to membrane-bound EpCAM over soluble EpCAM can be readily determined using binding assays.

[0097] In some embodiments, the EpCAM-binding protein is quite small, and in some embodiments, it is contemplated that the EpCAM-binding protein is 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In some examples, the EpCAM-binding protein, when a peptide or small molecule entity, is 5 kDa or less.

[0098] In other embodiments, the EpCAM-binding proteins described herein comprise small molecular entity (SME) binders for EpCAM. SME binders are small molecules, averaging approximately 500-2000 Da in size, that are attached to the EpCAM-binding protein by known methods, such as sortase ligation or conjugation. In these examples, the EpCAM-binding protein comprises a domain containing a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 376). To bind the SME binder to an EpCAM-binding protein containing a sortase recognition sequence, the protein is incubated with a sortase and an SME binder, whereby the sortase binds the SME binder to the recognition sequence. In yet other embodiments, the EpCAM-binding proteins described herein comprise knottin peptides for binding to EpCAM. Knottins are disulfide-stabilized peptides with a cysteine ​​knot scaffold and have an average size of approximately 3.5 kDa. Knottins are intended to bind to specific tumor molecules, such as EpCAM. In a further embodiment, the EPCAM-binding proteins described herein comprise the natural EpCAM ligand.

[0099] In some embodiments, the EpCAM-binding protein is a single polypeptide design that includes more than one domain and has flexible domain connections. This allows for easy production and manufacturing of the EpCAM-binding protein, as it is encoded by a single cDNA molecule and can be easily incorporated into a vector. Furthermore, in some embodiments in which the EpCAM-binding protein described herein is a single monomeric polypeptide chain, there is no problem with chain pairing or the requirement for dimer formation. In such embodiments, it is contemplated that the EpCAM-binding protein described herein has a reduced tendency to aggregate.

[0100] In EpCAM-binding proteins containing more than one domain, the domains are linked by one or more internal linkers. In certain embodiments, the internal linker is "short," i.e., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long," i.e., consisting of 15, 20, or 25 amino acid residues. In some embodiments, the internal linker consists of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues. Regarding the amino acid composition of the internal linker, the peptide is selected for its properties of providing flexibility to the EpCAM-binding protein, not interfering with the binding domain, and resisting cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of internal linkers suitable for linking domains in EpCAM-binding proteins include, but are not limited to, (GS) n (SEQ ID NO: 365), (GGS) n (SEQ ID NO: 366), (GGGS) n (SEQ ID NO: 367), (GGSG) n (SEQ ID NO: 368), (GGSGG) n (SEQ ID NO: 369), (GGGGS) n (SEQ ID NO: 370), (GGGGG) n (SEQ ID NO: 371), or (GGG) n (SEQ ID NO:372), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:373), (GGGGSGGGGSGGGGS) (SEQ ID NO:374), or (GGGGSGGGS) (SEQ ID NO:375).

[0101] In some cases, when an EpCAM-binding protein contains more than one domain, domains within the EpCAM-binding protein are conjugated using enzymatic site-specific conjugation methods, including the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of relatively pure enzymes in large quantities, its ease of use, and its lack of regulation by calcium or guanosine-5'-triphosphate (GTP) have made mTG the primary cross-linking enzyme used in both the food industry and biotechnology. Currently, mTG is used in many applications to conjugate proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. See, for example, Pavel Strp, "Veracity of microbial transglutaminase," Bioconjugate Chem. 25, 5, 855-862.

[0102] In some examples, an EpCAM-binding protein is provided that includes more than one domain, wherein one of the domains includes an acceptor glutamine in the constant region, which can be conjugated to another domain via a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, including alkylamines, oxoamines), wherein conjugation occurs exclusively at one or more acceptor glutamine residues present in the targeting portion outside the antigen-binding site (e.g., outside the variable region, within the constant region). Thus, conjugation does not occur at glutamines within the variable region, for example, at least partially surface-exposed glutamines. In some examples, the EpCAM-binding protein is formed by reacting one of the domains with a lysine-based linker in the presence of TGase.

[0103] In some embodiments, when one or more domains within an EpCAM-binding protein are directly linked, a hybrid vector is created in which the DNA encoding the directly linked domains is itself directly ligated to each other. In some embodiments, when a linker is used, a hybrid vector is created in which the DNA encoding one domain is ligated to the DNA encoding the linker at one end and the DNA encoding another domain is ligated to the other end of the linker.

[0104] In some embodiments, the EpCAM-binding protein is a single-chain variable fragment (scFv), a single-domain antibody, e.g., the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of a camelid-derived single-domain antibody. In other embodiments, the EpCAM-binding protein is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the EpCAM-binding protein is a ligand or peptide that binds to or associates with EpCAM. In further embodiments, the EpCAM-binding protein is a knottin. In further embodiments, the binding domain to EpCAM is a small molecule entity.

[0105] In certain examples, the EpCAM-binding proteins of the present disclosure can be incorporated into trispecific proteins that target EpCAM. In some embodiments, the trispecific protein comprises a CD3-binding domain, a half-life extending domain, and an EpCAM-binding domain of the present disclosure. In some embodiments, the EpCAM-binding trispecific protein comprises a trispecific antibody.

[0106] Multispecific EpCAM binding targeting proteins, trispecific proteins targeting EpCAM (also referred to herein as TriTAC proteins or molecules targeting EpCAM). In one aspect, described herein are multispecific or multivalent proteins comprising the EpCAM-binding proteins of the present disclosure. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3. In some embodiments, the multispecific protein further comprises a domain that specifically binds to human CD3. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3 gamma. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3 delta. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3 epsilon.

[0107] In further embodiments, the multispecific protein further comprises a domain that specifically binds to a T cell receptor (TCR). In some embodiments, the multispecific protein further comprises a domain that specifically binds to the alpha chain of the TCR. In some embodiments, the multispecific protein further comprises a domain that specifically binds to the beta chain of the TCR.

[0108] In certain embodiments, the CD3-binding domain of the multispecific protein not only exhibits strong CD3-binding affinity to human CD3, but also exhibits excellent cross-reactivity with the respective cynomolgus monkey CD3 proteins. In some examples, the CD3-binding domain of the multispecific protein is cross-reactive with CD3 from cynomolgus monkeys. In some examples, the ratio of human KD:cynomolgus monkey KD (hKd:cKd) for CD3 binding is between 20:1 and 1:2.

[0109] In some embodiments, the CD3-binding domain of the multispecific protein is any domain that binds to CD3, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment of a CD3-binding antibody, such as a single-domain antibody (sdAb), Fab, F(ab'), and Fv fragments, fragments composed of one or more CDRs, single-chain antibodies (e.g., single-chain Fv fragments (scFv)), disulfide-stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some instances, it is beneficial for the CD3-binding domain to be derived from the same species as the multispecific protein, including the single-domain serum albumin-binding proteins described herein, will ultimately be used. For example, for use in humans, it may be beneficial for the CD3-binding domain of a multispecific protein, including the EpCAM-binding proteins described herein, to include human or humanized residues from the antigen-binding domain of an antibody or antibody fragment. An exemplary amino acid sequence for the CD3-binding domain of a multispecific (e.g., trispecific) EpCAM-targeting TriTAC protein of the present disclosure is provided as SEQ ID NO:379, or a sequence at least about 75% to 100% identical to SEQ ID NO:379, e.g., 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:379.

[0110] In some embodiments, the serum albumin binding domain (also referred to herein as a half-life extending domain) of a multispecific protein, including the EpCAM binding proteins described herein, may be any domain that binds to serum albumin, including, but not limited to, a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the serum albumin-binding domain is a single-domain antibody, such as a single-chain variable fragment (scFv), e.g., the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of a camelid-derived sdAb; or an antigen-binding fragment of an HSA-binding antibody, e.g., Fab, F(ab'), and Fv fragments, fragments composed 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, peptides, ligands, or small molecule entities specific for serum albumin. In certain embodiments, the HSA-binding domain is a single-domain antibody. In other embodiments, the serum albumin-binding domain is a peptide. In further embodiments, the serum albumin-binding domain is a small molecule. The serum albumin binding domain of multispecific binding proteins, including single-chain variable fragment CD3 binding proteins, is contemplated to be fairly small, in some embodiments, 25 kD or less, 20 kD or less, 15 kD or less, or 10 kD or less. In certain examples, when the serum albumin binding is a peptide or small molecule entity, the serum albumin binding is 5 kD or less. An exemplary amino acid sequence for the serum albumin binding domain of a multispecific (e.g., trispecific) EpCAM-targeting TriTAC protein of the present disclosure is provided as SEQ ID NO:378, or a sequence at least about 75% to 100% identical to SEQ ID NO:378, e.g., 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:378.

[0111] As described herein, a half-life prolonging domain of a multispecific binding protein comprising a single-chain variable fragment CD3 binding protein results in altered pharmacokinetics and pharmacodynamics of the single-chain variable fragment CD3 binding protein itself. As described above, the half-life prolonging domain extends the elimination half-life. The half-life prolonging domain further alters the pharmacodynamic properties of the single-chain variable fragment CD3 binding protein, including altering its tissue distribution, penetration, and diffusion. In some embodiments, the half-life prolonging domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissues, and enhanced efficacy compared to proteins without the half-life prolonging domain. In one embodiment, a treatment method effectively and efficiently utilizes lower amounts of the multispecific binding protein comprising a single-chain variable fragment CD3 binding protein, resulting in reduced side effects, such as reduced off-target toxicity, such as non-tumor cytotoxicity.

[0112] Furthermore, the binding affinity of the half-life prolonging domain is selected in some embodiments to target a specific terminal half-life of a particular multispecific binding protein, including the EpCAM binding protein described herein. Thus, in some embodiments, the half-life prolonging domain has a high binding affinity. In other embodiments, the half-life prolonging domain has a moderate binding affinity. In still other embodiments, the half-life prolonging domain has a low or slight binding affinity. Exemplary binding affinities include a K of 10 nM or less. d (high), K between 10 nM and 100 nM d (moderate), and K >100 nM d As noted above, the binding affinity to serum albumin is determined by known methods such as surface plasmon resonance (SPR).

[0113] In some embodiments, the EpCAM-targeting multispecific protein of the present disclosure comprises: (A) a first domain that binds to CD3; (B) a second domain that is a half-life extending domain; and (C) a third domain that is an EpCAM-binding protein described herein. In some embodiments, the first domain comprises an scFv that specifically binds CD3. The CD3 is, for example, a human CD3 protein. In some embodiments, the second domain comprises an sdAb that specifically binds a bulk serum protein. In some examples, the bulk serum protein is albumin, for example, serum albumin, for example, human serum albumin.

[0114] Domains (A), (B), and (C) are, in some embodiments, linked by linkers L1 and L2 in any one of the following orientations: H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(C)-L1-(B)-L2-(A)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(B)-(C)-L2-COOH, or H2N-(B)-(C)-(A)-COO.

[0115] Multispecific proteins that target EpCAM of the present disclosure, in some embodiments, comprise an amino acid sequence that is at least about 70% to about 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 153-206 and 210-212. In some embodiments, a multispecific protein targeting EpCAM of the present disclosure comprises an amino acid sequence 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 a sequence selected from the group consisting of SEQ ID NOs: 153-206 and 210-212.

[0116] A conditionally active multispecific EpCAM-targeting protein, such as a conditionally active EpCAM-targeting trispecific protein (referred to herein as an EpCAM-targeting ProTriTAC or trispecific promoting protein or molecule). One embodiment of the present disclosure provides a conditionally active multispecific protein comprising an EpCAM-binding domain disclosed herein (e.g., in some embodiments, the present disclosure provides a trispecific promoting / ProTriTAC protein that targets EpCAM, comprising an EpCAM-binding domain of the present disclosure).

[0117] In some embodiments, the conditionally active multispecific protein further comprises a domain that specifically binds to CD3 and a binding moiety that specifically binds to a bulk serum protein, such as human serum albumin. In some embodiments, the binding moiety can mask the interaction of the EpCAM-binding domain or the CD3-binding domain with their targets. In some embodiments, the binding moiety of the present disclosure comprises a masking moiety and a cleavable linker, such as a protease-cleavable linker. Exemplary sequences for masking moieties within the binding moiety are provided in SEQ ID NOs: 380-424, or sequences comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 380-424. In some embodiments, the binding moiety comprises a modified non-CDR loop sequence and a cleavable linker. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 425-471, 503-506, 508-550, and 581, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 425-425-471, 503-506, 508-550, and 581. In some embodiments, the masking moiety comprises a modified non-CDR loop sequence and a non-cleavable linker. In some embodiments, the non-cleavable linker comprises the sequence set forth in SEQ ID NO: 507, or a sequence comprising one or more substitutions relative to SEQ ID NO: 507. In some embodiments, the binding moiety comprises a sequence 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 a sequence selected from the group consisting of SEQ ID NOs: 472-473 and 482-483. In some embodiments, the CD3 binding domain of EpCAM ProTriTAC of the present disclosure comprises a sequence 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: 474.

[0118] In some embodiments, the EpCAM-targeting ProTriTAC of the present disclosure comprises, from N-terminus to C-terminus, a binding moiety that is an anti-ALB domain containing a non-CDR loop with a binding site for a CD3-binding domain (e.g., the sequence of SEQ ID NO: 474, or a CD3-binding domain having at least about 75% identity thereto), a cleavable linker, the CD3-binding domain, and an anti-EpCAM-binding domain on the C-terminus. The EpCAM-binding domain of ProTriTAC, in some embodiments, has 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% identity to an amino acid sequence selected from SEQ ID NOs: 1-38, 207-209, and 496-497. , 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.

[0119] In some embodiments, the EpCAM-targeting ProTriTAC of the present disclosure comprises an amino acid sequence 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 a sequence selected from the group consisting of SEQ ID NOs: 495, 498, 499, 500, 502, 569, 570, 572, 573, 575, 576, 577, and 578. In some embodiments, the EpCAM-targeting ProTriTAC of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 576, pharmaceutical compositions comprising the amino acid sequences, and methods of using the amino acid sequences to treat diseases, such as neoplastic diseases, as described herein.

[0120] In some embodiments, the EpCAM-targeted ProTriTAC of the present 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 a sequence selected from the group consisting of SEQ ID NOs: 495 and 502.

[0121] Exemplary sequences for active EpCAM-targeting drugs (CTs) as described herein are those that are 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 NOs: 153-179, 180-206, 210-212, 494, 571, and 574.

[0122] The binding moiety can synergistically extend the therapeutic window of a conditionally active EpCAM-targeting trispecific promoting protein through both steric and specific masking. In some embodiments, the binding moiety combines both steric masking (e.g., via binding to bulk serum albumin) and specific masking (e.g., via non-CDR loops that bind to the CDRs of the anti-EpCAM domain or anti-CD3 scFv domain). In some cases, modification of the non-CDR loops within the binding moiety does not affect albumin binding. A protease-cleavable linker optionally allows activation of the EpCAM-targeting trispecific promoting protein in a single proteolytic event, thereby allowing more efficient transduction of the trispecific promoting molecule in the tumor microenvironment. Furthermore, tumor-associated proteolytic activation optionally reveals an active T cell engager with minimal off-tumor activity after activation. The present disclosure provides, in some embodiments, an extended half-life T cell engager format (ProTriTAC) comprising an EpCAM-binding moiety described herein, which, in some cases, represents a novel and improved approach for designing conditionally active T cell engagers.

[0123] The half-life of the EpCAM-binding domain in the conditionally active trispecific-promoting format is, in some embodiments, extended in the systemic circulation by using the above-described binding moiety, which acts as a safety switch, maintaining the multispecific protein in an inactive, pro-format until it reaches the tumor microenvironment where it can be conditionally activated by cleavage of the linker and bind to its target antigen. The safety switch, in some instances, provides several advantages, some of which include: (i) extending the therapeutic window of the conditionally active EpCAM-targeting protein; (ii) reducing target-mediated pharmacokinetics by maintaining the conditionally active EpCAM-targeting protein in the systemic circulation; (iii) reducing the concentration of undesired activated proteins in the systemic circulation, thereby minimizing the spread of chemistry, manufacturing, and control-related impurities, such as pre-activated drug products, endogenous viruses, host cell proteins, DNA, leachables, antifoams, antibiotics, toxins, solvents, and heavy metals; and (iv) reducing the concentration of undesired activated proteins in the systemic circulation, thereby preventing oxidation, deamidation, denaturation, and degradation of the C-terminus in MAbs. (v) minimizing the spread of impurities, aggregates, degradation products, and product variants associated with the Lys loss product; (v) preventing aberrant activation in the circulation; (vi) reducing toxicity associated with leakage of reactive species from abnormal tissues or other pathophysiological conditions, such as tumors, autoimmune diseases, inflammation, viral infections, tissue remodeling events (e.g., myocardial infarction, skin wound healing), or external insults (e.g., X-rays, CT scans, UV exposure); and (vii) reducing nonspecific binding of the conditionally active EpCAM-targeting protein. Furthermore, after activation, or in other words, after destruction of the safety switch, the conditionally active EpCAM-targeting protein is separated from the safety switch, providing it with an extended half-life and thus being removed from the circulation.For example, if a drug is inadvertently activated outside the tumor environment, or if the drug leaks out of the tumor environment after activation, the drug will be quickly cleared and unlikely to cause damage to normal tissue, thus reducing toxicity.

[0124] In some embodiments, the conditionally active multispecific EpCAM binding proteins described herein have an improved therapeutic index compared to the therapeutic index of a constitutively active, but not conditionally active, EpCAM binding protein. For example, EpCAM ProTriTAC, in some embodiments, has an increased therapeutic index over EpCAM TriTAC. The increase in therapeutic index is, in some embodiments, at least about 2-fold to about 1000-fold, e.g., 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, or about 50-fold to about 100-fold. The increase in therapeutic index, in some embodiments, results from conjugation of the EpCAM-binding domain to a binding moiety described above using a non-CDR loop and a cleavable linker.

[0125] The "therapeutic index" (TI) (also called the "therapeutic window"), in some embodiments, is the minimum amount of a therapeutic agent (e.g., EpCAM TriTAC, EpCAM ProTriTAC, EpCAM CAR, EpCAM ProCAR) that results in a therapeutic effect (e.g., improved survival in patients with EpCAM-expressing cancer treated with the therapeutic agent) compared to the minimum maximum tolerated dose. An example of an expanded therapeutic index for ProTriTAC is shown in Table 14. In some examples, an improved therapeutic index is due to the improved EC of EpCAM TriTAC compared to EpCAM ProTriTAC in T cell-mediated killing of cancer cells. 50 This is made clear in terms of:

[0126] In some embodiments, conditionally active EpCAM-targeting protein formats confer a significantly longer serum half-life to the EpCAM-binding domain, reducing the likelihood of its unwanted activation in the circulation, thereby producing a "bio-better" version.

[0127] The binding moieties described herein comprise at least one non-CDR loop. In some embodiments, the non-CDR loop provides a binding site for the binding moiety to bind to the EpCAM-binding domain of the present disclosure. In some cases, the binding moiety masks the binding of the EpCAM-binding domain to its target antigen, for example, through steric occlusion, through specific intermolecular interactions, or a combination of both.

[0128] In some embodiments, the binding moieties described herein further comprise a complementarity-determining region (CDR) specific for binding to, for example, a bulk serum protein (e.g., human serum albumin). In some examples, the binding moieties of the present disclosure are domains derived from immunoglobulin molecules (Ig molecules). Ig may be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chains of Ig molecules fold into a series of parallel β-strands connected by loops. In the variable region, three of the loops constitute "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, or antigen-binding fragments thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are hypervariable in sequence and / or involved in antigen recognition and / or usually form structurally defined loops, interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0129] In some embodiments, the binding moiety of the present disclosure is a heavy-chain-only antibody. As shown in Figure 26, the variable domain of a heavy-chain-only antibody has several β-strands arranged in two sheets. The variable domain of a heavy-chain-only antibody comprises three hypervariable loops, or complementarity-determining regions (CDRs), and framework regions FR1, FR2, FR3, and FR4. The three CDRs of the variable domain (CDR1, CDR2, CDR3) are clustered at one end of a β-barrel. The CDRs are the loops connecting β-strands BC, C'-C'', and FG of the immunoglobulin fold, while the bottom loop connecting β-strands AB, CC', C''-D, and EF of the immunoglobulin fold and the top loop connecting the DE strand of the immunoglobulin fold are non-CDR loops.

[0130] In some embodiments of the present disclosure, at least a portion or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR loops" of a binding moiety described herein, such as a binding moiety that is a heavy chain-only antibody. In some embodiments of the present disclosure, at least some amino acid residues of the constant domains CH1, CH2, or CH3 are part of the "non-CDR loops" of a binding moiety described herein. In some embodiments, the non-CDR loops include one or more of the AB, CD, EF, and DE loops of a C1 set domain of an Ig or Ig-like molecule; the AB, CC', EF, FG, BC, and EC' loops of a C2 set domain of an Ig or Ig-like molecule; or the DE, BD, GF, A(A1A2)B, and EF loops of an I (intermediate) set domain of an Ig or Ig-like molecule.

[0131] Within the variable domain, CDRs are thought to be responsible for antigen recognition and binding, while FR residues are thought to act as scaffolds for the CDRs. However, in some instances, some FR residues play an important role in antigen recognition and binding. Framework region residues that affect antigen binding can be divided into two categories. First, there are FR residues that connect the antigen and are therefore part of the binding site; some of these residues are in turn close to the CDRs. Other residues are in turn distant from the CDRs but are close to the CDRs in the 3D structure of the molecule (e.g., loops in the heavy chain).

[0132] In some embodiments, the non-CDR loops are modified to generate an antigen-binding site specific for a bulk serum protein such as albumin. In some embodiments, the non-CDR loops are modified to generate an antigen-binding site specific for the EpCAM-binding domain described herein. In some embodiments, the non-CDR loops are modified to generate an antigen-binding site specific for the CD3-binding domain described herein.

[0133] It is contemplated that various techniques, such as site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid foreign to the amino acid sequence of the non-CDR loop, or amino acid substitution, can be used to modify the non-CDR loop. In some instances, an antigenic peptide is inserted into the non-CDR loop. In some instances, an antigenic peptide is substituted for the non-CDR loop. The modification may be in only one non-CDR loop to generate an antigen-binding site. In other instances, more than one non-CDR loop is modified. For example, the modification may be in any one of the non-CDR loops shown in Figure 26 (i.e., AB, CC', C''D, EF, and DE). In some instances, the modification is in the DE loop. In other instances, the modification is in all four of the AB, CC', C''D, and EF loops.

[0134] In some examples, the binding moieties described herein are attached to the EpCAM-binding domain via their AB, CC', C"D, or EF loops and to a bulk serum protein, such as albumin, via their BC, C'-C" or FG loops. In some examples, the binding moieties are attached to the EpCAM-binding domain via their AB, CC', C"D, and EF loops and to a bulk serum protein, such as albumin, via its BC, C'C" and FG loops. In some examples, the binding moieties are attached to the EpCAM-binding domain via one or more of the AB, CC', C"D, and EF loops and to a bulk serum protein, such as albumin, via one or more of the BC, C'C" and FG loops. In some examples, the binding moieties are attached to the EpCAM-binding domain via its AB, CC', C"D, or EF loops and to a bulk serum protein, such as albumin, via its BC, C'C" or FG loops. In some examples, a binding moiety is bound to a bulk serum protein, such as albumin, via its AB, CC', C"D, and EF loops and is bound to an EpCAM-binding domain via its BC, C'C" and FG loops. In some examples, a binding moiety is bound to a bulk serum protein, such as albumin, via one or more of the AB, CC', C"D, and EF loops and is bound to an EpCAM-binding protein via one or more of the BC, C'C" and FG loops. In some examples, a binding moiety described herein is bound to a CD3-binding domain via its AB, CC', C"D, or EF loops and is bound to a bulk serum protein, such as albumin, via its BC, C'-C" or FG loops. In some examples, a binding moiety described herein is bound to a bulk serum protein, such as albumin, via its AB, CC', C"D, or EF loops and is bound to a CD3-binding domain via its BC, C'-C" or FG loops.In some examples, the binding moieties described herein are attached to the CD3 binding domain via their AB, CC', C"D, or EF loops and to the EpCAM binding domain via their BC, C'-C" or FG loops. In some examples, the binding moieties described herein are attached to the EpCAM binding domain via their AB, CC', C"D, or EF loops and to the CD3 binding domain via their BC, C'-C" or FG loops.

[0135] Bulk serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine-knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin binding module, or DNA or RNA aptamer scaffold.

[0136] In some cases, the binding moiety comprises a binding site for a bulk serum protein. In some embodiments, the CDRs within the binding moiety provide the binding site for the bulk serum protein. The bulk serum protein, in some instances, is globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the binding moiety comprises a binding site for an immunoglobulin light chain. In some embodiments, the CDRs provide the binding site for the immunoglobulin light chain. The immunoglobulin light chain, in some instances, is an Igκ free light chain or an Igλ free light chain.

[0137] In further embodiments, the binding moiety is any type of polypeptide. For example, in certain instances, the binding moiety is a natural peptide, a synthetic peptide, or a fibronectin scaffold or an engineered bulk serum protein. In some instances, the binding moiety comprises any type of binding domain, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a soluble TCR fragment, or a single-domain antibody, such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of a camelid-derived nanobody. In other embodiments, the binding moiety is a non-Ig binding domain, i.e., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, and monobodies.

[0138] It is contemplated herein that the binding moieties described herein comprise at least one cleavable linker. In one aspect, the cleavable linker comprises a polypeptide having a sequence that is recognized and cleaved in a sequence-specific manner. The binding moieties described herein optionally comprise a protease-cleavable linker that is recognized and cleaved in a sequence-specific manner. In some embodiments, the protease-cleavable linker is recognized in a sequence-specific manner by a matrix metalloprotease (e.g., MMP9) (MMP). In some cases, the protease-cleavable linker recognized by MMP9 comprises a polypeptide having the amino acid sequence PR(S / T)(L / I)(S / T). In some cases, the protease-cleavable linker recognized by MMP9 comprises a polypeptide having the amino acid sequence LEATA. In some cases, the protease-cleavable linker is recognized in a sequence-specific manner by MMP11.

[0139] Protease is a protein that cleaves proteins, sometimes in a sequence-specific manner.Proteases include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, and stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidin, bromelain, calpain, caspase, caspase-3, These include Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteinases (MMPs), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase-1, and dipeptidyl peptidase-IV (DPPIV / CD26).

[0140] [Table 2-1]

[0141] [Table 2-2]

[0142] Proteases are known to be secreted by some abnormal cells and tissues, such as tumor or cancer cells, creating a protease-rich or protease-enriched microenvironment. In some instances, a subject's blood is rich in proteases. In some cases, cells surrounding a tumor secrete proteases into the tumor microenvironment. Tumor-surrounding cells that secrete proteases include, but are not limited to, 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 leukocytes, and other cells. In some cases, proteases are present in a subject's blood (e.g., proteases targeting amino acid sequences found in microbial peptides). This feature allows targeted therapeutic agents, such as antigen-binding proteins, to have additional specificity, as T cells are not bound by antigen-binding proteins except in the protease-rich microenvironment of the target cell or tissue.

[0143] Other non-limiting examples of linkers that may be utilized in the constructs described herein are provided in the sequence listing below.

[0144] Incorporation into chimeric antigen receptors (CARs) In some examples, the EpCAM-binding proteins of the present disclosure can be incorporated into chimeric antigen receptors (CARs), or ProCARs. Engineered immune effector cells, such as T cells or NK cells, can be used to express CARs containing EpCAM-binding proteins containing, for example, the anti-EpCAM single-domain antibodies described herein. In one embodiment, a CAR containing an EpCAM-binding protein described herein is linked via a hinge region to a transmembrane domain and to a costimulatory domain, e.g., a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain, such as 4-1BB and / or CD3 zeta. Exemplary sequences for ProCAR containing the EpCAM-binding domain are provided in SEQ ID NOs: 485-491, or a sequence at least about 75% to 100% identical to a sequence selected from SEQ ID NOs: 485-491, e.g., 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.

[0145] The conditionally active receptors described herein comprise at least one binding moiety comprising a non-CDR loop. In one aspect, the binding moiety masks the binding of the EpCAM-binding domain until activation. The cleavable linker comprises, for example, a protease cleavage site or a pH-dependent cleavage site. In certain instances, the cleavable linker is cleaved only within the tumor microenvironment. Thus, in some instances, a binding moiety connected to a cleavable linker and further bound to the EpCAM-binding domain maintains the EpCAM-binding domain in an inactive state in circulation until the cleavable linker is cleaved within the tumor microenvironment. In some embodiments, the binding moiety binds to the EpCAM-binding domain. In some embodiments, a non-CDR loop provides a binding site for the moiety to bind to the EpCAM-binding domain. In some embodiments, the binding moiety masks the binding of the EpCAM-binding domain to its target antigen through specific intramolecular interactions, such as interactions within various domains of a polypeptide comprising the binding moiety, e.g., through steric occlusion. In some embodiments, the binding moiety further comprises a complementarity-determining region (CDR).

[0146] In some examples, the binding portion of a CAR or proCAR described herein is a domain derived from an immunoglobulin molecule (Ig molecule), as described above in the section of this disclosure corresponding to the conditionally active multispecific EpCAM-targeting protein.

[0147] Figure 27 shows a schematic diagram of a portion of an exemplary cleavable conditionally active receptor of the present disclosure. The conditionally active receptor comprises an EpCAM-binding domain (aTarget1), a cleavable linker, and a binding moiety (aTarget2). The EpCAM-binding domain has specificity for a first target (EpCAM), while the binding moiety has specificity for a second target. The binding moiety also has a modified non-CDR loop that inhibits binding of the EpCAM-binding domain to its target. Once cleaved at the cleavable linker, the binding moiety is released, allowing binding of the EpCAM-binding domain.

[0148] Figure 28 shows a schematic diagram of activation of an exemplary conditionally active receptor of the present disclosure. The inactive receptor (inactive ProCAR) comprises an EpCAM-binding domain (anti-tumor target sdAb or scFv) connected to a binding moiety (anti-target 2 sdAb) via a linker containing 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 EpCAM antigen-binding domain. In some embodiments, the binding moiety has specificity for a given target, as further described elsewhere herein. The receptor further comprises a transmembrane domain and an intracellular signaling domain. The receptor is provided in T cells (CART). Upon exposure to the tumor environment, the protease cleavage site is cleaved by tumor-associated proteases, thereby activating the receptor and generating an active receptor that does not contain the binding moiety. The receptor contains an active antigen-binding domain. When the receptor is internalized by the cell, a new receptor is generated that contains the binding moiety and is inactive.

[0149] The cleavable linker of the binding moiety, in some embodiments, comprises a protease cleavage site similar to those described above for the conditionally active multispecific proteins comprising the EpCAM-binding domain of the present disclosure. For example, the cleavable linker, in some embodiments, comprises a sequence selected from Table 1 or other linker sequences provided in the Sequence Listing.

[0150] Transmembrane domain The conditionally active chimeric antigen receptor, T cell receptor fusion protein, and T cell receptor of the present disclosure comprise a transmembrane domain for insertion into a eukaryotic cell membrane. In some embodiments, the transmembrane domain is inserted between the EpCAM-binding domain and the intracellular domain. In some embodiments, the transmembrane domain is inserted between the EpCAM-binding domain and the costimulatory domain.

[0151] Any transmembrane (TM) domain for inserting a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. As one non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 551) may be used. Further non-limiting examples of suitable TM sequences include: a) CD8β-derived: GLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO: 552); b) CD4-derived: ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 553); c) CD3 zeta-derived: LCYLLDGILFIYGVILTALFLRV (SEQ ID NO: 554); d) CD28-derived: WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 555); e) CD134 (OX40)-derived: AAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO: 556); and f) CD7-derived: ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO: 557).

[0152] Hinge Area In some cases, the conditionally active chimeric antigen receptors, T cell receptor fusion proteins, and T cell receptors of the present disclosure comprise a hinge region (also referred to herein as a "spacer"), wherein the hinge region is inserted between the EpCAM-binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a receptor-derived hinge region polypeptide (e.g., a hinge region from CD8).

[0153] The hinge region may have a length of about 4 amino acids to about 50 amino acids (aa), for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

[0154] Suitable spacers may be readily selected from or be 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 from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0155] Exemplary spacers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GGGS)n, and (GSGGS)n, where n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used; both Gly and Ser are relatively amorphous and can therefore function as neutral tethers between components. Glycine polymers may also be used; glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary spacers include amino acid sequences, including, but not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, and the like.

[0156] The amino acid sequences of immunoglobulin hinge regions are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge region can comprise one of the following amino acid sequences: DKTHT; CPPC; CPEPKSCDTPPPCPR (SEQ ID NO: 558); see, e.g., Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO: 559); KSCDKTHTCP (SEQ ID NO: 560); KCCVDCP (SEQ ID NO: 561); KYGPPCP (SEQ ID NO: 562); EPKSCDKTHTCPPCP (SEQ ID NO: 563); human IgG1 hinge; ERKCCVECPPCP (SEQ ID NO: 564); human IgG2 hinge; ELKTPLGDTTHTCPRCP (SEQ ID NO: 565); human IgG3 hinge; SPNMVPHAHHAQ (SEQ ID NO: 566); human IgG4 hinge);

[0157] In some embodiments, the hinge region comprises the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4. The hinge region may comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may be substituted with Tyr, such that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 567). See, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891.

[0158] In some embodiments, the hinge region comprises an amino acid sequence derived from human CD8; for example, the hinge region comprises the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 568), or a variant thereof.

[0159] Conditionally active chimeric antigen receptors In one embodiment, the present disclosure provides a conditionally active chimeric antigen receptor (CAR). CARs generally comprise 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 that binds to EpCAM, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a signaling domain of a protein, including, but not limited to, ZAP70, CD3 zeta, and 4-1BB.

[0160] In some embodiments, the conditionally active chimeric antigen receptor comprises a costimulatory domain, which 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 or more modifications, up to 20, 10, or five or fewer modifications.

[0161] In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein, including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof with one to twenty modifications.

[0162] In one aspect, the disclosure provides a cell (e.g., a T cell) engineered to express a CAR. In one aspect, the cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell is capable of stably expressing the CAR. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell is capable of transiently expressing the CAR.

[0163] Conditionally active T cell receptor fusion proteins In one example, the present disclosure provides a conditionally active T cell receptor fusion protein. As used herein, "T cell receptor (TCR) fusion protein" or "TFP" typically includes recombinant polypeptides derived from various polypeptides, including a TCR, that, when co-located in or on the surface of a T cell, are generally capable of: i) binding to a surface antigen on a target cell; and ii) interacting with other polypeptide components of an intact TCR complex.

[0164] The conditionally active TFP comprises a binding moiety, an EpCAM-binding domain, and a T cell receptor subunit. In some embodiments, the T cell receptor subunit further comprises an extracellular domain of a T cell receptor, a transmembrane domain, and at least a portion of an intracellular domain of a T cell receptor. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a functional fragment thereof, or an amino acid sequence having one, two, or three or more modifications up to 20, 10, or five or fewer modifications.

[0165] In some embodiments, the intracellular domain of the T cell receptor comprises a stimulatory domain, which may be from a T cell receptor subunit, including but not limited to, an alpha subunit, a beta subunit, a delta subunit, a gamma subunit, an epsilon subunit, or a combination thereof. In some embodiments, the stimulatory domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) or portion thereof, including, but not limited to, a CD3 zeta TCR subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a TCR zeta chain, an Fc epsilon receptor 1 chain, an Fc epsilon receptor 2 chain, an Fc gamma receptor 1 chain, an Fc gamma receptor 2a chain, an Fc gamma receptor 2b 1 chain, an Fc gamma receptor 2b 2 chain, an Fc gamma receptor 3a chain, an Fc gamma receptor 3b chain, an Fc beta receptor 1 chain, TYROBP (DAP12), CDS, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences having 1, 2, or 3 or more modifications up to 20, 10, or 5 modifications.

[0166] In some embodiments, the conditionally active TFP further comprises a costimulatory domain, hi some embodiments, the costimulatory domain is a functional signaling domain of a protein, including but not limited to, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and an amino acid sequence having at least 1, 2, or 3 or more modifications, up to 20, 10, or 5 or fewer modifications.

[0167] In some embodiments, the EpCAM-binding domain is connected to the extracellular domain of the T cell receptor by a linker sequence. In some examples, the encoded linker sequence comprises (G4S)n, where n=1-4. In some examples, the encoded linker sequence comprises a long linker (LL) sequence. In some examples, the encoded long linker sequence comprises (G4S)n, where n=2-4. In some examples, the encoded linker sequence comprises a short linker (SL) sequence. In some examples, the encoded short linker sequence comprises (G4S)n, where n=1-3.

[0168] In one aspect, the present disclosure provides a cell (e.g., a T cell) engineered to express a conditionally active T cell receptor fusion protein (TFP). In one aspect, the cell is transformed with the conditionally active TFP, and the conditionally active TFP is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding the conditionally active TFP. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the conditionally active TFP. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the conditionally active TFP. In some such embodiments, the cell can transiently express the conditionally active TFP.

[0169] Conditionally active T cell receptors In one embodiment, the present disclosure provides a conditionally active T cell receptor. T cell receptors generally comprise multiple subunits, including alpha, beta, delta, gamma, epsilon, and zeta subunits. The conditionally active T cell receptor of the present disclosure comprises a binding moiety. In some embodiments, the binding moiety is bound to a T cell receptor subunit, including, but not limited to, an alpha subunit, a beta subunit, or a combination thereof.

[0170] In some embodiments, the binding moiety can mask binding of a T cell receptor to its target. In some embodiments, the binding moiety binds to a T cell receptor. In some embodiments, the non-CDR loops provide a binding site for the moiety to bind to the T cell receptor. In some embodiments, the non-CDR loops provide a binding site specific for T cell receptor alpha, T cell receptor beta, or a combination thereof. In some embodiments, the binding moiety masks binding of a T cell receptor to its target through specific intermolecular interactions, e.g., through steric occlusion.

[0171] In one aspect, the present disclosure provides a cell (e.g., a T cell) engineered to express a conditionally active T cell receptor (TCR). In one aspect, the cell is transformed with the conditionally active TCR, and the conditionally active TCR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding the conditionally active TCR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the conditionally active TCR. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the conditionally active TCR. In some such embodiments, the cell can transiently express the conditionally active TCR.

[0172] cell In one embodiment, the present disclosure provides a cell comprising a chimeric antigen receptor or 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.

[0173] Suitable mammalian cells include primary cells and immortalized cell lines, including human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL-1651), and the like. No. CRL1573), HLHepG2 cells, HuT-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0174] In some instances, the cells are not immortalized cell lines, but instead are cells (e.g., primary cells) obtained from an individual. For example, in some instances, the cells are immune cells obtained from an individual. In one example, the cells are T lymphocytes obtained from an individual. In another example, the cells are cytotoxic cells obtained from an individual. In another example, the cells are stem or progenitor cells obtained from an individual.

[0175] In recent studies, CAR constructs have been used to target natural killer (NK) cell activity, as 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-expressing constructs and used to elicit immune responses. Because NK cells do not require HLA matching, they can be used as allogeneic effector cells (Harmanson & Kaufman, 2015). Furthermore, therapeutically useful peripheral blood NK cells (PB-NK) can be isolated from donors by simple blood draw. Useful CAR constructs may contain elements similar to those used to create CAR T cells.

[0176] Thus, in some embodiments, the present disclosure provides cells, including NK cells, comprising a chimeric antigen receptor, a conditionally active chimeric antigen receptor, a conditionally active T cell receptor fusion protein, or a conditionally active T cell receptor of the present disclosure.

[0177] As noted above in the context of conditionally active EpCAM-binding proteins (e.g., EpCAM ProTriTAC), in some embodiments, the conditionally active chimeric antigen receptors described herein have an improved therapeutic index compared to the therapeutic index of a chimeric antigen receptor that contains the same EpCAM-binding domain as a conditionally active variant but is constitutively active instead of conditionally active. For example, EpCAM ProCAR, in some embodiments, has an increased therapeutic index relative to EpCAM CAR. The increase, in some embodiments, is at least about 2-fold to about 1000-fold, e.g., 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, or about 50-fold to about 100-fold. The increased therapeutic index, in some embodiments, results from conjugation of the EpCAM-binding domain to the binding moiety described above via a non-CDR loop and a cleavable linker.

[0178] Methods for generating cells containing conditionally active receptors The present disclosure provides methods for generating cells containing a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor. The methods generally involve genetically modifying mammalian cells containing a nucleotide sequence encoding a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor of the present disclosure using an expression vector or RNA (e.g., in vitro transcribed RNA). The genetic alteration can be performed in vitro, ex vivo, or ex vivo. The cells can be, for example, immune cells (e.g., T lymphocytes or NK cells), stem cells, or progenitor cells.

[0179] In some cases, the genetic modification is performed ex vivo, for example, T lymphocytes, stem cells, or NK cells are obtained from an individual, and the cells obtained from the individual are genetically modified to express a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor of the present disclosure.

[0180] T cell source In some embodiments, the T cell source is obtained from a subject. The term "subject," as used throughout this disclosure, is intended to include a living organism (e.g., a mammal) in which an immune response may be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including, but not limited to, allogeneic T cells (e.g., CAR T cells from an allogeneic donor), natural killer cells (e.g., natural killer cells from a donor), peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cells available in the art may be used. In certain embodiments of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis are washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the present disclosure, cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many, but not all, divalent cations. In the absence of calcium, the initial activation step may result in escalated activation. As those skilled in the art will readily appreciate, the wash step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free PBS, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer.Alternatively, unwanted apheresis sample components may be removed and the cells resuspended directly in culture medium.

[0181] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysis of red blood cells and removal of monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. In one embodiment, this period is approximately 30 minutes. In further embodiments, the period is 30 minutes to 36 hours or more, and all integer values ​​therebetween. In further embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet other embodiments, the period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. Longer incubations may be used to isolate T cells in any situation where T cells are scarce relative to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times can improve the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time period, T cells can bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells may be preferentially positively or negatively selected for or against at the initiation of culture or at other time points during treatment. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells may be preferentially positively or negatively selected for or against at the initiation of culture or at other desired time points. Multiple rounds of selection may also be used within the scope of the present disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected cells" during the activation and expansion process. The "unselected" cells may also be subjected to further selection.

[0182] Enrichment of T cell populations by negative selection can be achieved by a combination of antibodies directed against surface markers specific to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich or positively select regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some embodiments, regulatory T cells are removed by anti-CD25-conjugated beads or other similar selection methods.

[0183] In one embodiment, a T cell population may be selected that expresses one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, for example, by the methods described in PCT International Publication No. WO2013 / 126712.

[0184] In isolating a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increasing the cell concentration) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In another embodiment, 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 million or 150 million cells / ml may be used. The use of higher concentrations may increase cell yield, cell activation, and cell expansion. Furthermore, the use of higher cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or from samples containing many tumor cells (e.g., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. For example, the use of higher cell concentrations may allow for more efficient selection of CD8+ T cells, which normally have weak CD28 expression.

[0185] In another embodiment, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), particle-cell interactions are minimized. This selects for cells that express high amounts of the desired antigen bound to the particles. For example, CD4+ T cells express higher levels of CD28 than CD8+ T cells at dilute concentrations and are captured more efficiently. In one embodiment, the cell concentration used is 5 x 10e6 / ml. In another embodiment, the concentration used is about 1 x 10 5 / ml ~ 1 × 10 6 / ml and any integer value therebetween.

[0186] In other embodiments, the cells may be incubated on a rotator at various speeds for various times at 2-10° C., or at room temperature.

[0187] T cells for stimulation may also be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing step provides a more uniform product by removing granulocytes and some monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium 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 medium containing, for example, Hespan and PlasmaLyte A, after which the cells are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen, may also be used.

[0188] In one embodiment, cryopreserved cells are thawed, washed as described herein, and allowed to stand at room temperature for 1 hour before being activated using the methods of the present disclosure.

[0189] It is further contemplated within the context of the present disclosure that a blood sample or apheresis product may be collected from a subject at the same time prior to the need for expanded cells as described herein. As such, a source of expanded cells may be collected at the desired time point, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is obtained from a generally healthy subject. In certain embodiments, a blood sample or apheresis is obtained from a generally healthy subject who is at risk for developing a disease but remains free of disease, and the desired cells are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and later used. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a particular disease as described herein but before treatment. In further embodiments, the cells are isolated from a subject's blood sample or apheresis prior to any number of relevant treatment modalities, including but not limited to, drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

[0190] In further embodiments of the present disclosure, T cells are obtained directly from a patient after treatment that leaves the subject with functional T cells. In this regard, following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of T cells obtained may be optimal or improved in terms of ex vivo expansion capacity immediately after treatment, during the period when the patient is normally recovering from the treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, within the context of the present disclosure, it is contemplated to harvest blood cells, including T cells, dendritic cells, or other hematopoietic cells, during this recovery phase. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and pre-transplant conditioning regimens may be used to create conditions for the subject that favor the repopulation, recirculation, regeneration, and / or expansion of specific cell types, particularly during a predetermined time frame after treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0191] T cell activation and expansion T cells can generally be activated and expanded using methods such as those described in, for example, U.S. Patent 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.

[0192] Generally, T cells of the present disclosure can be expanded by contacting the surface to which they adhere with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody, an antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. To costimulate accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. To stimulate proliferation of either CD4+ or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody are used. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), as well as other methods well 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).

[0193] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells may be provided by different protocols. For example, the agents providing each signal may be in solution or bound to a surface. When bound to a surface, the agents may be bound to the same surface (i.e., in a "cis" configuration) or to different surfaces (i.e., in a "trans" configuration). Alternatively, one agent may be bound to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to the cell surface, and the agent providing the primary activation signal is in solution or bound to a surface. In some embodiments, both agents may be in solution. In one embodiment, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor or antibody, or other binding agent that binds the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, for artificial antigen-presenting cells (aAPCs) contemplated for use in activating and expanding T cells in the present disclosure.

[0194] In one embodiment, the two agents are immobilized either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof. Both agents are immobilized on the same bead with comparable molecular weights. In one embodiment, a 1:1 ratio of each antibody bound to the beads is used for CD4+ T cell expansion and T cell growth. In certain embodiments of the present disclosure, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one particular embodiment, an increase of about 1 to about 3-fold is observed compared to the expansion observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to the beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one embodiment of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the present disclosure, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to the beads is used. In one embodiment, a 1:75 CD3:CD28 ratio of antibody bound to the beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to the beads is used. In one embodiment, a 1:30 CD3:CD28 ratio of antibody bound to the beads is used. In one embodiment, a 1:10 CD3:CD28 ratio of antibody bound to the beads is used. In one embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In one embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

[0195] Particle-to-cell ratios of 1:500 to 500:1, and all integer values ​​therebetween, may be used to stimulate T cells or other target cells. As one skilled in the art will readily appreciate, the particle-to-cell ratio may depend on the particle size relative to the target cells. For example, small beads can bind only a small number of cells, while larger beads can bind a large number of cells. In certain embodiments, cell-to-particle ratios ranging from 1:100 to 100:1, and all integer values ​​therebetween, and in further embodiments, ratios ranging from 1:9 to 9:1, and all integer values ​​therebetween, may be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells resulting in T cell stimulation can vary as described above, with particular values ​​including 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 to T cells. In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In one particular embodiment, the particle-to-cell ratio is 1:5. In further embodiments, the particle-to-cell ratio can vary depending on the day of stimulation. For example, in one embodiment, the particle to cell ratio is 1:1 to 10:1 on day 1, and additional particles are added daily or every other day thereafter at a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition) for up to 10 days. In one particular embodiment, the particle to cell ratio is 1:1 on day 1 of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In one embodiment, particles are added daily or every other day at a ratio of 1:1 on day 1 of stimulation and 1:5 on days 3 and 5 of stimulation. In one embodiment, the particle to cell ratio is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In one embodiment, particles are added daily or every other day at a ratio of 1:1 on day 1 of stimulation and 1:10 on days 3 and 5 of stimulation. Those skilled in the art will recognize that various other ratios may be suitable for use in the present disclosure.Specifically, the ratio varies depending on the size of the particle and the size and type of cell.

[0196] In a further embodiment of the present disclosure, cells such as T cells are combined with drug-coated beads, followed by separating the beads and cells, and culturing the cells. In an alternative embodiment, the drug-coated beads and cells are not separated but are cultured together prior to culturing. In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0197] By way of example, cell surface proteins may be ligated by allowing anti-CD3 and anti-CD28 conjugated paramagnetic beads (3x28 beads) to contact the T cells. In one embodiment, cells (e.g., 10 4 ~10 9T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a 1:1 ratio) are combined in a buffer, such as PBS (without divalent cations such as calcium or magnesium). Again, one of skill in the art can readily recognize that any cell concentration may be used. For example, target cells may be very rare in a sample and comprise as little as 0.01% of the sample, or the entire sample (i.e., 100%) may contain the desired target cells. Thus, any cell number is within the scope of this disclosure. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / ml is used. In one embodiment, greater than 100 million cells / ml is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml are used. In another embodiment, 75, 80, 85, 90, 95, or 100 million cells / ml are used. In further embodiments, concentrations of 125 or 150 million cells / ml may be used. The use of higher concentrations may increase cell yield, cell activation, and cell expansion. Furthermore, the use of higher cell concentrations may more efficiently capture cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are desirable in certain embodiments. For example, the use of higher cell concentrations may more efficiently select CD8+ T cells, which normally have weak CD28 expression.

[0198] In one embodiment of the present disclosure, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value in between. In one embodiment, the mixture may be cultured for 21 days. In one embodiment of the present disclosure, the beads and T cells are cultured together for about 8 days. In one embodiment, the beads and T cells are cultured together for 2-3 days. Multiple stimulations may also be desired, so that the T cell culture time can be 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimal Essential Media or RPMI Media 1640, or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal, bovine, or human serum), interleukin 2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, TNF-α, or other cell growth additives known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, or Oprimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a predetermined set of hormones and / or cytokines in amounts sufficient for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and are not included in cultures of cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air and 5% CO2).

[0199] T cells exposed to various stimulation times may exhibit different characteristics. For example, a typical blood or apheresed peripheral blood mononuclear cell product has a larger helper T cell population (TH, CD4+) than a cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population consisting of TH cells before approximately day 8-9, but after approximately day 8-9, the T cell population contains a progressively larger proportion of TC cells.

[0200] EpCAM-binding protein modification The EpCAM-binding proteins described herein include EpCAM-binding domains (e.g., the EpCAM-binding sdAbs of the present disclosure) and multispecific proteins that target EpCAM (e.g., trispecific or trispecific promoting proteins that target EpCAM as described herein), and encompass derivatives or analogs in which (i) amino acids are substituted with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused to other compounds such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader sequence, secretory sequence, or sequence for protein purification.

[0201] Exemplary 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 cross-links, 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.

[0202] Modifications may occur anywhere in the EpCAM-binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Specific peptide modifications commonly used to modify EpCAM-binding proteins include glycosylation of glutamic acid residues, lipid attachment, sulfation, gamma-carboxylation, hydroxylation, blocking of amino or carboxyl groups, or both, in the polypeptide by covalent modification, and ADP-ribosylation.

[0203] In some embodiments, derivatives of EpCAM binding proteins as described herein include immunoreactive modulator derivatives and antigen-binding molecules that contain one or more modifications.

[0204] In some embodiments, the EpCAM-binding proteins of the present disclosure are monovalent or multivalent (bivalent, trivalent, etc.). As used herein, the term "valency" refers to the number of possible target binding sites associated with an antibody. Each target binding site specifically binds to one target molecule or a specific position or locus on a target molecule. When an antibody is monovalent, each binding site on the molecule specifically binds to a single antigen position or epitope. When an antibody contains more than one target binding site (multivalent), each target binding site may specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes or positions on the same antigen).

[0205] In some embodiments, an EpCAM-binding protein as described above is fused to an Fc region from any species, including, but not limited to, human immunoglobulins such as human IgG1, human IgG2, human IgG3, and human IgG4, to generate an Fc-fusion EpCAM-binding protein. In some embodiments, the Fc-fusion EpCAM-binding protein of the present disclosure has an increased half-life compared to an otherwise identical EpCAM-binding protein. In some embodiments, the Fc-fusion EpCAM-binding protein of the present disclosure includes substitutions, mutations, and / or modifications of one or more additional amino acid residues, e.g., in the Fc region, among other things, resulting in a binding protein with favorable characteristics, including, but not limited to, improved pharmacokinetics and extended serum half-life.

[0206] In some embodiments, such Fc-fusion EpCAM-binding proteins provide a long half-life in mammals, such as humans, 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. In some cases, the increased half-life results in higher serum titers, which allow for less frequent administration of the EpCAM-binding protein and / or a lower concentration of the administered antibody. In vivo binding to human FcRn and serum half-life of human FcRn high-affinity-binding polypeptides are, in some examples, assayed in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with different Fc regions are administered.

[0207] In some cases, the EpCAM-binding proteins are differentially modified during or after production, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to antibody molecules or other cellular ligands, etc. Any of a number of chemical modifications may be achieved by techniques including, but not limited to, specific chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0208] Additionally, various post-translational modifications of EpCAM-binding proteins encompassed by the present disclosure include, for example, N- or O-linked carbohydrate chains, N- or C-terminal processing, conjugation of chemical moieties to the amino acid backbone, chemical modification of N- or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. Additionally, EpCAM-binding proteins are optionally modified with a detectable label, such as an enzymatic, fluorescent, radioisotope, or affinity label, to allow for detection and isolation of modulators.

[0209] Polynucleotides encoding EpCAM-binding proteins Also provided in some embodiments are polynucleotide molecules encoding the EpCAM-binding proteins described herein. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0210] The polynucleotide molecule is constructed by known methods, such as by combining genes encoding a single-domain EpCAM-binding protein or genes encoding various domains of an EpCAM-binding protein containing more than one domain. In some embodiments, the genes encoding the domains are separated by a peptide linker, or in other embodiments, directly linked by a peptide bond, into a single gene construct that is operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressed in bacteria or other suitable expression systems, such as CHO cells. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected to drive expression of the polynucleotide in the respective host cell.

[0211] In some embodiments, a polynucleotide encoding an EpCAM-binding protein as described herein is inserted into a vector, preferably an expression vector, representing a further embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0212] A variety of expression vector / host systems may be utilized to contain and express the polynucleotides encoding the described EpCAM-binding protein polypeptides. Examples of expression vectors for expression in E. coli include pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells. Thus, in some embodiments, EpCAM-binding proteins as described herein are produced by introducing a vector encoding such a protein into a host cell and culturing the host cell under conditions in which the protein domain is expressed, may be isolated, and optionally further purified.

[0213] Pharmaceutical Composition Some embodiments also provide pharmaceutical compositions comprising the EpCAM-binding proteins described herein, vectors containing polynucleotides encoding the EpCAM-binding protein polypeptides, or host cells transformed with the vectors and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the components and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and administered to a subject in an appropriate dosage. Preferably, the compositions are sterile. These compositions may further include adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents. Further embodiments provide one or more of the above-mentioned EpCAM-binding proteins packaged in lyophilized form or in an aqueous medium.

[0214] In some embodiments of the pharmaceutical composition, the EpCAM-binding protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the EpCAM-binding protein is bound to a liposome. In some examples, the EpCAM-binding protein is conjugated to the surface of the liposome. In some examples, the EpCAM-binding protein is encapsulated within the shell of the liposome. In some examples, the liposome is a cationic liposome.

[0215] The EpCAM-binding proteins described herein are intended for use as drugs. Administration can be achieved by various methods, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound included in the pharmaceutical composition. The administration regimen will be determined by the attending physician and other clinical factors. The dose for a single patient will depend on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time of administration, the route of administration, the type of treatment, health status, and other drugs administered concomitantly. An "effective amount" refers to the amount of active ingredient sufficient to affect the course and severity of the disease, resulting in a reduction or remission of such symptoms, and may be determined using known methods.

[0216] In some embodiments, the EpCAM-binding protein of the disclosure is administered at a dose of up to 10 mg / kg once a week. Optionally, the dose ranges from about 1 ng / kg to about 10 mg / kg, e.g., from about 1 ng / kg to about 70 ng / kg. In some embodiments, the dosage ranges from about 1 ng / kg to about 10 ng / kg, about 5 ng / kg to about 15 ng / kg, about 12 ng / kg to about 20 ng / kg, about 18 ng / kg to about 30 ng / kg, about 25 ng / kg to about 50 ng / kg, about 35 ng / kg to about 60 ng / kg, about 45 ng / kg to about 70 ng / kg, about 65 ng / kg to about 85 ng / kg, about 80 ng / kg to about 1 μg / kg, about 0.5 μg / kg ~ approx. 5 μg / kg, approx. 2 μg / kg ~ approx. 10 μg / kg, approx. 7 μg / kg ~ approx. 15 μg / kg, approx. 12 μg / kg ~ approx. 25 μg / kg, approx. 20 μg / kg ~ approx. 50 μg / kg, approx. 35 μg / kg kg to about 70 μg / kg, about 45 μg / kg to about 80 μg / kg, about 65 μg / kg to about 90 μg / kg, about 85 μg / kg to about 0.1 mg / kg, and about 0.095 mg / kg to about 10 mg / kg. In some embodiments, 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 administration frequency, in some embodiments, is about less than daily, every other day, less than once daily, twice weekly, weekly, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once monthly. In some embodiments, the administration frequency is weekly. Optionally, the administration frequency is weekly, and the dose is up to 10 mg / kg. Optionally, the administration period is from about 1 day to about 4 weeks or longer.

[0217] Treatment Methods and Tumor Growth Reduction Properties In certain embodiments, there is also provided a method of treating a disease associated with malignant cells expressing EpCAM in a subject, the method comprising administering to a subject in need thereof an effective amount of an EpCAM-binding domain of the present disclosure or a multispecific protein (optionally including an active multispecific protein) comprising the EpCAM-binding domain, a CAR or ProCAR comprising an EpCAM-binding protein as described herein, or a pharmaceutical composition comprising any of the foregoing. In some embodiments, the disease is cancer.

[0218] In another aspect, the present disclosure provides a method for inhibiting tumor growth or progression in a subject harboring malignant cells expressing EpCAM, comprising administering to a subject in need thereof an effective amount of an EpCAM-binding domain of the present disclosure or a multispecific protein comprising the EpCAM-binding domain, a CAR comprising an EpCAM-binding protein as described herein, or a pharmaceutical composition comprising the same. In another aspect, the present disclosure provides a method for inhibiting metastasis of malignant cells expressing EpCAM in a subject, comprising administering to a subject in need thereof an effective amount of an EpCAM-binding domain of the present disclosure or a multispecific protein comprising the EpCAM-binding domain, or a pharmaceutical composition comprising the same. In another aspect, the present disclosure provides a method for inducing tumor regression in a subject harboring malignant cells expressing EpCAM, comprising administering to a subject in need thereof an effective amount of an EpCAM-binding domain of the present disclosure or a multispecific protein comprising the EpCAM-binding domain, or a pharmaceutical composition comprising the same. In some embodiments, the methods described herein further comprise administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is a biological drug, such as an antibody. In some embodiments, the second therapeutic agent is a cytokine, TNFa (tumor necrosis factor alpha), a PAP (phosphatidic acid phosphatase) 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 therapeutic, a TLR (toll-like receptor) agonist (e.g., TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine.

[0219] In certain embodiments, an EpCAM-binding protein of the present disclosure reduces tumor cell proliferation in vivo when administered to a subject harboring tumor cells that express EpCAM. Measurement of reduced tumor cell proliferation can be determined by several different methods well known in the art. Non-limiting examples include direct measurement of tumor size, measurement of a resected tumor mass compared to a control, and measurement via imaging techniques (e.g., CT or MRI) with or without the use of isotopes or luminescent molecules (e.g., luciferase) to enhance analysis. In specific embodiments, administration of an EpCAM-binding protein of the present disclosure reduces in vivo tumor cell proliferation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control antigen-binding agent, with a reduction of about 100% tumor growth indicating complete tumor remission and disappearance. In further embodiments, administration of an EpCAM binding protein of the present disclosure reduces in vivo tumor cell proliferation by about 50-100%, about 75-100%, or about 90-100% compared to a control antigen binding agent. In further embodiments, administration of an EpCAM binding protein of the present disclosure reduces in vivo tumor cell proliferation by about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% compared to a control antigen binding agent.

[0220] In some embodiments, the EpCAM-binding proteins of the present disclosure are administered to treat a neoplastic disease, which in some embodiments is a benign or malignant, solid tumor or other hematologic neoplasia, and in some embodiments is selected from the group including, but not limited to, adrenal gland tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, autonomic ganglionic tumors, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocoelic disorders, bone cancer (adamantinoma, aneurysmal bone cyst, osteochondroma, osteosarcoma), brain and spinal cord cancer, metastatic brain tumors, breast cancer, including triple-negative breast cancer, carotid globe tumors, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, epithelial disorders, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibroplastic osteogenesis imperfecta ossium), fibrous dysplasia of bone, gallbladder and bile duct cancer, stomach cancer, gastrointestinal cancer, gestational trophoblastic disease, germ cell tumors, glandular disorders, head and neck cancer, hypothalamic tumors, intestinal cancer, islet cell tumors, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma / benign fatty tumors, liposarcoma / malignant fatty tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), macrophage disorders disorders), medulloblastoma, melanoma, meningioma, multiple endocrine adenoma, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, phaeochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare blood disorders, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, stromal disorders, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer (cervical cancer, endometrial cancer, and leiomyoma).

[0221] In certain embodiments, the EpCAM binding proteins of the present disclosure are used as first-line therapy and are administered to subjects who have not previously been treated for cancerous disease. In other embodiments, the EpCAM binding proteins of the present disclosure are used to treat subjects who have been previously treated (with the EpCAM binding proteins of the present disclosure or other anti-cancer agents) or who have relapsed or are deemed refractory to previous treatment. In some embodiments, the EpCAM binding proteins of the present disclosure are used to treat subjects whose tumors have recurred.

[0222] In some embodiments, an EpCAM binding protein as described herein, including a multispecific protein, a CAR, or a ProCAR as described herein, is administered to treat cancers with widespread EpCAM expression and prevalence, including, but not limited to, colon, prostate, neuroendocrine, thyroid, lung (both non-small cell and small cell lung cancer), gastric, ovarian, endometrial, pancreatic, bile duct, gallbladder cancer, esophageal, breast, and all adenocarcinomas.

[0223] In some embodiments, the EpCAM-binding proteins of the present disclosure are administered to treat proliferative disorders, including solid tumors, including, but not limited to, adrenal gland, liver, kidney, bladder, breast, stomach, ovary, cervix, uterus, esophagus, colon, prostate, pancreas, lung (both non-small cell lung and small cell lung), thyroid, carcinoma, sarcoma, glioblastoma, and various head and neck masses.

[0224] In some embodiments, the EpCAM-binding protein of the present disclosure is administered to a subject with melanoma. In some embodiments, the EpCAM-binding protein of the present disclosure is used to diagnose, monitor, treat, or prevent melanoma. The term "melanoma," as used herein, includes any type of melanoma, including but not limited to primary melanoma, malignant melanoma, cutaneous melanoma, extradermal melanoma, superficial spreading melanoma, polypoid melanoma, melanoma, acanthosis nigricans, melanosarcoma, melanomatous in situ nodular malignant melanoma, lentigo maligna melanoma, lentigo melanoma, lentigo malignant melanoma, mucosal lentigo melanoma, mucosal melanoma, acral lentigo melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical lentigo and melanoma (FAM-M) syndrome, fibrosclerosing malignant melanoma, or uveal melanoma.

[0225] In some embodiments, candidate indications for administration of the EpCAM-binding protein of the present disclosure or a pharmaceutical composition comprising the same are tumor diseases, particularly epithelial cancers / carcinomas such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, genitourinary cancer, e.g., ovarian cancer, endometrial cancer, uterine cancer, and kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer. In some embodiments, administration of the EpCAM-binding protein of the present disclosure or a pharmaceutical composition comprising the same is indicated for minimal residual disease, characterized by local and non-local recurrence of tumors caused by single-cell survival, such as early-stage solid tumors, advanced solid tumors, or metastatic solid tumors.

[0226] In selected embodiments, the EpCAM binding proteins of the present disclosure are incorporated into chimeric antigen receptors (CARs), and the EpCAM CARs are administered in CAR-based therapeutics effective in treating cancers such as epithelial cancers / carcinomas such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, genitourinary cancers, e.g., ovarian cancer, endometrial cancer, uterine cancer, and kidney cancer, lung cancer, stomach cancer, cancer of the small intestine, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer, 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), and large cell neuroendocrine tumors (LCNEC).

[0227] Chimeric antigen receptors are generally artificially constructed hybrid proteins or polypeptides that encompass or contain the antigen-binding domain of an antibody linked to a signaling domain (e.g., a T cell signaling or T cell activation domain). In some embodiments, CARs comprising the EpCAM-binding proteins of the present disclosure have the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T cells) to EpCAM-positive target cells in a non-MHC-restricted manner by utilizing the antigen-binding properties of an antibody or its antigen-binding fragment. Non-MHC-restricted antigen recognition confers oncogenic EpCAM-CAR-expressing T cells the ability to recognize tumorigenic EpCAM independently of antigen processing, thereby circumventing a major tumor escape mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) α and β chains.

[0228] In some embodiments, the disclosed EpCAM binding proteins are administered to refractory patients (i.e., patients whose disease recurs during or shortly after completing an initial course of treatment), sensitive patients (i.e., patients whose recurrence is greater than 2-3 months after first-line treatment), or patients who tolerate platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, the disclosed EpCAM CAR treatment is effective in treating ovarian cancer, including serous ovarian carcinoma and papillary serous ovarian carcinoma.

[0229] In another embodiment, the EpCAM binding protein, EpCAM CAR, or EpCAM-sensitized lymphocytes of the present disclosure, or any combination thereof, is used in maintenance therapy to reduce or eliminate the possibility of tumor recurrence after the initial symptoms of the disease. Optionally, the disorder is treated, and the initial tumor mass is eliminated, reduced, or otherwise improved so that the patient is asymptomatic or in remission. At that time, the subject is administered one or more pharmaceutically effective doses of the EpCAM binding protein, EpCAM CAR, or EpCAM-sensitized lymphocytes of the present disclosure, or any combination thereof, regardless of whether there is little or no indication of disease using standard diagnostic procedures. In some embodiments, the EpCAM binding protein, EpCAM CAR, or EpCAM-sensitized lymphocytes of the present disclosure, 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 every year, to reduce the possibility of disease recurrence. Moreover, in some embodiments, such treatment is continued for weeks, months, years, or even indefinitely, depending on patient response and clinical and diagnostic parameters.

[0230] In yet other embodiments, the disclosed EpCAM binding protein, EpCAM CAR, or EpCAM-sensitized lymphocytes, or any combination thereof, are used prophylactically or as adjuvant therapy to prevent or reduce the likelihood of tumor metastasis following a debulking procedure. As used in this disclosure, "debulking procedure" refers to a procedure, technique, or method that eliminates, reduces, treats, or ameliorates a tumor or its growth. Exemplary debulking procedures include, but are not limited to, surgery, radiation therapy (i.e., beam radiation), chemotherapy, immunotherapy, or ablation. In some embodiments, at the appropriate time, the disclosed EpCAM binding protein, EpCAM CAR, or EpCAM-sensitized lymphocytes, or any combination thereof, is administered as indicated by a clinical, diagnostic, or theranostic procedure to reduce tumor metastasis. In some embodiments, the administration regimen is accompanied by appropriate diagnostic or monitoring techniques that allow for its modification.

[0231] Still other embodiments of the present disclosure include administering an EpCAM-binding protein, EpCAM CAR, or EpCAM-sensitized lymphocyte of the present disclosure, or any combination thereof, to a subject who is asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, an EpCAM-binding protein, EpCAM CAR, or EpCAM-sensitized lymphocyte of the present disclosure, or any combination thereof, is used in a prophylactic sense and is administered to a patient who has been examined or tested for and has one or more noted risk factors (e.g., genomic indications, family history, in vivo or in vitro test results, etc.) but has not yet developed neoplasia. In such cases, one of skill in the art can determine an effective dosing regimen through empirical observation or accepted clinical practice.

[0232] In some embodiments of the methods described herein, an EpCAM-binding protein or composition as described herein is administered in conjunction with an agent for the treatment of a particular disease, disorder, or condition. Agents include, but are not limited to, antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiation therapy (directed delivery of gamma rays, X-rays, and / or radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapeutic treatments. In some embodiments, an EpCAM-binding protein as described herein is administered in combination with an antidiarrheal, antiemetic, analgesic, opioid, and / or nonsteroidal anti-inflammatory drug. In some embodiments, an EpCAM-binding protein as described herein is administered in combination with an anti-cancer agent. Non-limiting examples of anti-cancer agents that can be used in various embodiments of the present disclosure, including the pharmaceutical compositions, dosage forms, and kits of the present disclosure, include: acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, aztomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, biceresin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cacti nomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelcin, cedefingal, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexoromaplatin, dezaguamine, dezaguamine mesylate, diazicon, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride,Elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etopurine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, foskidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride lido, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansin , mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitodilline, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxislan, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perphos Famid, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimastine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurine, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromastine, spiroplatin, streptonigrin, streptozocin,Surofenal, tallysomycin, tecogalan sodium, tegafur, trexatrone hydrochloride, temoporfin, teniposide, teloxylone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestrone acetate, tricibirine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurodin sulfate, vinorelbine tartrate, vinzoquidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride. Examples of other anti-cancer agents include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, akylfulvene, adecipenol, adozelesin, aldesleukin, ALL-TK antagonist, altretamine, ambamastine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1 protein-1), antiandrogens, antiestrogens, antineoplastons, antisense oligonucleotides for prostate cancer, aphidicolin glycinate, apoptosis gene modulators, cell death regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimastine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxins, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzotyroline, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistraten A, biceresin, brephrateBropirimine, budotitanium, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canaripox IL-2, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage-derived inhibitor, carzelcin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogs, clotrimazole, colismycin A, colismycin B, combretastatin A4, combretastatin analogs, conagenin, crambecidin 816, crisnatol, kryptof Cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentaanthraquinone, cycloplatam, sipemycin, cytarabine octophosphate, cytotoxic factors, cytostatin, daclizumab, decitabine, dehydrodidemin B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diazicon, dididemin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol 9-, dioxamycin, diphenylspiromastine, docetaxel, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epristeride, estramustine analogs, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole , fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunornithine hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texapyrrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hapsulfame, heregulin, hexamethylene bisacetamide,Hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilmostat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor I receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol 4-, ilopract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, Kahala Leptolstatin F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprolide, levamisole, liarozole, linear polyamine analogs, lipophilic disaccharide peptides, lipophilic platinum compounds, lissoclinamide, lobaplatin, lombricine, lometerexol, lonidamine, losoxantrone, HMG-CoA reductase inhibitors (limited (including but not limited to lovastatin, pravastatin, fluvastatin, statins, simvastatin, and atorvastatin), loxoribine, raltotecan, lutetium texapyrin, lisofylline, cytolytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, melbarone, metalarelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, mirimostim, mismatched double-stranded RNA, mitoguazone , Mitolactol, Mitomycin analogue, Mitonafide, Mitotoxin fibroblast growth factor-saporin, Mitoxantrone, Mofalotene, Molgramostim, Monoclonal antibody, Human placental gonadotropin, Monophosphoryl lipid A + Myobacterial cell wall sk, Mopidamol, Multidrug resistance gene inhibitor, Multiple tumor suppressor gene 1-based therapeutic agent, Mustard anticancer drug, Mycaperoxide B, Mycobacterium cell wall extract, Mirapolon, N-acetyldinaline, N-substituted benzamide, Nafarelin, Nagrestipp, Naloxone + Pentazocine,Napavine, naphterpin, nartograstim, nedaplatin, nemorubicin, nylidronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidants, nitrulline, O6-benzylguanine, octreotide, oxenon, oligonucleotides, , onapristone, ondansetron, ondansetron, oracin, oral cytokine inducers, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronate, panaxytriol, panomyphen, parabactin, pazelliptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, penttrozole, perflubron, perfosfamide, peryl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen Activator inhibitors, platinum complexes, platinum compounds, platinum triamine complexes, porfimer sodium, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, microalgae protein kinase C inhibitors, tyrosine phosphatase protein inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridines, pyridoxylated hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitors, demethylated reterliptin, rhenium Re 186 Etidronate, Rhizoxin, Ribozyme, RII retinamide, Rogletimide, Rohitukin, Romurtide, Roquinimex, Ravidinone B1, Lavoxil, Safingol, Saintpin, SarCNU, Sarcophytol A, Sargramostim, Sdi1 mimics, semustine, senescence-derived inhibitors 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single-stranded antigen binding proteins, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, salvalor, somatomedin binding proteins, sonarmin, sparfosic acid, spicamycin D, spiromastine, splenopentin, spongistatin 1, squalamine, stem cell inhibitors, stem cell division inhibitors, stipiamid, stromelysin inhibitors, sulfinodine, superactive vasoactive intestinal peptide antagonists, saladista, suramin, swainsonine, synthetic glycosaminoglycans, talimustine, tamoxifen methiodide, tauromastine, tazarotene, tecogalan sodium, tegafur, terlapyrium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazolium thrombopoietin, saliblastin, thiocoraline, thrombopoietin, thrombopoietin mimetics, thymalfadine, thymopoietin receptor agonist, thymotrinan, thyroid-stimulating hormone, tin ethyl etioproprine, tirapazamine, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, tricibirine, trimetrexate, triptorelin, tropisetron, turosteride , tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitors, urokinase receptor antagonists, vapreotide, variolin B, vector systems, erythroid gene therapy drugs, veraresol, veramine, turicola, verteporfin, vinorelbine, vinzartine, Vitaxin®, vorozole, zanoterone, zeniplatin, zilascorub, and zinostatin stimalamer. Additional anticancer drugs include 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods utilizing thalidomide and topoisomerase inhibitors. In some embodiments, the EpCAM binding protein of the present disclosure is combined with gemcitabine. In some embodiments, the EpCAM binding protein as described herein is administered before, during, or after surgery.

[0233] Methods for detecting EpCAM expression and diagnosing EpCAM-associated cancers According to another embodiment of the present disclosure, a kit for detecting EpCAM expression in vitro or in vivo is provided. The kit includes the EpCAM-binding protein (e.g., an EpCAM-binding protein containing a labeled anti-EpCAM single-domain antibody or antigen-binding fragment thereof) and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0234] In some cases, EpCAM expression is detected in biological sample.Sample can be any sample, including but not limited to tissue from biopsy, autopsy and pathological specimen.Biological sample also includes tissue section, for example, frozen section taken for histological purpose.Biological sample also includes body fluids such as blood, serum, plasma, sputum, cerebrospinal fluid or urine.Biological sample is generally obtained from mammals such as human or non-human primates.

[0235] In one embodiment, a method is provided for determining whether a subject has cancer by contacting a sample from the subject with an anti-EpCAM single domain antibody as disclosed herein and detecting binding of the single domain antibody to the sample. If binding of the antibody to the sample is increased compared to binding of the antibody to a control sample, the subject is identified as having cancer.

[0236] In another embodiment, a method is provided for confirming a diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with an anti-EpCAM single domain antibody as disclosed herein and detecting binding of the antibody to the sample. If binding of the antibody to the sample is increased compared to binding of the antibody to a control sample, the diagnosis of cancer in the subject is confirmed.

[0237] In some examples of the disclosed method, the EpCAM single domain antibody is directly labeled. In some examples, the method further includes contacting the sample with a second antibody that specifically binds to the anti-EpCAM single domain antibody and detecting the binding of the second antibody. If the binding of the second antibody to the sample is increased compared to the binding of the second antibody to a control sample, cancer is detected in the subject or the diagnosis of cancer in the subject is confirmed. In some cases, the cancer is neuroendocrine cancer, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, intrahepatic cholangiocarcinoma, triple-negative breast cancer or ovarian cancer (such as epithelial ovarian carcinoma), or any other type of cancer that expresses EpCAM. In some examples, the control sample is a sample from a subject without cancer. In certain examples, the sample is a blood or tissue sample.

[0238] In some cases, the antibody that binds (e.g., specifically binds) EpCAM is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) EpCAM (the first antibody) is unlabeled, and a second antibody or other molecule capable of binding to the antibody that specifically binds EpCAM is labeled. The second antibody is selected to specifically bind to the specific species and class of the first antibody. For example, if the first antibody is a llama IgG, the second antibody may be an anti-llama IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for antibodies or second antibodies are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol, a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive materials include 125I, 131I, 35S, or 3H.

[0239] In an alternative embodiment, EpCAM can be assayed in a biological sample by a competitive immunoassay that utilizes an EpCAM standard labeled with a detectable substance and an unlabeled antibody that specifically binds to EpCAM. In this assay, the biological sample, the labeled EpCAM standard, and the antibody that specifically binds to EpCAM are combined, and the amount of labeled EpCAM standard that is bound to the unlabeled antibody is determined. The amount of EpCAM in the biological sample is inversely proportional to the amount of labeled EpCAM standard that is bound to the antibody that specifically binds to EpCAM.

[0240] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, an antibody that specifically binds to EpCAM may be used to detect the production of EpCAM in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of EpCAM in a biological sample, such as a tissue sample, a blood sample, or a serum sample. In some examples, the EpCAM is cell-surface EpCAM. In other examples, the EpCAM is soluble EpCAM (e.g., EpCAM in a cell culture supernatant or soluble EpCAM in a body fluid sample, such as a blood sample or a serum sample).

[0241] In one embodiment, a kit is provided for detecting EpCAM in a biological sample, such as a blood sample or a tissue sample. For example, to confirm a subject's cancer diagnosis, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained and the presence of soluble EpCAM protein or fragments can be detected. Kits for detecting polypeptides generally include a single domain antibody according to the present disclosure that specifically binds to EpCAM. In some embodiments, the kit includes an antibody fragment, such as an scFv fragment, a VH domain, or a Fab. In further embodiments, the antibody is labeled (e.g., with a fluorescent label, a radiolabel, or an enzyme label).

[0242] In one embodiment, the kit includes instructional materials disclosing means for using antibodies that bind to EpCAM. The instructional materials can be written in electronic format (e.g., computer disk or compact disk), viewable (e.g., video file), or provided via an electronic network over the Internet, World Wide Web, intranet, or other network. The kit can also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit can additionally include means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a second antibody, etc.). The kit can additionally include buffers and other reagents routinely used in practicing a particular method. Such kits and suitable contents are well known to those of skill in the art.

[0243] In one embodiment, the diagnostic kit comprises an immunoassay. While the details of the immunoassay may vary depending on the particular format utilized, methods for detecting EpCAM in a biological sample generally involve contacting the biological sample with an antibody that specifically reacts with an EpCAM polypeptide under immunologically reactive conditions. 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.

[0244] Methods for determining the presence or absence of cell surface markers are well known in the art. For example, antibodies may be conjugated to other compounds, including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorescent dyes, metal compounds, radioactive compounds, or drugs. Antibodies can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemistry assays. Antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS utilizes multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels to sort or select cells, especially at more sophisticated detection levels. See U.S. Patent No. 5,061,620. Any of the single-domain antibodies that bind to EPCAM as disclosed herein can be used in these assays. Thus, antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation. [Example]

[0245] Example 1: Screening of a phage display library for identification of EpCAM-binding domains

[0246] Llamas were immunized with purified EpCAM protein expressed in Expi293 cells. A phage display library for expression of heavy variable antibody domains was constructed from circulating B cells. See van der Linden, de Geus, Stok, Bos, van Wassenaar, Verrips, and Frenken. 2000. J Immunol Methods 240:185-195. Phage clones were screened for binding to EpCAM by expressing anti-EpCAM protein in E. coli and preparing periplasmic extracts. Proteins were screened for human and cynomolgus EpCAM binding activity using colorimetric ELISA. Thirty-eight unique heavy chain-only sequences (SEQ ID NOS: 1-38) that generated signals in ELISA screening relative to controls with human and / or cynomolgus EpCAM protein (as shown in Table 2) were identified. The CDR1, CDR2, and CDR3 sequences for these heavy variable domains are SEQ ID NOs: 39 to 76, 77 to 114, and 115 to 152, respectively.

[0247] [Table 3]

[0248] Example 2: Incorporation of only a single domain antibody of the EpCAM-binding heavy chain into a fusion protein and T-cell dependent cytotoxicity assay Only the anti-EpCAM heavy chain single domain antibodies selected from Example 1 were cloned into DNA constructs for recombinant protein expression. All of these expression constructs encoded a signal peptide. One set of anti-EpCAM constructs (SEQ ID NOs: 153-179) was designed to express a fusion protein with a humanized anti-CD3 scFv domain at the N-terminus of the mature secreted fusion protein, followed by a llama anti-EpCAM domain, the two domains linked by the sequence GGGGSGGGS, and HHHHHHH at the C-terminus. Another set of anti-EpCAM constructs (SEQ ID NOs: 180-1206) was designed to express a fusion protein with a llama anti-EpCAM domain at the N-terminus of the mature secreted fusion protein, followed by a humanized anti-CD3-scFv domain, the two domains linked by the sequence GGGGSGGGS, and HHHHHHH at the C-terminus.

[0249] These anti-EpCAM / anti-CD3 (N- to C-terminus) or anti-CD3 / anti-EpCAM (N- to C-terminus) fusion protein constructs were transfected into Expi293 cells. The amount of anti-EpCAM / anti-CD3 fusion protein in the conditioned medium from transfected Expi293 cells was quantified using an Octet instrument with streptavidin, and biotinylated CD3-Fc fusion protein was loaded using an anti-CD3 fusion protein of equivalent molecular weight to the anti-EpCAM / anti-CD3 protein as a standard.

[0250] The conditioned medium described above was tested in a T cell-dependent cytotoxicity assay. See Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27. In this assay, luciferase-labeled NCI-H508 cells expressing EpCAM were combined with purified human T cells and titrated with anti-EpCAM / anti-CD3 fusion protein or anti-CD3 / anti-EpCAM. It was hypothesized that if the fusion protein directed T cells to kill NCI-H508 cells, the signal in the luciferase assay performed 48 hours after the start of the experiment would decrease. Figures 1-4 provide the TDCC data in graphical format.

[0251] ECs from TDCC assay 50 The values ​​are shown in Table 3 (EC for SEQ ID NOs: 153 to 179) 50 (Listing data) and Table 4 (EC for SEQ ID NOS: 180-206) 50 The EC of the most potent molecule (EPL13) is listed in the table below. 50 The value was approximately 1.6 pM. Some of the anti-EpCAM binding proteins were active only when present in an anti-CD3 / anti-EpCAM configuration. One anti-EPCAM sequence, EPL34, was active only in an anti-EpCAM / anti-CD3 configuration. The negative control for the TDCC assay was anti-GFP / anti-CD3 protein, which did not induce T cells to kill NCI-H508 cells (data not shown).

[0252] [Table 4]

[0253] [Table 5]

[0254] The binding affinity of the fusion proteins to human and cynomolgus monkey EpCAM proteins was measured using conditioned medium with known concentrations of anti-EpCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion proteins. The K was determined by loading biotinylated human or cynomolgus monkey EpCAM proteins into an Octet device equipped with a streptavidin tip and measuring the on- and off-rates of binding of anti-EpCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion proteins to the biotinylated EpCAM protein. D K values ​​were calculated using a single 50 nM concentration of anti-EPCAM / anti-CD3 or anti-CD3 / anti-EpCAM fusion protein. D Measurements were performed, which allowed for a ranking of potencies. The measured relative affinities are listed in Table 5. All fusion proteins bound to cynomolgus EpCAM, with K D The K values ​​ranged from 1.6 to 56 nM. Most, but not all, of the fusion proteins bound to human EpCAM. D The values ​​were 0.8-74 ​​nM.

[0255] [Table 6]

[0256] Example 3: Humanization of EpCAM-binding heavy chain single domain antibodies only and T cell dependent cytotoxicity assay Three of the llama anti-EpCAM antibody sequences identified in Example 1 were humanized (SEQ ID NOs: 207-209) by grafting their CDR sequences onto a human germline sequence antibody while retaining some llama framework sequences to ensure the antibody did not lose activity.

[0257] As described in Example 2, these sequences were cloned into an expression construct for expression of an anti-EpCAM / anti-CD3 fusion protein (SEQ ID NOs: 210-212) in Expi293 cells.

[0258] The amount of anti-EpCAM / anti-CD3 fusion protein present in the conditioned medium was quantified as described in Example 2. The affinity of these humanized proteins for human, cynomolgus, and mouse EpCAM was measured as described in Example 2. The relative K calculated from these measurements D The values ​​are listed in Table 6. All three sequences bind to human and cynomolgus EpCAM, with relative K D The K values ​​ranged from about 0.3 to about 18 nM. Two of these sequences further bound to mouse EpCAM, D The values ​​were approximately 1.4 to 1.8 nM.

[0259] [Table 7]

[0260] The T cell killing potential of the anti-EpCAM / anti-CD3 fusion protein present in the conditioned medium was evaluated as described in Example 2. The results are provided in Table 7 and FIG.

[0261] [Table 8]

[0262] Example 4: Xenograft tumor model The EpCAM-targeting fusion proteins of the present disclosure (e.g., fusion proteins that are trispecific proteins containing only an anti-EpCAM heavy chain single domain antibody, an anti-CD3 scFv, and an anti-albumin domain) are evaluated in xenograft models. To determine the efficacy of exemplary EpCAM-targeting fusion proteins in vivo, multiple xenograft tumor models are used. Examples of common tumor cell lines used in xenograft tumor studies include A549 (non-small cell lung cancer) cells, DU-145 (prostate) cells, MCF-7 (breast) cells, Colo205 (colon) cells, 3T3 / GF-IR (mouse fibroblast) cells, NCI H441 cells, HEP G2 (stem cell) cells, MDA-MB 231 (breast) cells, HT-29 (colon) cells, MDA-MB-435s (breast) cells, U266 cells, SH-SYSY cells, Sk-Mel-2 cells, NCI-H929, RPM18226, and A431 cells. Immunodeficient NOD / SCID mice were sublethally irradiated (2 Gy) and 1X10 6 Tumor cells (e.g., NCI H441 cells) are inoculated subcutaneously into the right dorsal flank. Tumors grow to 100–200 mm. 3 When the number of mice reached 1.5x10, the mice were assigned to three treatment groups: groups 2 and 3, 7 of activated human T cells are injected intraperitoneally. Three days later, animals in group 3 are subsequently administered an exemplary trispecific antigen binding protein targeting EpCAM. Groups 1 and 2 are administered vehicle only. Body weights and tumor volumes are determined for 30 days, starting at least 5 days after administration of the exemplary trispecific protein targeting EpCAM.

[0263] Mice treated with exemplary EpCAM-targeting trispecific proteins are expected to experience a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0264] Example 5: Proof-of-concept clinical trial protocol for administration of the trispecific antigen binding protein targeting EpCAM of Example 4 to patients with ovarian cancer This study is a Phase I / II clinical trial to test an exemplary EpCAM-targeting trispecific antigen binding protein of the present disclosure as a treatment for epithelial ovarian cancer.

[0265] Study outcomes:

[0266] Primary outcome: Maximum tolerated dose of exemplary EpCAM-targeting trispecific protein.

[0267] Secondary Outcomes: Determine whether the in vitro response of exemplary EpCAM-targeting trispecific proteins correlates with clinical response.

[0268] Phase I

[0269] The maximum tolerated dose (MTD) will be determined in the Phase I portion of this study. 1.1 The maximum tolerated dose (MTD) will be determined in the Phase I portion of this study. 1.2 Patients who meet the selection criteria will be enrolled in a trial against the trispecific protein targeting EpCAM of the previous example. 1.3 The goal is to identify the highest dose of the trispecific protein targeting EpCAM from the previous example that can be safely administered to participants without causing severe or unmanageable side effects. The dose administered will depend on the number of participants previously enrolled in the study and how well the dose is tolerated. Not all participants will receive the same dose.

[0270] Phase II 2.1 Subsequent Phase II entries will target whether treatment with exemplary EpCAM-targeted trispecific protein therapeutics results in a response rate of at least 20%, with treatment at the MTD. Primary outcome of Phase II: Determine whether at least 20% of patients achieve a clinical response (blast response, minor response, partial response, or complete response) with treatment with the trispecific protein targeting EpCAM.

[0271] Eligibility: Patients with histologically or cytologically confirmed epithelial ovarian cancer. Patients with recurrent epithelial ovarian cancer or disease progression after failure of frontline platinum-based chemotherapy with no more than one prior platinum-based regimen. Patients with adequate bone marrow, renal, liver, and echocardiographic tests.

[0272] Phase III

[0273] 3.1 Subsequent Phase III studies will be conducted with exemplary EpCAM-targeting trispecific proteins, which will assess secondary endpoints such as response rate (RR), patient-recorded outcomes (PROs), progression-free survival (PFS), progression-free survival, time to progression (TIP), overall survival, health-related quality of life assessment, number of overall surviving participants, duration of response, time to response, number of responding participants, and time to tumor growth.

[0274] Example 6: Construction and testing of exemplary multivalent target binding proteins construct Figure 6: The EpCAM binder sequences provided herein were used to generate the following ProCAR constructs: An exemplary construct comprising an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 485); An exemplary construct comprising an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 486); and An exemplary construct comprising an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 487). An exemplary construct comprising an anti-human serum albumin sdAb, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 488). An exemplary construct comprising an anti-human serum albumin sdAb, protease cleavage site 3, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 489). An exemplary construct comprising an anti-human serum albumin sdAb, protease cleavage site 3, an anti-human EpCAM sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 490). An exemplary construct comprising an anti-GFP sdAb, a FLAG epitope, a CD8 hinge / transmembrane domain, a 4-1BB intracellular domain, and a CD3 zeta intracellular domain (SEQ ID NO: 491).

[0275] EpCAM mask 1 interferes with ProCAR EpCAM-binding activity 300,000 primary human T cells isolated from healthy donors were infected with 1 mL of lentiviral supernatant prepared from the constructs shown in Figure 6 to generate anti-EpCAM CAR-T cells. The cells were then stained with anti-FLAG antibody and EpCAM-Fc along with the indicated secondary antibodies. Data were analyzed by flow cytometry. Figure 7 provides histograms of EpCAM-Fc / Alexa Fluor 647 staining of CAR-T cells grouped into low (Figure 7A), moderate (Figure 7B), or high (Figure 7C) CAR expression based on anti-FLAG staining.

[0276] This data demonstrates the effectiveness of EpCAM mask 1 in preventing ProCAR EpCAM binding activity.

[0277] EpCAM mask 2 interferes with ProCAR EpCAM-binding activity 300,000 primary human T cells isolated from healthy donors were infected with 1 mL of lentiviral supernatant made from the constructs shown in Figure 21 to generate anti-EpCAM CAR-T cells. The cells were then stained with anti-FLAG antibody and EpCAM-Fc along with the indicated secondary antibodies. Data were analyzed by flow cytometry.

[0278] Figure 8 provides histograms of EpCAM-Fc / Alexa Fluor 647 staining of the CAR-T cells from Figure 6 grouped into low (Figure 8A), moderate (Figure 8B), or high (Figure 8C) CAR expression based on anti-FLAG staining demonstrating the effectiveness of EpCAM mask 2 in preventing ProCAR EpCAM binding activity.

[0279] Masking of anti-EpCAM sdAb H90 Anti-EpCAM CAR-T cells were generated by infecting 300,000 primary human T cells isolated from healthy donors with 1 mL of lentiviral supernatant prepared from the indicated constructs in Figure 1. These cells were then co-cultured with EpCAM-expressing cancer cells stably expressing luciferase at various ratios (CAR-T:target cells). Luciferase activity was measured after 72 hours as a surrogate for cancer cell viability and normalized to the anti-GFP control CAR-T cells, C1081.

[0280] The data presented in Figure 9 demonstrates the masking of anti-EpCAM sdAb H90. SEQ ID NO: 485 is a "naked" CAR, i.e., without the anti-ALB domain. Addition of the anti-ALB domain (SEQ ID NO: 486) has little effect on cell killing activity. Addition of a mask to the CC' loop of the anti-ALB domain has a significant effect on cell killing activity due to specific blocking of EpCAM binding.

[0281] Steric blocking of anti-EpCAM sdAb H90 by HSA Anti-EpCAM CAR-T cells were generated by infecting 300,000 primary human T cells isolated from healthy donors with 1 mL of lentiviral supernatant prepared from the indicated constructs in Figure 6. These cells were then co-cultured with EpCAM-expressing cancer cells stably expressing luciferase in various ratios (CAR-T:target cells) with or without human serum albumin (HSA). Luciferase activity was measured after 72 hours as a surrogate for cancer cell viability and normalized to the anti-GFP control CAR-T cells, C1081.

[0282] The data presented in Figure 10 demonstrate steric blocking of anti-EpCAM sdAb H90 by HSA.

[0283] Example 7: EpCAM ProCAR protease site-dependent cell killing Chimeric antigen receptor-expressing T cells (CAR T cells) were generated by infecting CD4 / CD8-positive T cells isolated from healthy donors with lentiviruses expressing the indicated constructs. Figure 6 shows a schematic diagram of the constructs used. Cells were incubated with Fc-tagged EpCAM extracellular domain (ECD). Cells were washed to remove unbound Fc-tagged EpCAM ESD. Cells were then incubated with a secondary antibody conjugated to DyLight650, which recognizes human Fc. Binding of the secondary antibody to the cells was measured by flow cytometry.

[0284] FIG. 11 demonstrates protease site-dependent activation of EpCAM ProCAR mask 2 cell killing activity.

[0285] Example 8: Protease activation of EpCAM Mask 1 ProCAR antigen binding activity To generate anti-EpCAM CAR-T cells, 300,000 primary human T cells isolated from healthy donors were infected with 1 mL of lentiviral supernatant prepared from the indicated constructs in Figure 1. The cells were then washed in PBS and treated with PBS or PBS containing 400 nM recombinant UPA protease for 1 hour at room temperature. They were then stained with anti-FLAG antibody and EpCAM-Fc along with anti-mouse BV421 and anti-human Fc Alexa Fluor 647 secondary antibodies and analyzed by flow cytometry.

[0286] Histograms of EpCAM-Fc staining of CAR-T cells grouped into low (Figure 12A), moderate (Figure 12B), or high (Figure 12C) CAR expression based on anti-FLAG staining. These results demonstrate protease activation of EpCAM-mask1 ProCAR antigen-binding activity.

[0287] Example 9: Protease activation of EpCAM Mask 2 ProCAR antigen binding activity To generate anti-EpCAM CAR-T cells, 300,000 primary human T cells isolated from healthy donors were infected with 1 mL of lentiviral supernatant prepared from the indicated constructs in Figure 1. The cells were then washed in PBS and treated with PBS or PBS containing 400 nM recombinant UPA protease for 1 hour at room temperature. They were then stained with anti-FLAG antibody and EpCAM-Fc along with anti-mouse BV421 and anti-human Fc Alexa Fluor 647 secondary antibodies and analyzed by flow cytometry.

[0288] Histograms of EpCAM-Fc staining of CAR-T cells grouped into low (Figure 13A), moderate (Figure 13B), or high (Figure 13C) CAR expression based on anti-FLAG staining. These results demonstrate protease activation of EpCAM-mask2 ProCAR antigen-binding activity.

[0289] Example 10: Demonstration of improved tolerability in mice conferred by exemplary EpCAM-targeted ProTriTAC molecules In this study, we evaluated the tolerability of an exemplary EpCAM-targeting molecule, ProTriTAC. Tumor-free, 7-week-old, female NSG mice were administered 2 × 10 mAbs at the start of the study, i.e., day 0. 71 mg / kg, and expanded human T cells were injected intraperitoneally. On day 2, mice were divided into various groups, and treatment was initiated by administering various concentrations of an exemplary EpCAM-targeting ProTriTAC molecule containing the linker sequence L040 (SEQ ID NO: 494), an EpCAM-targeting TriTAC molecule (SEQ ID NO: 492), an EpCAM-targeting ProTriTAC molecule containing a non-cleavable linker (EpCAM ProTriTAC (NCLV) (SEQ ID NO: 495), and a control GFP TriTAC molecule (SEQ ID NO: 493). These molecules were administered once daily for 10 days at doses of 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg. Starting on day 2, mouse weights were recorded daily. As shown in Figures 14A-14C, the EpCAM-targeting ProTriTAC molecule containing a non-cleavable linker (ProTriTAC (NCLV)) and GFP TriTAC molecule (SEQ ID NO: 493) were administered once daily for 10 days. TriTAC (used as a negative control) was well tolerated in mice even at the highest dose of 1 mg / kg. The EpCAM-targeted ProTriTAC molecule containing the L040 linker sequence was well tolerated at a dose of 1 mg / kg, whereas EpCAM-targeted TriTAC was well tolerated at 0.1 mg / kg. Thus, we observed that EpCAM-targeted ProTriTAC containing the L040 linker sequence provided mice with at least approximately 10-fold improved tolerability compared to EpCAM-targeted TriTAC.

[0290] Example 11: Affinity of EpCAM-targeting ProTriTAC protein to human, cynomolgus monkey, or mouse EpCAM The affinity of various exemplary EpCAM-targeting ProTriTAC proteins for human, cynomolgus monkey, or mouse EpCAM was measured in binding assays, and the binding kinetics of the interaction between the tested ProTriTAC proteins and human EpCAM were also examined. The binding kinetics are shown in Figures 15A, 15B, and 15C.

[0291] A summary of affinities for ProTriTAC proteins targeting EpCAM, including exemplary EpCAM binding domains H13 (SEQ ID NO: 207), H90 (SEQ ID NO: 209), or H90.2 (SEQ ID NO: 497), is provided in Table 8.

[0292] [Table 9]

[0293] Example 12: T cell engagement potency and specificity of exemplary EpCAM-targeting proteins in TriTAC format, non-cleavable prodrug format, or active drug format Three exemplary humanized EpCAM-targeting ProTriTAC proteins, including the exemplary EpCAM-binding domains described herein, H13 (SEQ ID NO: 207), H90 (SEQ ID NO: 209), or H90.2 (SEQ ID NO: 497), were tested in a T cell-dependent cytotoxicity (TDCC) assay (see Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20: 519-27) using EpCAM-expressing colon cancer cells HC116. The EpCAM-binding domain sequences of H90.2 and H138.2 were highly similar to the EpCAM-binding domain sequences H90 and H138, respectively, but the first amino acid of framework 4 was modified (see SEQ ID NO: 582). Specifically, the EpCAM-binding domain sequences H90 and H138 contained Asn deamidation (NG) sites that were modified to WG to remove the Asn deamidation, with the intention of improving protein stability (potentially favoring improved drug manufacturability).

[0294] The results are shown in Figures 16A (H13), 16B (H90.2), and 16C (H138.2). The EpCAM-binding proteins tested contained an EpCAM-binding domain (H13, H90.2, or H138.2), an albumin-binding domain (anti-ALB), and a CD3-binding domain (anti-CD3). The anti-ALB domain further contained a non-cleavable linker (NCLV) (non-cleavable prodrug format) or a non-cleavable linker and masking domain (ACT). The activated EpCAM-binding protein (CT) (active drug format) contained an EpCAM-binding domain (H13, H90.2, or H138.2) and a CD3-binding domain (anti-CD3).

[0295] In this assay, luciferase-labeled HCT116 cells were combined with purified human T cells and exposed to a titration of exemplary EpCAM-binding proteins (NCLV, CT, and ACT) as described above. It was hypothesized that if the EpCAM-binding proteins led T cells to kill EpCAM-expressing HCT116 cells, the viability of these cells would be reduced, as determined by performing a luciferase assay 48 hours after the start of the experiment. A similar assay was also performed using EpCAM-negative NCI-H929 myeloma cells, and as shown in Figures 17A, 17B, and 17C, none of the proteins tested were able to engage T cells in killing NCI-H929 cells.

[0296] Figures 16A, 16B, and 16C show representative TDCC data from assays using HCT116 cells. EC from TDCC assays 50 The values ​​are listed in Table 9. 50 As shown by the values, binding proteins encompassing any of the three EpCAM-binding domains (H13, H90.2, or H138.2) functioned as T cell engagers in EpCAM-expressing HCT116 colon cancer cells. Furthermore, binding proteins with a cleavable linker and masking domain (ACT) were able to bind to ECs. 50The values ​​were approximately 7-10 times (H13 and H138.2) lower compared to the conjugated protein with a non-cleavable linker (NCLV) (non-cleavable prodrug format), while for the active drug (CT), the EC 50 The EC values ​​were up to approximately 350-fold (H138.2) lower than those of NCLV. 50 The values ​​were approximately 37 pM, approximately 11 pM for the H90.2 binding domain, and approximately 107 pM for the H138.2 binding domain.

[0297] [Table 10]

[0298] To further evaluate the EpCAM-specific cell killing ability of the test EpCAM-binding proteins, a TDCC assay as described above was performed using HCT116 colon cancer cells expressing EpCAM (wild-type) or HCT116 cells not expressing EpCAM (EpCAM-KO). EpCAM TriTAC proteins encompassing the EpCAM-binding domains H13 or H90.2 were tested in this assay, with GFP TriTAC protein and EGFR TriTAC protein as control proteins. The results of this assay are shown in Figures 18A and 18B, and EC 50 The values ​​are listed in Table 10.

[0299] The EpCAM-binding proteins demonstrated differential cell killing based on EpCAM expression. Specificity of binding was also confirmed by flow cytometry, as shown in Figure 19. In the flow cytometry assay, cells were stained with 300 nM H13 or H90.2 in CT format (active drug) and detected with the 11D3-AF650 antibody.

[0300] [Table 11]

[0301] Additionally, TDCC assays were performed using various other cell lines to compare the cell-killing ability of the two EpCAM-binding proteins in either CT format (active drug comprising the EpCAM-binding domain of either H13 or H90.2 and the anti-CD3 domain) or in non-cleavable prodrug format (comprising either H13 or H90.2, anti-CD3, and anti-HSA with the non-cleavable linker NCLV). The results are provided in Table 11. The plot in Figure 20 is for a TDCC assay using SKBR3 (human breast cancer cells).

[0302] We observed that active drugs containing the EpCAM-binding domain H13 were approximately three times more potent than active drugs containing the EpCAM-binding domain H90.2 in in vitro TDCC assays. These proteins showed similar 400- to 600-fold masking in TDCC assays using multiple EpCAM-expressing cells when compared with the active drug format and the non-cleavable prodrug format (CT vs. NCLV). Representative plots demonstrating the masking effect achieved by the non-cleavable prodrug format are shown in Figures 21A and 21B.

[0303] [Table 12]

[0304] Additional TDCC assays were performed using cell lines (including CAPAN2, DMS53, HepG2, KMRC3, MDAPCA2b, and SKBE3) to compare the EpCAM-binding proteins NCLV (non-cleavable prodrug) containing H13, ProTriTAC (with cleavable linker L040), and the active drug CT. 50 Values ​​are provided in Table 12. Representative plots are shown in Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, and 22H.

[0305] [Table 13-1]

[0306] [Table 13-2]

[0307] Example 13: Antitumor efficacy and tolerability of EpCAM-targeted ProTriTAC compared to EpCAM-targeted TriTAC The in vivo efficacy of EpCAM ProTriTAC, which contains the EpCAM-binding domain H13, was evaluated in an established HT29 colon tumor model and compared with EpCAM TriTAC, which also contains the EpCAM-binding domain H13.

[0308] On day 5 after tumor implantation, mice were injected with either 0.3 mg / kg or 0.1 mg / kg of test EpCAM H13 ProTriTAC, 0.3 mg / kg of test EpCAM H13 TriTAC, or equivalent doses of control TriTAC, and tumor volumes were measured for approximately 20 days. As shown in Figures 23A and 23B, EpCAM H13 ProTriTAC was more effective than EpCAM H13 TriTAC in the mouse tumor model.

[0309] In a separate study, cynomolgus monkeys were injected with equivalent doses (30 μg / kg) of EpCAM H13 ProTriTAC or EpCAM H13 TriTAC, and cytokine levels were measured (IFN-γ, IL-2, IL-6, and IL-10). The results are shown in Figures 24A, 24B, 24C, and 24D. These figures show that EpCAM H13 ProTriTAC demonstrated significantly reduced cytokine production compared to EpCAM H13 TriTAC in a single-dose cytotoxicity study at 30 μg / kg.

[0310] Example 14: Pharmacokinetic properties of exemplary EpCAM-binding proteins in cynomolgus monkeys Binding proteins comprising EpCAM binding domain H13 or H90.2 (in TriTAC format, ProTriTAC NCLV format, and ProTriTAC format with cleavable linker L040) were administered to cynomolgus monkeys to evaluate their pharmacokinetic properties. Figure 25A and Figure 25B show the plasma concentration of the tested proteins over time in cynomolgus monkeys. In addition, Table 13 summarizes the pharmacokinetic parameters.

[0311] [Table 14]

[0312] Example 15: Evaluation of the therapeutic index (efficacy and toxicity) of ProTriTAC / TriTAC proteins targeting EpCAM In this study, mice bearing the LoVo tumor model (a colon cancer model) were injected with various doses of EpCAM-targeted TriTAC protein, including the EpCAM-binding domain H90, or ProTriTAC protein, or control TriTAC protein. The results are shown in Figures 29A-29K. The safety and tolerability of ProTriTAC and TriTAC were also tested, and the results are shown in Figures 30A-30E. Survival rates are shown in Figures 30A-30B, and clinical chemistry parameters (ALT, AST, and total bilirubin) are shown in Figures 30C, 30D, and 30E. Overall, the ProTriTAC format was approximately 30-fold better tolerated than the TriTAC format in mice bearing the same LoVo tumors. This observation was further supported by histopathological examination, a summary of which is provided in Figure 31 and Table 14.

[0313] Thus, this study demonstrated an approximately 10-fold expansion of the therapeutic index in the ProTriTAC format based on drug efficacy and safety in the same animals.

[0314] [Table 15]

[0315] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein are available for practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0316] [Table 16-1]

[0317] [Table 16-2]

[0318] [Table 16-3]

[0319] [Table 16-4]

[0320] [Table 16-5]

[0321] [Table 16-6]

[0322] [Table 16-7]

[0323] Table 16-8

[0324] Table 16-9

[0325] Table 16-10

[0326] Table 16-11

[0327] Table 16-12

[0328] Table 16-13

[0329] Table 16-14

[0330] Table 16-15

[0331] Table 16-16

[0332] Table 16-17

[0333] Table 16-18

[0334] Table 16-19

[0335] Table 16-20

[0336] Table 16-21

[0337] Table 16-22

[0338] Table 16-23

[0339] Table 16-24

[0340] Table 16-25

[0341] Table 16-26

[0342] Table 16-27

[0343] Table 16-28

[0344] Table 16-29

[0345] Table 16-30

Claims

1. An EpCAM binding domain comprising complementarity determining regions (CDR) 1, CDR2, and CDR3, wherein the CDR1 comprises the sequence of SEQ ID NO: 39, the CDR2 comprises the sequence of SEQ ID NO: 77, and the CDR3 comprises the sequence of SEQ ID NO: 115, wherein the EpCAM binding domain is a single domain antibody or a VHH domain.

2. The EpCAM binding domain of claim 1 , comprising the amino acid sequence of SEQ ID NO:

1.

3. The EpCAM binding domain of claim 1 or 2, comprising a single domain antibody.

4. The EpCAM binding domain of claim 1, which binds to EpCAM with a binding affinity (Kd) of about 0.001 nM to about 500 nM.

5. The EpCAM binding domain of claim 4, which binds to human EpCAM, mouse EpCAM, cynomolgus monkey EpCAM, or a combination thereof.

6. A polynucleotide encoding the EpCAM binding domain of claim 1.

7. A pharmaceutical composition comprising the EpCAM binding domain of claim 1 or the polynucleotide of claim 6, and a pharmaceutically acceptable excipient.

8. A multispecific protein comprising: (a) an EpCAM binding domain according to any one of claims 1 to 5; (b) a CD3ε binding domain; and (c) a bulk serum protein binding domain that binds to human serum albumin protein.

9. The multispecific protein of claim 8 , wherein the CD3ε binding domain comprises the amino acid sequence of SEQ ID NO:

474.

10. The multispecific protein of claim 8 , wherein the bulk serum protein binding domain comprises the amino acid sequence of SEQ ID NO:

472.

11. The multispecific protein of claim 8 , wherein the CD3ε binding domain and the bulk serum protein binding domain are linked by a linker comprising the amino acid sequence of SEQ ID NO:

503.

12. A polynucleotide encoding the multispecific protein of claim 8.

13. A pharmaceutical composition comprising the multispecific protein of claim 8 or the polynucleotide of claim 12, and a pharmaceutically acceptable excipient.

14. Use of the EpCAM binding domain of claim 1, the multispecific protein of claim 8, or the polynucleotide of claim 6 or 12 in the manufacture of a medicament for treating cancer in a subject in need of cancer treatment.

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