TROP2-targeted trispecific proteins for the treatment of cancer

Abstract

Provided herein are TROP2-binding proteins, pharmaceutical compositions containing such proteins or fragments thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such TROP2-binding proteins. Methods of using the disclosed TROP2-binding proteins in the prevention and / or treatment of diseases, conditions, and disorders are also disclosed.

Description

[Technical Field]

[0001] The present invention relates to TROP2-targeting (i.e., TROP2-targeting) trispecific proteins for the treatment of cancer (hereinafter referred to as cancer).

[0002] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 478,640, filed January 5, 2023, and U.S. Provisional Patent Application No. 63 / 496,159, filed April 14, 2023, each of which is incorporated by reference in its entirety.

[0003] 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]

[0004] Background to the disclosure TROP2 is a protein encoded by the TACSTD2 gene in humans. This antigen is a member of a family containing at least two type I membrane proteins. It transduces intracellular calcium signals and functions as a cell surface receptor. Studies have shown that abnormal TROP2 protein overexpression is associated with several carcinomas, including colorectal cancer, pancreatic cancer, gastric cancer, oral squamous cell carcinoma, ovarian cancer, and breast cancer. Cancers with high TROP2 expression are associated with increased disease recurrence and drug resistance, and are a poor prognostic factor for survival.

[0005] There is a need for greater treatment options that allow physicians to select the treatment with the best side effect profile for each individual patient. The present disclosure provides novel polypeptide and protein therapeutics that are useful in therapeutic methods, particularly for the treatment of conditions associated with aberrant expression of TROP2. Summary of the Invention

[0006] Disclosure Overview Provided herein is a TROP2-binding domain comprising a complementarity-determining region 1 (CDR1), CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58 to 114 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58 to 114; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115 to 171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115 to 171; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172 to 228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172 to 228. In some embodiments, CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58 to 114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115 to 171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172 to 228. In some embodiments, the CDR1 comprises an amino acid sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57. In some embodiments, the TROP2-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 form. 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: 493 or 549. In some embodiments, the CD3-binding domain comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 494. In some embodiments, the multispecific protein comprises a sequence at least about 75% identical to the sequence set forth in SEQ ID NOs: 229-264.In some embodiments, the bulk serum protein binding domain is a binding moiety comprising a linker and a masking moiety, wherein the masking moiety can or does mask binding of the TROP2-binding domain or the CD3-binding domain to their respective targets. In some embodiments, the multispecific protein comprises a non-cleavable prodrug form. In some embodiments, the masking moiety comprises a sequence selected from the group consisting of SEQ ID NO: 550 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NO: 550. In some embodiments, the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497-543. 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 NO: 493. In some embodiments, the CD3-binding domain comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 494. 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: 229-264. In some embodiments, the multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264. In some embodiments, the active drug comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0007] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a TROP2-binding domain according to the present specification or a pharmaceutical composition comprising the same. In some embodiments, the subject is a human.

[0008] Provided herein is a conditionally active TROP2-binding protein comprising a single polypeptide chain comprising (a) a binding moiety comprising a non-CDR loop and a cleavable linker, and (b) complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58 to 114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58 to 114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115 to 171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115 to 171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172 to 228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172 to 228, wherein the binding moiety can mask or masks binding of the TROP2-binding domain to its target.

[0009] In some embodiments, the TROP2 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0010] Provided herein is a conditionally active TROP2-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 a TROP2-binding domain, and the TROP2-binding domain comprises complementarity-determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 58 to 114 or a sequence within the group consisting of SEQ ID NOs: 58 to 114. In some embodiments, the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171, and the CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228, wherein the non-CDR loops are capable of binding to the TROP2-binding domain or the second target antigen-binding domain, and the binding moiety can or does mask binding of the TROP2-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: 550 and 558-560, or a sequence containing one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence containing one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 497-543. In some embodiments, the binding moiety comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NO: 493. 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: 494.

[0011] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease, comprising administering to a subject a conditionally active chimeric antigen receptor according to the present disclosure or a pharmaceutical composition comprising the same. In some embodiments, the subject is a human.

[0012] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a conditionally active TROP2-binding protein according to any of the preceding claims or a pharmaceutical composition comprising the same. In some embodiments, the subject is a human.

[0013] 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, VHH domain, scFv, VH domain, VL domain, Fab, Fab', non-Ig domain, ligand, knottin, or small molecule. In some embodiments, the binding domain comprises a single-domain antibody. In some embodiments, the binding domain binds to TROP2 with a binding affinity (KD) of about 0.001 nM to about 500 nM. In some embodiments, the binding domain binds to human TROP2, mouse TROP2, cynomolgus monkey TROP2, or a combination thereof.

[0014] Provided herein are multispecific proteins comprising a TROP2-binding domain, wherein the TROP2-binding domain is as described herein. In some embodiments, a TROP2-binding domain (anti-TROP2 domain) and a CD3-binding domain (anti-CD3 domain) as described herein. In some embodiments, the anti-TROP2 domain and the anti-CD3 domain are present in an anti-TROP2:anti-CD3 orientation. In some embodiments, the anti-TROP2 domain and the anti-CD3 domain are present in an anti-CD3:anti-TROP2 orientation. In some embodiments, a TROP2-binding domain (anti-TROP2 domain), a CD3-binding domain (anti-CD3 domain), and an albumin-binding domain (anti-ALB domain) as described herein. In some embodiments, the anti-CD3 domain comprises the amino acid sequence set forth in SEQ ID NO:494. In some embodiments, the anti-ALB domain comprises the amino acid sequence set forth in SEQ ID NO:493. In some embodiments, the anti-TROP2 domain, anti-CD3 domain, and anti-ALB domain are present in the orientation of anti-CD3:anti-ALB:anti-TROP2. In some embodiments, the anti-TROP2 domain, anti-CD3 domain, and anti-ALB domain are present in the orientation of anti-TROP2:anti-ALB:anti-CD3. In some embodiments, the anti-TROP2 domain, anti-CD3 domain, and anti-ALB domain are present in the orientation of anti-ALB:anti-TROP2:anti-CD3. In some embodiments, the anti-TROP2 domain, anti-CD3 domain, and anti-ALB domain are present in the orientation of anti-CD3:anti-TROP2:anti-ALB. In some embodiments, the anti-TROP2 domain, anti-CD3 domain, and anti-ALB domain are present in the orientation of anti-ALB:anti-CD3:anti-TROP2. In some embodiments, the anti-TROP2 domain, anti-CD3 domain, and anti-ALB domain are present in the orientation of anti-TROP2:anti-CD3:anti-ALB.

[0015] Provided herein is a multivalent protein comprising a sequence set forth in any one of SEQ ID NOs: 229 to 264.

[0016] Provided herein is an active agent comprising a sequence set forth in any one of SEQ ID NOs: 229-264.

[0017] Provided herein is an active drug comprising a sequence set forth in any one of SEQ ID NOs: 1-57.

[0018] Provided herein are pharmaceutical compositions comprising (i)(a) a TROP2-binding domain described herein, (i)(b) a conditionally active TROP2-binding protein described herein, (i)(c) a multispecific protein described herein, (i)(d) a multivalent protein described herein, or (i)(e) an active drug described herein, and (ii) a pharmaceutically acceptable carrier.

[0019] The present specification provides a method for producing the TROP2-binding domain described herein, which method comprises culturing a host transformed or transfected with a vector containing a nucleic acid sequence encoding the TROP2-binding domain under conditions that allow expression of the TROP2-binding domain, and further recovering and purifying the produced protein from the culture.

[0020] Provided herein are methods for producing a multispecific protein according to the description herein, the methods comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of a multispecific TROP2 binding protein according to the description herein under conditions that allow expression of the multispecific protein, and recovering and purifying the protein produced from the culture.

[0021] Provided herein is a method for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a TROP2-binding domain as described herein or a pharmaceutical composition as described herein.

[0022] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a multispecific protein described herein, a multivalent protein described herein, an active agent described herein, or a pharmaceutical composition described herein. In some embodiments, the subject is human. In some embodiments, the method further comprises administering a substance in combination with a TROP2-binding domain described herein, a multispecific protein described herein, a multivalent protein described herein, an active agent described herein, or a pharmaceutical composition described herein. In some embodiments, the TROP2-binding domain selectively binds to tumor cells expressing TROP2. 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 oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T-cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof. In some embodiments, the method further comprises administering a substance in combination with, or a pharmaceutical composition comprising, any one of the conditionally active TROP2-binding proteins described herein. In some embodiments, the TROP2-binding domain selectively binds to tumor cells expressing TROP2. 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 colorectal cancer, oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof.

[0023] Provided herein is a method for producing a conditionally active TROP2-binding protein as described herein, the method comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of a conditionally active TROP2-binding protein as described herein under conditions that allow expression of the conditionally active TROP2-binding protein, and recovering and purifying the protein produced from the culture.

[0024] Provided herein are methods for producing a multivalent protein according to the description herein, the methods 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 description herein under conditions that allow expression of the multivalent protein, and recovering and purifying the produced protein from the culture.

[0025] Provided herein is a method for producing an active drug according to the description herein, the method comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of an active drug according to the description herein under conditions allowing expression of the active drug, and recovering and purifying the drug produced from the culture.

[0026] Provided herein are conditionally active TROP2-binding proteins, wherein the conditionally active TROP2-binding protein has a greater therapeutic index than a TROP2-binding protein that does not include the binding moiety but is otherwise identical to the conditionally active TROP2-binding protein. In some embodiments, the conditionally active TROP2-binding protein has a therapeutic index that is at least about 5- to about 100-fold greater than that of a TROP2-binding protein that does not include the binding moiety but is otherwise identical to the conditionally active TROP2-binding protein. 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: 229-264. In some embodiments, the protein comprises a sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0027] Provided herein are pharmaceutical compositions comprising (i) a conditionally active TROP2-binding protein according to the description herein, and (ii) a pharmaceutically acceptable carrier.

[0028] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a conditionally active chimeric antigen receptor according to the present specification or a pharmaceutical composition according to the present specification.

[0029] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease, comprising administering to a subject a conditionally active TROP2-binding protein as described herein or a pharmaceutical composition as described herein. In some embodiments, the subject is a human. In some embodiments, the neoplastic disease includes at least one of oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T-cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof.

[0030] Provided herein is a method for increasing the therapeutic index of a TROP2-binding domain, the method comprising conjugating (binding) the TROP2-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 TROP2-binding domain, the TROP2-binding domain is masked from binding to its target by the binding moiety, and the TROP2-binding domain can bind to its target upon cleavage of the cleavable linker. In some embodiments, the TROP2-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: 58-114 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the TROP2-binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, and 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 NO: 493. In some embodiments, the CD3-binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 494. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497-543. In some embodiments, the TROP2-binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57. 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: 229-264.In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264. In some embodiments, the TROP2-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: 58 to 114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58 to 114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115 to 171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115 to 171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172 to 228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172 to 228. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NO: 493. In some embodiments, the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57. In some embodiments, the TROP2 binding domain comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0031] Provided herein is a method for increasing the therapeutic index of a TROP2-binding protein comprising a first target antigen-binding domain and a second target antigen-binding domain, wherein at least one of the first and second target antigen-binding domains comprises a TROP2-binding domain, the method comprising conjugating the first or second target antigen-binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for the first or second target antigen-binding domain, wherein at least one of the first or second target antigen-binding domain is masked from binding to its target by the binding moiety, and wherein the masked first or second target antigen-binding domain can bind to its target upon cleavage of the cleavable linker. In some embodiments, the TROP2-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: 58-114 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the non-CDR loop comprises a binding site specific for the TROP2-binding domain. In some embodiments, at least one of the first or 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: 494. 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 NO: 493. In some embodiments, the TROP2 binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57.In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497-543. 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: 229-264. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0032] Provided herein are methods for increasing the therapeutic index of a TROP2-binding protein comprising a TROP2-binding domain and a CD3-binding domain, the methods 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 TROP2-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: 58-114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the CD3 binding domain comprises a sequence at least about 75% identical to SEQ ID NO: 494. 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 NO: 493. In some embodiments, the TROP2 binding domain comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497-543. 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: 229-264. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264. In some embodiments, the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0033] Provided herein is a conditionally active TROP2-targeting multispecific protein comprising a TROP2-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 TROP2-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: 58 to 114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58 to 114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115 to 171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115 to 171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172 to 228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172 to 228. 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 NO: 494. In some embodiments, the TROP2 binding domain comprises a sequence at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57. In some embodiments, the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 496-543 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497-543. 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: 229-264. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264. In some embodiments, the conditionally active multispecific protein comprises a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0034] Provided herein is a pharmaceutical composition comprising a conditionally active TROP2-targeting multispecific protein in accordance with the description herein, which in some embodiments further comprises a pharmaceutically acceptable carrier.

[0035] Provided herein is a method for producing a conditionally active TROP2-targeted multispecific protein according to the description herein, the method comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding domains of a conditionally active TROP2-targeted multispecific protein according to the description herein under conditions allowing expression of the conditionally active TROP2-targeted multispecific protein, and recovering and purifying the produced protein from the culture.

[0036] Provided herein are methods for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a conditionally active TROP2-targeting multispecific protein as described herein or a pharmaceutical composition as described herein. 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 oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T-cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof. [Brief explanation of the drawings]

[0037] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, which are briefly described below. [Figure 1]FIG. 1 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL3, 2TRL92, 3TRL77, 2TRL4 and 2TRL76 and H292 cells. [Figure 2] FIG. 2 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL3, 2TRL92, 3TRL77, 2TRL4 and 2TRL76 and H292 cells. [Figure 3] FIG. 3 shows the results of a TDCC assay using an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequences 3TRL56, 3TRL87, 2TRL33, 2TRL1, and 2TRL5 and H292 cells. [Figure 4] FIG. 4 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL8, 2TRL27, 2TRL46, 2TRL69 and 2TRL94 and H292 cells. [Figure 5] FIG. 5 shows the results of a TDCC assay using H292 cells and an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequences 2TRL79, 2TRL81, 2TRL18, 3TRL27 and a GFP negative control. [Figure 6] Figure 6 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL28, 3TRL39, 3TRL82, 2TRL68, 2TRL64 and 3TRL53 and H292 cells. [Figure 7] FIG. 7 shows the results of a TDCC assay using H1376 cells and an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequences 3TRL3, 2TRL92, 3TRL77, 2TRL4 and 2TRL76. [Figure 8]FIG. 8 shows the results of a TDCC assay using H1376 cells and an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequences 3TRL16, 2TRL29, 3TRL58, 2TRL31 and 2TRL70. [Figure 9] FIG. 9 shows the results of a TDCC assay using H1376 cells and an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequences 3TRL56, 3TRL87, 2TRL33, 2TRL1 and 2TRL5. [Figure 10] FIG. 10 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL8, 2TRL27, 2TRL46, 2TRL69 and 2TRL94 and H1376 cells. [Figure 11] FIG. 11 shows the results of a TDCC assay using H1376 cells and an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequences 2TRL79, 2TRL81, 2TRL18, 3TRL27 and a GFP negative control. [Figure 12] FIG. 12 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL28, 3TRL39, 3TRL82, 2TRL68, 2TRL64 and 3TRL53 and H1376 cells. [Figure 13] FIG. 13 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL53, 3TRH53, 2TRL76 and 2TRH76 and H292 cells. [Figure 14] FIG. 14 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRL81 and 2TRH81 and H292 cells. [Figure 15]FIG. 15 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL53, 3TRH53, 2TRL76 and 2TRH76 and HT1376 cells. [Figure 16] FIG. 16 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRL81 and 2TRH81 and HT1376 cells. [Figure 17] FIG. 17 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL53, 3TRH53, 2TRL76 and 2TRH76 and HCC70 cells. [Figure 18] FIG. 18 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRL81 and 2TRH81 and HCC70 cells. [Figure 19] FIG. 19 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRH79B and 2TRH79B L040 ProTriTAC and HCC70 cells. [Figure 20] Figure 20 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequence and the humanized anti-TROP2 sequences 2TRL79, 2TRH79, 2TRH79B and 2TRH79B L040 ProTriTAC and HPAF-II cells. [Figure 21] Figure 21 shows the results of a TDCC assay using anti-CD3 / anti-TROP2 fusion proteins containing the humanized anti-TROP2 sequences 2TRH79B and 2TRH79B L040 ProTriTAC and CAL27 cells. [Figure 22]Figure 22 shows the results of a mixed therapy xenograft rodent study using an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequence, 2TRL79 ProTriTAC, and HCC70 cells. [Figure 23] Figure 23 shows the results of a mixed therapy xenograft rodent study using an anti-CD3 / anti-TROP2 fusion protein containing the llama anti-TROP2 sequence, 2TRL79 ProTriTAC, and HCC70 cells. [Figure 24] Figure 24 shows the results of a mixed therapy xenograft rodent study using an anti-CD3 / anti-TROP2 fusion protein containing the humanized anti-TROP2 sequence, 2TRH79 ProTriTAC, and HCC70 cells. [Figure 25] Figure 25 shows the results of a mixed therapy xenograft rodent study using an anti-CD3 / anti-TROP2 fusion protein containing the humanized anti-TROP2 sequence, 2TRH79B ProTriTAC, and CAL27 cells. [Figure 26] Figure 26 shows the results of a combined therapy xenograft rodent study using an anti-CD3 / anti-TROP2 fusion protein containing the humanized anti-TROP2 sequence, 2TRH79B ProTriTAC, and HPAF-II cells. [Figure 27] FIG. 27 shows that molar equivalents of the prodrug and active drug exhibited equally potent antitumor activity in HCC70 cells. [Figure 28A] Figures 28A-28B show that anti-CD3 / anti-TROP2 fusion proteins containing humanized 2TRH79B bind to human (Figure 28A) and cynomolgus monkey (cyno) Trop2 (Figure 28B) with similar affinity in the presence or absence of calcium (47-51 nM or 50 nM, respectively). [Figure 28B] Figures 28A-28B show that anti-CD3 / anti-TROP2 fusion proteins containing humanized 2TRH79B bind to human (Figure 28A) and cynomolgus monkey (cyno) Trop2 (Figure 28B) with similar affinity in the presence or absence of calcium (47-51 nM or 50 nM, respectively). [Figure 29] FIG. 29 shows the experimental design for the one-month dose escalation / maximum tolerated dose study. [Figure 30] FIG. 30 shows that anti-CD3 / anti-TROP2 fusion proteins containing humanized 2TRH79B exhibit favorable pharmacokinetics, half-life, and systemic accumulation.

[0038] Detailed Description of Disclosure Provided herein are trispecific proteins that target TROP2, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the disclosed TROP2-targeting trispecific proteins in the prevention and / or treatment of diseases, conditions, and disorders. The TROP2-targeting trispecific proteins are capable of specifically binding to TROP2 and CD3 and have a half-life-extending domain, e.g., a domain that binds to human albumin (ALB).

[0039] TROP2-binding protein Described herein are proteins that bind to TROP2, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for producing such proteins. Also provided are methods of using the disclosed TROP2-binding proteins in the prevention and / or treatment of diseases, conditions, and disorders. In some embodiments, the TROP2-binding protein is part of a multispecific (e.g., trispecific) protein that includes a TROP2-binding domain as described herein.

[0040] Trophoblast cell surface antigen 2 (Trop2), also known as tumor-associated calcium signaling factor 2 (TACSTD2), epithelial glycoprotein 1 (EGP-1), pancreatic cancer marker protein GA733-1, GP50, or membrane component 1 surface marker 1 (M1S1), is a 323-amino acid, widely expressed, 35-kDa type I transmembrane glycoprotein with four N-linked glycosylation sites encoded by the TACSTD2 gene. It is a member of the tumor-associated calcium signaling factor (TACSTD) family and is structurally related to epithelial cell adhesion molecule (EpCAM). Trop-2 contains a large extracellular domain, a single transmembrane domain, and an intracellular tail. The crystal structure of the TROP2 ectodomain revealed a small subunit composed of three domains: the N-terminal domain (ND), the thyroglobulin type 1 domain (TY), and the C-terminal domain (CD). The TROP2 extracellular domain can form dimers.

[0041] TROP2 has claudin-interacting ability similar to its paralog, EpCAM, and both are involved in signal transduction induced by proteolytic cleavage within the ectodomain. TROP2 plays essential roles in embryonic development, placental tissue formation, embryo implantation, stem cell proliferation, and organogenesis. TROP2 is a stem / progenitor cell marker, and low basal levels of TROP2 are found on the surface of multiple normal epithelial tissues, including skin and oral mucosa. Overexpression of TROP2 has been observed in many types of malignant epithelial tumors, including gastric cancer, thyroid cancer, papillary thyroid cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer, e.g., lung adenocarcinoma), breast cancer (e.g., ductal carcinoma), pancreatic cancer, ovarian cancer, prostate cancer, bladder cancer, gallbladder cancer, cervical cancer, uterine serous papillary carcinoma, uterine and ovarian carcinosarcoma, endometrial cancer, nasopharyngeal carcinoma, portal vein bile duct carcinoma, oral squamous cell carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, laryngeal squamous cell carcinoma, liver fluke-associated cholangiocarcinoma, lung adenocarcinoma, hepatocellular carcinoma, cervical squamous cell carcinoma, head and neck squamous cell carcinoma, and esophageal squamous cell carcinoma. High expression of Trop2 has also been found in tumors of non-epithelial origin, such as melanoma, nasal NK / T-cell lymphoma, glioma and glioblastoma, and osteosarcoma. Overexpression of TROP2 has also been associated with pituitary adenoma. Although Trop2 is frequently overexpressed during tumorigenesis, genetic analysis has shown that point mutations and copy number alterations in the TACSTD2 gene are relatively rare in human tumors. TROP2 overexpression is associated with poor prognosis for survival and drug resistance.

[0042] An exemplary protein sequence for TROP2 is set forth in UniProtkB ID No. P09758 and RefSeq NP_002344 (SEQ ID NO: 546). In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein comprising the amino acid sequence set forth in UniProtkB ID No. P09758 or RefSeq NP_002344. In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein comprising the amino acid sequence set forth in UniProtkB ID No. Q8BGV3 or RefSeq NP_064431. In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein comprising the amino acid sequence set forth in Refseq XP_005543292.2. In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein encoded by the nucleic acid set forth in RefSeq NM_002353. In some embodiments, the TROP2 binding proteins of the present disclosure bind to a TROP2 protein encoded by a nucleic acid set forth in RefSeq NM_0200047. In some embodiments, the TROP2 binding proteins of the present disclosure bind to a TROP2 protein comprising the amino acid sequence set forth in SEQ ID NO: 546 or 547.

[0043] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGA FNHSDLDDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL (SEQ ID NO: 546).

[0044] HTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRL FRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL (SEQ ID NO: 547).

[0045] In some embodiments, the TROP2 binding domain binds to the extracellular domain of the mature TROP2 protein. In some embodiments, the TROP2 binding domain binds to the transmembrane domain of the mature TROP2 protein. In some embodiments, the TROP2 binding domain binds to the intracellular tail of the mature TROP2 protein.

[0046] In some embodiments, the TROP2-binding domain binds to a protein comprising a truncated sequence compared to SEQ ID NO: 546 or 547. In some embodiments, the TROP2-binding domain binds to a protein comprising the sequence of SEQ ID NO: 546 or 547. In some embodiments, the TROP2-binding domains disclosed herein recognize full-length TROP2. In certain cases, the TROP2-binding domains disclosed herein recognize an epitope within TROP2; for example, in some cases, the TROP2-binding protein interacts with one or more amino acids present within a domain of human TROP2. The epitope to which the antibody binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of TROP2. Alternatively, an epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within a domain of TROP2.

[0047] In some embodiments, the TROP2-binding domains disclosed herein recognize full-length TROP2. In certain cases, the TROP2-binding domains disclosed herein recognize an epitope within TROP2; for example, in some cases, the TROP2-binding protein interacts with one or more amino acids present within a domain of human TROP2. The epitope to which the antibody can bind can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of TROP2. Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within a domain of TROP2.

[0048] In some embodiments, the TROP2-binding proteins of the present disclosure bind to the full-length TROP2 protein or fragments thereof, e.g., epitope-containing fragments within the full-length TROP2 protein, as described above. In some cases, the epitope-containing fragments include antigenic or immunogenic fragments of the TROP2 protein and derivatives thereof. In some embodiments, epitope-containing fragments, including antigenic or immunogenic fragments, are 12 amino acids or more, e.g., 20 amino acids or more, 50 amino acids or more, or 100 amino acids or more. In some embodiments, the TROP2 fragment comprises 95% or more of the length of the full-length protein, or 90% or more, 75%, 50%, 25%, 10% or more of the length of the full-length protein. In some embodiments, epitope-containing fragments of TROP2, including antigenic or immunogenic fragments, can elicit a relevant immune response in a patient. Derivatives of TROP2 include, in some embodiments, variants of the TROP2 sequence set forth in SEQ ID NO: 546 or 547 in which one or more (e.g., 1 to 20, e.g., 15 amino acids, or up to 20%, e.g., up to 10%, 5% or 1% of the total amino acids in the full length of the protein) amino acids have been deleted, inserted or substituted.

[0049] In some embodiments, substitutions include conservative substitutions. In some instances, derivatives and variants have essentially the same biological function as the protein from which they are derived. For example, derivatives and variants of TROP2 may in some cases be equally antigenic or immunogenic as the protein from which they are derived, have either or preferably both the ligand binding activity or the ability to form an active receptor complex of the protein from which they are derived, and exhibit the same tissue distribution as TROP2.

[0050] In some embodiments, the TROP2-binding protein specifically binds to TROP2 with an affinity comparable to or better than that of a reference TROP2-binding protein. In such embodiments, the TROP2-binding protein comprises an affinity-matured TROP2-binding molecule, which is derived from a TROP2-binding parent molecule and contains one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) compared to the TROP2-binding parent molecule. In some embodiments, the affinity-matured TROP2-binding molecule has superior stability to a selected destabilizing agent compared to the reference TROP2-binding parent molecule. In some embodiments, the affinity-matured TROP2-binding molecule is identified in a process comprising panning one or more preliminary candidate TROP2-binding molecules derived from one or more TROP2-binding parent molecules expressed in a phage display library against a TROP2 protein (e.g., a human TROP2 protein). In some embodiments, the preliminary candidate TROP2-binding molecule contains amino acid substitutions in variable region, CDR, or framework residues compared to the parent molecule.

[0051] As used herein, "phage display" refers to a technique for displaying mutant polypeptides as fusion proteins to at least a portion of a coat protein on the surface of a phage (e.g., 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. Display of 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 fusion 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 methods, 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, so that phage particles display only one or no fusion proteins. Compared to polyvalent phage, avidity effects are reduced so that selection occurs based on specific ligand affinity, and phagemid vectors are used, simplifying DNA manipulation. See, e.g., Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).

[0052] In some embodiments, panning involves using different binding times and concentrations to identify TROP2-binding molecules with increased or decreased on-rates (association rates) from the candidate TROP2 molecules. In some embodiments, panning involves using different wash times to identify TROP2-binding molecules with increased or decreased off-rates (dissociation rates) from the candidate TROP2 molecules. In some embodiments, panning involves using both different binding times and different wash times. In some embodiments, one or more stabilizing mutations are combined to enhance the stability of affinity-matured TROP2-binding molecules. This is done, for example, by shuffling such mutants to generate a second-stage combinatorial library and performing a second round of panning and subsequent binding selection.

[0053] In some embodiments, affinity-matured TROP2-binding molecules have affinity for TROP2 protein (e.g., human TROP2 protein) that is comparable to or better than the parent v-binding molecule, but have reduced, or in some embodiments, enhanced, cross-reactivity to selected substances (e.g., ligands, proteins, antigens, etc.) other than the TROP2 epitope for which the parent TROP2-binding molecule is specific or designed to be specific. Regarding the latter, in some embodiments, affinity-matured TROP2-binding molecules are more successfully tested in animal models when they are reacted with both human TROP2 and the corresponding target in an animal model (e.g., mouse TROP2 or cynomolgus monkey (cyno) TROP2). In some embodiments, the parent TROP2-binding molecule binds to human TROP2 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, or 10 nM or less, and to cynomolgus monkey TROP2 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 TROP2-binding molecule identified after one round of panning binds to human TROP2 with an affinity of about 5 nM or less, e.g., 1 nM or less, and to cynomolgus monkey TROP2 with an affinity of about 7.5 nM or less, e.g., 1 nM or less. In some embodiments, affinity-matured TROP2-binding molecules identified after two rounds of panning bind to human TROP2 with an affinity of about 2.5 nM or less and to cynomolgus monkey TROP2 with an affinity of about 3.5 nM or less.

[0054] In some embodiments, the TROP2-binding protein comprises an antigen-specific binding domain polypeptide that specifically binds to a target (e.g., a target on a diseased cell or on another cell that supports the pathology, e.g., a target on a stromal cell that supports tumor growth, or a target on an immune cell that supports disease-mediated immunosuppression). In some examples, the antigen-specific binding domain includes 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 mimics (e.g., 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).

[0055] In some embodiments, the TROP2-binding domain is an anti-TROP2 antibody or antigen-binding fragment thereof, or an antibody variant of the TROP2-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 described herein. In certain embodiments, amino acid sequence variants of the anti-TROP2 antibodies or antigen-binding fragments described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of the anti-TROP2 antibodies or antigen-binding fragments described herein are contemplated to improve their binding affinity and / or other biological properties. Exemplary methods for producing amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding fragment thereof.

[0056] Any combination of deletion, insertion, and substitution can be made to obtain the final construct, as long as the final construct possesses the desired properties, e.g., antigen binding. In certain embodiments, variants with one or more amino acid substitutions are provided. Targeted sites for substitutional mutagenesis include CDRs and framework regions. Examples of such substitutions are described below. Amino acid substitutions can be introduced into an antibody or antigen-binding fragment of interest, and the products can 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). Both conservative and non-conservative amino acid substitutions are contemplated for producing antibody variants.

[0057] In another example of substitutions to generate mutant anti-TROP2 antibodies or antigen-binding fragments thereof, one or more hypervariable (hypermutable) region residues of a parent antibody are substituted. Typically, variants are then selected based on the desired improved property compared to the parent antibody or antigen-binding fragment thereof, such as increased affinity, decreased affinity, decreased immunogenicity, or increased pH-dependence of binding.

[0058] In some embodiments, the TROP2-binding domain is a single-domain antibody (sdAb), such as a heavy chain variable domain (VH), a variable domain (VHH) of a llama-derived sdAb, a peptide, a ligand, or a small molecule, specific for TROP2. In some embodiments, the TROP2-binding domain described herein is any domain that binds to TROP2, including, but not limited to, domains derived from monoclonal, polyclonal, recombinant, human, and humanized antibodies. In certain embodiments, the TROP2-binding domain is a single-domain antibody. In other embodiments, the TROP2-binding domain is a peptide. In yet other embodiments, the TROP2-binding domain is a small molecule.

[0059] It should be noted that, in general, the term "single domain antibody" as used herein, in its broadest sense, is not limited to a particular biological origin or a particular production method. Single domain antibodies are antibodies having complementarity determining regions that are 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 conventional 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 any future single domain antibody. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, or cow. For example, in some embodiments, a single domain antibody of the disclosure is obtained by: (1) isolating a VHH domain of a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanizing" a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain; (4) "camelizing" a naturally occurring VH domain from any animal species, particularly a mammalian species, especially human, or expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" a "domain antibody" or "Dab" or expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques to produce a protein, polypeptide or other amino acid sequence; (7) producing a nucleic acid encoding a single domain antibody using techniques for nucleic acid synthesis known in the art, followed by expressing the nucleic acid so obtained; and / or (8) any combination of one or more of the foregoing.

[0060] In one embodiment, the single domain antibody corresponds to the VHH domain of a naturally occurring heavy chain antibody against TROP2. As described in further detail herein, such VHH sequences may generally be produced or obtained by suitably immunising a species of llama with TROP2 (i.e. to elicit an immune response and / or heavy chain antibodies against TROP2), obtaining a suitable biological sample from the llama (e.g. a blood sample, serum sample or B cell sample), and producing the VHH sequence against TROP2 from said sample using any suitable technique known in the art.

[0061] In another embodiment, such naturally occurring VHH domains against TROP2 are obtained from a naive library of camelid VHH sequences, for example, by screening such a library with TROP2 or at least a portion, fragment, antigenic determinant, or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naive VHH libraries are used, such as VHH libraries obtained from naive VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described, for example, in WO 00 / 43507.

[0062] In another embodiment, yet another technique for obtaining VHH sequences against TROP2 involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., to elicit an immune response and / or heavy chain antibodies against TROP2), obtaining a suitable biological sample (e.g., a blood sample, serum sample, or B cell sample) from the transgenic mammal, and then generating VHH sequences against TROP2 from the sample using any suitable technique known in the art. For example, rats or mice expressing heavy chain antibodies and the further methods and techniques described in WO 02 / 085945 and WO 04 / 049794 may be used for this purpose.

[0063] In some embodiments, the anti-TROP2 single-domain antibodies of the present disclosure include single-domain antibodies having an amino acid sequence that corresponds to that of a non-human antibody and / or a naturally occurring VHH domain, e.g., a llama anti-TROP2 antibody, but that has been "humanized." "Humanization" is achieved by substituting one or more amino acid residues in the amino acid sequence of the non-human anti-TROP2 and / or naturally occurring VHH sequence (and particularly the framework sequence) with one or more amino acid residues present 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 achieved by methods known in the art, for example, as will be apparent to those skilled in the art based on the further detailed description herein. Again, it should be noted that such humanized anti-TROP2 single-domain antibodies of the present disclosure can be obtained by any suitable method known per se [i.e., the methods described in (1) to (8) above] and are therefore not strictly limited to polypeptides obtained using a polypeptide containing a naturally occurring VHH domain as a starting material. In some additional embodiments, the single-domain anti-TROP2 antibodies described herein include single-domain antibodies having an amino acid sequence that corresponds to that of a naturally occurring VH domain, but that has been "camelized." The "camelization" is achieved by substituting one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody with one or more amino acid residues present at the corresponding positions in the VHH domain of a heavy-chain antibody. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-VL interface and / or at so-called Camelidae hallmark residues. See, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996). Preferably, the VH sequence used as the starting material or starting point for generating or designing a camelized single domain is preferably a VH sequence of mammalian origin, more preferably a human VH sequence, e.g., a VH3 sequence.However, it should be noted that, in certain embodiments, such camelized anti-TROP2 single domain antibodies of the present disclosure can be obtained by any suitable method known in the art (i.e., the methods described in (1) to (8) above) and are therefore not strictly limited to polypeptides obtained using nonhuman anti-TROP2 antibodies and / or naturally occurring polypeptides containing a VH domain as starting materials. For example, as described in further detail herein, both "humanization" and "camelization" are performed by preparing 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. This nucleic acid can then be expressed to obtain the desired anti-TROP2 single domain antibody of the present disclosure. Alternatively, in other embodiments, the amino acid sequence of the desired humanized or camelized anti-TROP2 single domain antibody of the present disclosure is designed based on the amino acid sequence of the naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using known techniques for peptide synthesis. In some embodiments, a nucleotide sequence encoding the desired humanized or camelized anti-TROP2 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, and the nucleic acid thus obtained is then expressed using known expression techniques to obtain the desired anti-TROP2 single domain antibody of the present disclosure.

[0064] Other suitable methods and techniques for obtaining an anti-TROP2 single domain antibody of the present disclosure and / or a nucleic acid encoding same starting from a naturally occurring VH sequence or VHH sequence include, for example, combining one or more parts of one or more naturally occurring VH sequences (e.g., one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more parts of one or more naturally occurring VHH sequences (e.g., one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in an appropriate manner to obtain an anti-TROP2 single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding it.

[0065] In some embodiments, the TROP2 binding domain is an anti-TROP2 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 TROP2-binding domain comprises any domain that binds to TROP2, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or antigen-binding fragments such as single-domain antibodies (sdAbs), Fab, Fab', F(ab)2, and Fv fragments, fragments composed of one or more CDRs, single-chain antibodies (e.g., single-chain Fv fragments (scFvs)), 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 composed of one heavy chain and one light chain. In some embodiments, the TROP2-binding domain is a single-domain antibody. In some embodiments, the anti-TROP2 single-domain antibody comprises the variable complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 of the heavy chain.

[0066] In some embodiments, the TROP2 binding domain is a polypeptide comprising an amino acid sequence consisting of four framework regions / sequences (f1-f4) interposed by three complementarity determining regions / sequences, which is 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 TROP2-binding proteins of the present 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 TROP2-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0067] In some embodiments, the binding proteins described herein comprise polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding protein has at least 70% to 95% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding protein has at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding protein has at least 70% to 95% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding protein has at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof.

[0068] In some embodiments, CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 58 to 114, or an amino acid sequence that contains one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 114. In some embodiments, CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 115 to 171, or an amino acid sequence that contains one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 115 to 171. In some embodiments, CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 172 to 228, or an amino acid sequence that contains one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 172 to 228.

[0069] In various embodiments, the TROP2-binding domain of the present disclosure has a sequence identity of at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical.

[0070] In various embodiments, the complementarity determining regions of the TROP2 binding domains 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 sequences set forth in SEQ ID NOs: 58-114.

[0071] In various embodiments, the complementarity determining region of the TROP2 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence set forth in SEQ ID NOs: 115-171.

[0072] In various embodiments, the complementarity determining region of the TROP2 binding domain of the present disclosure is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the amino acid sequence set forth in SEQ ID NOs: 172-228.

[0073] In some embodiments, the TROP2-binding domain is cross-reactive with human, cynomolgus monkey, and mouse TROP2. In some embodiments, the TROP2-binding domain is specific for human TROP2. In certain embodiments, the TROP2-binding domains disclosed herein are specific for human TROP2. D (h K D In certain embodiments, the TROP2-binding domains disclosed herein bind to cynomolgus monkey TROP2 at the cynomolgus monkey K D (c K D In certain embodiments, the TROP2-binding domains disclosed herein bind to cynomolgus monkey TROP2 at the mouse K D (m K D In certain embodiments, the TROP2-binding domains disclosed herein bind to both cynomolgus monkey TROP2 and human TROP2 at the cynomolgus monkey K2 domain. D (c K D) and human K D (h K D In certain embodiments, the TROP2-binding domains disclosed herein bind to cynomolgus monkey TROP2, mouse TROP2, and human TROP2 at the cynomolgus monkey K2 domain, respectively. D (c K D ), Mouse K D (m K D ) and human K D (h K D In some embodiments, the TROP2 binding protein binds to human, mouse, and cynomolgus monkey TROP2 with comparable binding affinity (i.e., hK D , m K D and c K D (Values ​​do not vary by more than ±10%). In some embodiments, the TROP2-binding domains disclosed herein bind to Trop2 in the presence of calcium. In some embodiments, the TROP2-binding domains disclosed herein bind to Trop2 in the absence of calcium.

[0074] In some embodiments, h D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 500 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 450 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 400 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 350 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 300 nM.D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 250 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 200 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 150 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 100 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 80 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 50 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 40 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 200 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 150 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.001 nM to about 100 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.1 nM to about 90 nM. D , m K Dand c K D In some embodiments, hK ranges from about 0.2 nM to about 80 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.3 nM to about 70 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.4 nM to about 50 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.5 nM to about 30 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.6 nM to about 10 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.7 nM to about 8 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.8 nM to about 6 nM. D , m K D and c K D In some embodiments, hK ranges from about 0.9 nM to about 4 nM. D , m K D and c K D is in the range of about 1 nM to about 2 nM.

[0075] In some embodiments, h k(1 / Ms)e5, c k(1 / Ms)e5, and m k(1 / Ms)e5 are in the range of about 0.001 to about 100, for example, about 0.1 to 1, or about 0.5 to 0.9. In some embodiments, h K(1 / s), c K(1 / s), and m K(1 / s) are about 1×10 -2 ~9×10 -6 , for example, about 1 × 10 -2 ~9×10 -3 , about 4×10 -3~6×10 -3 The range is.

[0076] In some embodiments, any of the TROP2-binding domains (e.g., the anti-TROP2 single-domain antibodies of SEQ ID NOs: 1-57) contains an affinity peptide tag to facilitate purification. In some embodiments, the affinity peptide tag is six consecutive histidine residues, also referred to as 6x-His (SEQ ID NO: 496).

[0077] In certain embodiments, the TROP2-binding domain of the present disclosure preferentially binds membrane-bound TROP2 over soluble TROP2. Membrane-bound TROP2 refers to TROP2 present in or on the cell membrane surface of a cell that expresses TROP2. Soluble TROP2 refers to TROP2 that is no longer present in or on the cell membrane surface of a cell that expresses or has expressed TROP2. In certain examples, soluble TROP2 is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the TROP2-binding domain binds membrane-bound TROP2 at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold more strongly than soluble TROP2. In one embodiment, the TROP2-binding protein of the present disclosure preferentially binds membrane-bound TROP2 30-fold more strongly than soluble TROP2. Whether an antigen-binding protein preferentially binds to membrane-bound TROP2 over soluble TROP2 can be readily determined using binding assays.

[0078] In some embodiments, the TROP2-binding protein is very small, contemplated to be 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less in some embodiments. In certain cases, the TROP2-binding protein, when it is a peptide or small molecule, is 5 kDa or less.

[0079] In other embodiments, the TROP2-binding proteins described herein comprise small molecule (SME) binders (conjugates) for TROP2. SME binders are small molecules with an average size of approximately 500-2000 Da that are attached to the TROP2-binding proteins by known methods, such as sortase ligation or conjugation. In these cases, the TROP2-binding proteins comprise a domain containing a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 548). To bind the SME binder to a TROP2-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 TROP2-binding proteins described herein comprise knottin peptides for binding to TROP2. Knottins are disulfide-stabilized peptides with a cysteine ​​knot backbone and have an average size of approximately 3.5 kDa. Knottins are believed to bind to specific tumor molecules, such as TROP2. In yet other embodiments, the TROP2 binding proteins described herein comprise natural TROP2 ligands.

[0080] In some embodiments, the TROP2-binding proteins comprise multiple domains and are designed as a single polypeptide with flexible linkages between the domains. This allows for easy production and manufacturing of the TROP2-binding proteins, as they can be encoded by a single cDNA molecule, allowing for easy integration into a vector. Furthermore, in some embodiments, in which the TROP2-binding proteins described herein are monomeric single polypeptide chains, there is no need for chain pairing or dimerization. In such embodiments, the TROP2-binding proteins described herein are believed to have a reduced tendency to aggregate.

[0081] In TROP2-binding proteins containing multiple domains, 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 cases, 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, a peptide is selected that confers flexibility to the TROP2-binding protein, does not inhibit the binding domain, and is resistant to cleavage by proteases. For example, glycine and serine residues generally confer protease resistance. Examples of internal linkers suitable for linking domains in TROP2-binding proteins include (GS) n (SEQ ID NO: 514), (GGS) n (SEQ ID NO: 515), (GGGS) n (SEQ ID NO: 516), (GGSG) n (SEQ ID NO: 517), (GGSGG) n (SEQ ID NO: 518), (GGGGS) n (SEQ ID NO: 519), (GGGGGG) n (SEQ ID NO: 520) or (GGG) n (SEQ ID NO:521) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the linker is (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:522), (GGGGSGGGSGGGGS) (SEQ ID NO:523) or (GGGGSGGGS) (SEQ ID NO:524).

[0082] In some cases where the TROP2-binding protein contains multiple domains, the domains within the TROP2-binding protein are conjugated using enzymatic site-specific conjugation methods involving 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, ease of use, and lack of regulation by calcium and guanosine-5'-triphosphate (GTP) have encouraged mTG to become a major cross-linking enzyme used in both the food industry and biotechnology. Currently, mTG is used in numerous 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.

[0083] In some examples, a TROP2-binding protein is provided that includes multiple domains, where one of the domains includes an acceptor glutamine within the constant region, which can then be conjugated to another domain via a lysine-based (i.e., lysine-based) linker (e.g., one containing any primary amine chain that is a substrate for TGase, such as an alkylamine or oxoamine), where conjugation occurs only at one or more acceptor glutamine residues present in a targeting site 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, e.g., at least partially surface-exposed glutamines. In some examples, the TROP2-binding protein is formed by reacting one of the domains with the lysine-based linker in the presence of TGase.

[0084] In some embodiments where one or more domains within a TROP2 binding protein are directly linked, a hybrid vector is created in which the DNA encoding the directly linked domains is itself directly linked to each other. In some embodiments where linkers are used, a hybrid vector is created in which the DNA encoding one domain is linked to the DNA encoding the other domain at the other end of the linker.

[0085] In some embodiments, the TROP2-binding protein is a single-chain variable fragment (scFv), a single-domain antibody, e.g., a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) (such as those of camelid-derived single-domain antibodies). In other embodiments, the TROP2-binding protein is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, AFFIMERS®, affitins, alphabodies, avimers, DARPINS®, phinomers, Kunitz domain peptides, and monobodies. In still other embodiments, the TROP2-binding protein is a ligand or peptide that binds to or associates with TROP2. In still other embodiments, the TROP2-binding protein is a knottin. In still other embodiments, the TROP2-binding domain is a small molecule.

[0086] In certain embodiments, a TROP2-binding protein according to the present disclosure may be incorporated into a TROP2-targeting trispecific protein. In some embodiments, the trispecific protein comprises a CD3-binding domain, a half-life-extending domain, and a TROP2-binding domain according to the present disclosure. In some embodiments, the TROP2-binding trispecific protein comprises a trispecific antibody.

[0087] Multispecific TROP2-targeting proteins, such as TROP2-targeting trispecific proteins (also referred to herein as TROP2-targeting TriTAC proteins or molecules) In one aspect, described herein are multispecific or multivalent proteins comprising a TROP2-binding protein according to 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.

[0088] In additional 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.

[0089] In certain embodiments, the CD3 binding domain of the multispecific protein not only exhibits strong CD3 binding affinity for human CD3, but also exhibits good cross-reactivity to the respective cynomolgus monkey CD3 protein. In some cases, the CD3 binding domain of the multispecific protein exhibits cross-reactivity to CD3 from cynomolgus monkeys. In some cases, the human:cynomolgus monkey K for CD3 binding is D ratio(h K D :c K D ) is 20:1 to 1:2.

[0090] In some embodiments, the CD3-binding domain of the multispecific protein is any domain that binds to CD3, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or antigen-binding fragments (of CD3-binding antibodies), such as single-domain antibodies (sdAbs), Fab, Fab', F(ab)2, 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 composed of one heavy chain and one light chain. In some cases, it is beneficial for the CD3-binding domain to be derived from the same species as the species in which the multispecific protein, including the single-domain serum albumin-binding proteins described herein, will ultimately be used. For example, when used in humans, it may be beneficial for the CD3-binding domain of a multispecific protein, including a TROP2-binding protein described herein, to comprise human or humanized residues from the antigen-binding domain of an antibody or antibody fragment. An exemplary amino acid sequence of the CD3-binding domain of a multispecific (e.g., trispecific) TROP2-targeting TriTAC protein of the present disclosure is provided as SEQ ID NO:494, or an amino acid sequence that is at least about 75% to 100% identical to SEQ ID NO:494, 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:494.

[0091] In some embodiments, the serum albumin binding domain (also referred to herein as a half-life extending domain) of the multispecific proteins, including the TROP2 binding proteins described herein, can be any domain that binds to serum albumin, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the serum albumin-binding domain is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) (such as that of a camelid-derived sdAb), or an antigen-binding fragment of an HSA-binding antibody, such as a Fab, F(ab'), or Fv fragment, a fragment consisting 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, a dimer consisting of one heavy chain and one light chain, a peptide, a ligand, or a small molecule substance (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 yet other 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 relatively small, and in some embodiments is contemplated to be 25 kDa or less, 20 kDa or less, 15 kDa or less, or 10 kDa or less. In certain cases, when the serum albumin binding domain is a peptide or small molecule substance, it is 5 kDa or less.Exemplary amino acid sequences of the serum albumin binding domain of a multispecific (e.g., trispecific) TROP2-targeting TriTAC protein of the present disclosure are provided as SEQ ID NO: 493 or 566, or an amino acid sequence that is at least about 75% to 100% identical to SEQ ID NO: 493 or 566, e.g., an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to SEQ ID NO: 493 or 549.

[0092] The half-life prolonging domain of the multispecific binding proteins described herein, including single-chain variable fragment CD3 binding proteins, results in altered pharmacodynamics and pharmacokinetics 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 also alters the pharmacodynamic properties of the single-chain variable fragment CD3 binding protein, including altered tissue distribution, penetration, and diffusion. In some embodiments, the half-life prolonging domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, tissue diffusion, and enhanced efficacy compared to the protein in the absence of the half-life prolonging domain. In one embodiment, a treatment method effectively and efficiently utilizes reduced amounts of the multispecific binding protein, including the single-chain variable fragment CD3 binding protein, resulting in reduced side effects, e.g., reduced off-target effects such as cytotoxicity against non-tumor cells.

[0093] Furthermore, in some embodiments, the binding affinity of the half-life prolonging domain is selected to target a particular terminal half-life of a particular multispecific binding protein, including the TROP2 binding proteins 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 marginal binding affinity. Exemplary binding affinities include K values ​​of 10 nM or less (high), 10 nM-100 nM (moderate), and greater than 100 nM (low). D As noted above, the binding affinity to serum albumin is determined by known methods, such as surface plasmon resonance (SPR).

[0094] In certain embodiments, the TROP2-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 a TROP2-binding protein described herein. In certain embodiments, the first domain comprises an scFv that specifically binds to CD3. CD3 is, for example, a human CD3 protein. In certain embodiments, the second domain comprises an sdAb that specifically binds to a bulk serum protein. In some cases, the bulk serum protein is albumin, for example, serum albumin, for example, human serum albumin. Domains (A), (B), and (C) are, in some embodiments, linked via linkers L1 and L2 in any of the following orientations: H2N-(A)-L1-(C)-L2-(B)-COOH, H2N-(B)-L1-(A)-L2-(C)-COOH, H2N-(C)-L1-(B)-L2-(A)-COOH, H2N-(C)-L1-(A)-L2-(B)-COOH, H2N-(A)-L1-(B)-(C)-L2-COOH, or H2N-(B)-(C)-(A)-COOH.

[0095] In some embodiments, the TROP2-targeting multispecific proteins of the present disclosure comprise an amino acid sequence that is at least about 70% to about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57 and 229-264. In some embodiments, a TROP2-targeting multispecific protein of the present disclosure comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 76%, at least about 77%, about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57 and 229-264.

[0096] Conditionally active multispecific TROP2-targeting proteins, such as conditionally active TROP2-targeting trispecific proteins (also referred to herein as TROP2-targeting ProTriTACs or pro-trispecific proteins or molecules). One embodiment of the present disclosure provides conditionally active multispecific proteins comprising a TROP2-binding domain disclosed herein (e.g., in some embodiments, the present disclosure provides TROP2-targeting pro-trispecific / PrOTriTAC proteins comprising a TROP2-binding domain of the present disclosure).

[0097] In some embodiments, the conditionally active multispecific protein further comprises a domain that specifically binds 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 or does mask the interaction of the TROP2-binding domain or the CD3-binding domain with their target. In some embodiments, the binding moiety of the present disclosure comprises a masking moiety and a cleavable linker, e.g., a protease-cleavable linker. Exemplary sequences of masking moieties within the binding moiety include a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560, or an amino acid sequence comprising one or more substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560. In some embodiments, the binding moiety comprises a modified non-CDR loop sequence and a cleavable linker. In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 497-543, or an amino acid sequence comprising one or more substitutions relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 497-543. 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 amino acid sequence set forth in SEQ ID NO:544 or 545, or an amino acid sequence comprising one or more substitutions relative to SEQ ID NO:544 or 545. In some embodiments, the binding moiety comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:549. In some embodiments, the CD3-binding domain of a TROP2 ProTriTAC of the present disclosure comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to SEQ ID NO:494.

[0098] In some embodiments, a TROP2-targeting ProTriTAC of the present disclosure comprises, in an N-terminal to C-terminal direction, a binding moiety that is an anti-ALB domain including a non-CDR loop containing a binding site for a CD3-binding domain (e.g., a CD3-binding domain having the sequence of SEQ ID NO: 549 or at least about 75% identity thereto), a cleavable linker, a CD3-binding domain, and an anti-TROP2 binding domain at the C-terminus. In some embodiments, the TROP2-binding domain of ProTriTAC is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0099] In some embodiments, a TROP2-targeting ProTriTAC of the present disclosure comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57 and 229-264. In some embodiments, a TROP2-targeting ProTriTAC of the present disclosure comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-57 and 229-264, and pharmaceutical compositions comprising same and methods of using same to treat diseases, such as neoplastic diseases, as described herein are provided.

[0100] In some embodiments, the TROP2-targeting ProTriTAC of the present disclosure, in its non-cleavable prodrug form, comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0101] Exemplary sequences of the active TROP2 targeting agents (CTs) described herein are amino acid sequences 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 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57 and 229-264.

[0102] The binding moiety may synergistically expand the therapeutic window of a conditionally active TROP2-targeting pro-trispecific protein by both steric and specific masking. In some embodiments, the binding moiety provides both steric masking (e.g., by binding to bulk serum albumin) and specific masking (e.g., by binding of non-CDR loops to the CDRs of the anti-TROP2 domain or anti-CD3 scFv domain). For example, the binding moiety can mask or mask the binding of the TROP2-binding domain (e.g., conceal the TROP2-binding domain and / or prevent premature binding) until activation, and the "masking" ("masking") ability of a sequence can be tested using an assay comparing activity in the presence and absence of a masking sequence (e.g., a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560, or a sequence comprising one or more substitutions in a sequence having a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560). Briefly, to test masking, ProCAR constructs containing a masking moiety, for example, in the CC' loop, and ProCAR constructs without the masking moiety are prepared.The lentivirus prepared from these constructs is used to infect T cells to prepare CAR-T cells, which are then stained with anti-FLAG antibody, TROP2-Fc, and fluorescently labeled secondary antibody, and analyzed by flow cytometry.The staining dot plots are prepared and compared.In some cases, the masking moiety is a masking peptide / moiety inserted into one or more non-CDR loops, so that the binding moiety binds to and inhibits the TROP2 antigen-binding domain until the construct is delivered to the tumor microenvironment.In some cases, modifying the non-CDR loop in the binding moiety does not affect albumin binding.In some cases, a protease-cleavable linker allows the activation of TROP2-targeting pro-trispecific proteins in a single proteolytic event, thereby enabling more efficient conversion of pro-trispecific molecules in the tumor microenvironment.Furthermore, in some cases, tumor-associated proteolytic activation results in the emergence of active T cell engagers with minimal extratumoral activity after activation. In some embodiments, the present disclosure provides extended half-life T cell engager formulations (ProTriTACs) comprising the TROP2-binding moieties described herein, which in some cases represent a novel and improved approach to designing conditionally active T cell engagers.

[0103] In some embodiments, the half-life of the TROP2-binding domain in the conditionally active pro-trispecific form is extended in the systemic circulation by using the binding moiety, which functions as a safety switch that maintains the pro-form of the multispecific protein in an inactive state until it reaches the tumor microenvironment, where it is conditionally activated by cleavage of the linker and becomes capable of binding to its target antigen. In certain cases, the safety switch provides several advantages, some examples of which include: (i) expanding the therapeutic window of the conditionally active TROP2-targeting protein; (ii) reducing target-mediated drug disposition by maintaining the conditionally active TROP2-targeting protein in the systemic circulation; (iii) reducing the concentration of undesired activated protein in the systemic circulation, thereby minimizing the spread of chemistry-, manufacturing-, and management-related impurities (e.g., pre-activated drug, endogenous viruses, host cell proteins, DNA, leachables, antifoaming agents, antibiotics, toxins, solvents, heavy metals); (iv) reducing the concentration of undesired activated protein in the systemic circulation, thereby minimizing the spread of chemistry-, manufacturing-, and management-related impurities (e.g., pre-activated drug, endogenous viruses, host cell proteins, DNA, leachables, antifoaming agents, antibiotics, toxins, solvents, heavy metals); (v) reducing the concentration of undesired activated protein in the systemic circulation, thereby minimizing the spread of chemistry-, manufacturing-, and management-related impurities (e.g., pre-activated drug, endogenous viruses, host cell proteins, DNA, leachables, antifoaming agents, antibiotics, toxins, solvents, heavy metals); (vi) reducing the concentration of undesired activated protein in the systemic circulation, thereby minimizing the spread of chemistry-, manufacturing-, and management-related impurities (e.g., pre-activated drug, endogenous viruses, host cell proteins, DNA, leachables, antifoaming agents, antibiotics, toxins, (iv) reduce the concentration of undesired activated proteins in the systemic circulation, thereby minimizing the spread of product-related impurities, aggregates, degradation products, and product variants due to oxidation, deamidation, denaturation, and loss of the C-terminal Lys of MAbs; (v) prevent aberrant activation in the circulation; (vi) reduce toxicity associated with leakage of activated species from diseased tissues or other pathophysiological conditions (e.g., tumors, autoimmune diseases, inflammation, viral infections, tissue remodeling events (e.g., myocardial infarction, skin wound healing), or trauma (e.g., X-rays, CT scans, UV exposure)); and (vii) reduce nonspecific binding of the conditionally active TROP2-targeting protein. Furthermore, after activation, i.e., after destruction of the safety switch, the conditionally active TROP2-targeting protein is separated from the safety switch, which resulted in an extended half-life, and eliminated from the circulation. For example, if a drug is accidentally activated outside the tumor environment or if it leaks out of the tumor environment after activation, it is rapidly eliminated, reducing the likelihood of causing damage to normal tissues and thereby reducing toxicity.

[0104] In some embodiments, the conditionally active multispecific TROP2-binding proteins described herein have an improved therapeutic index compared to constitutively active, but not conditionally active, TROP2-binding proteins. For example, in some embodiments, TROP2 ProTriTAC has an increased therapeutic index relative to TROP2 TriTAC. In some embodiments, the increase in therapeutic index 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. In some embodiments, the increase in therapeutic index results from conjugation of the TROP2-binding domain to the binding moiety in the presence of a non-CDR loop and a cleavable linker.

[0105] In some embodiments, the "therapeutic index" (TI) (also referred to as the "therapeutic window") is the comparison of the minimum tolerated dose with the minimum amount of a therapeutic agent (e.g., TROP2 TriTAC, TROP2 ProTriTAC, TROP2 CAR, TROP2 ProCAR) that provides a therapeutic benefit (e.g., improved survival in patients with a TROP2-expressing cancer treated with said therapeutic agent). In some instances, an improved therapeutic index is the EC2 of TROP2 TriTAC compared to TROP2 ProTriTAC in T cell-mediated killing of cancer cells. 50 This will manifest itself in the form of improvements in

[0106] In some embodiments, the conditionally active TROP2-targeting protein form confers a significantly longer serum half-life on the TROP2-binding domain, reducing the likelihood of its unwanted activation in the circulation, thereby resulting in a "biobetter" form.

[0107] 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 the TROP2-binding domain of the present disclosure. In some cases, the binding moiety masks the binding of the TROP2-binding domain to its target antigen, for example, by steric occlusion, specific intermolecular interactions, or a combination of both.

[0108] 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 cases, the binding moieties of the present disclosure are domains derived from immunoglobulin molecules (Ig molecules). Ig can 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 beta 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 chains (H chains) and two light chains (L chains), or antigen-binding fragments thereof, inter-connected 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, namely, 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 are further subdivided into hypervariable (highly mutable) regions called complementarity-determining regions (CDRs), which are hypervariable in sequence and involved in antigen recognition and / or usually form structurally defined loops, interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed 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.

[0109] In some embodiments, the binding moiety of the present disclosure is a heavy-chain-only antibody. In some embodiments, the variable domain of a heavy-chain-only antibody has several beta strands arranged in two sheets. The variable domain of a heavy-chain-only antibody comprises three hypervariable loops, i.e., complementarity-determining regions (CDRs), and framework regions FR1, FR2, FR3, and FR4. The three CDRs (CDR1, CDR2, CDR3) of the variable domain are clustered at one end of a beta barrel. The CDRs are the loops connecting beta strands BC, C'-C" and FG of the immunoglobulin fold, while the lower loops connecting beta strands AB, CC', C"-D and EF of the immunoglobulin fold and the upper loop connecting the DE strand of the immunoglobulin fold are non-CDR loops.

[0110] 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, e.g., 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 domain, 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.

[0111] In the variable domain, CDRs are considered to be responsible for antigen recognition and binding, while FR residues are considered to be the scaffold of the CDRs. However, in some cases, some FR residues play an important role in antigen recognition and binding. Framework region residues that affect antigen binding are divided into two categories. One is FR residues that contact the antigen and are therefore part of the binding moiety, and some of these residues are located near the CDRs in sequence. Other residues are residues that are located far from the CDRs in sequence but are nearby in the three-dimensional structure of the molecule, such as loops in the heavy chain.

[0112] 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 a TROP2-binding domain described herein. In some embodiments, the non-CDR loops are modified to generate an antigen-binding site specific for a CD3-binding domain described herein.

[0113] It is contemplated that various techniques may be used to modify non-CDR loops, including, for example, site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid foreign to the non-CDR loop amino acid sequence, and amino acid substitution. In some examples, an antigenic peptide is inserted into a non-CDR loop. In some examples, an antigenic peptide is substituted for a non-CDR loop. In some cases, the modification to generate an antigen-binding site is in only one non-CDR loop. In other examples, multiple non-CDR loops are modified. For example, the modification is in any one of the non-CDR loops AB, CC', C"D, EF, and DE. In some examples, the modification is in the DE loop. In other examples, the modification is in all four of the AB, CC', C"D, and EF loops.

[0114] In certain examples, the binding moieties described herein bind to the TROP2 binding domain via their AB, CC', CD'D, or EF loops and bind to a bulk serum protein, such as albumin, via their BC, C'-C" or FG loops. In certain examples, the binding moieties bind to the TROP2 binding domain via their AB, CC', CD'D, and EF loops and bind to a bulk serum protein, such as albumin, via its BC, C'C" and FG loops. In certain examples, the binding moieties bind to the TROP2 binding domain via one or more of the AB, CC', CD'D, and EF loops and bind to a bulk serum protein, such as albumin, via one or more of the BC, C'C" and FG loops. In certain examples, the binding moieties bind to a bulk serum protein, such as albumin, via its AB, CC', CD'D, or EF loops and bind to the TROP2 binding domain via its BC, C'C" or FG loops. In certain examples, the binding moieties bind to a bulk serum protein, such as albumin, via its AB, CC', CD'D, and EF loops, and to a TROP2-binding domain via its BC, C'C" and FG loops. In certain examples, the binding moieties of the first embodiment bind to a bulk serum protein, such as albumin, via one or more of its AB, CC', CD'D, and EF loops, and to a TROP2-binding protein via one or more of its BC, C'C" and FG loops. In certain examples, the binding moieties described herein bind to a CD3-binding domain via their AB, CC', CD'D, or EF loops, and to a bulk serum protein, such as albumin, via their BC, C'-C" or FG loops. In certain examples, the binding moieties described herein bind to a bulk serum protein, such as albumin, via their AB, CC', CD'D, or EF loops, and to a CD3-binding domain via their BC, C'-C" or FG loops.In certain examples, the binding moieties described herein bind to the CD3 binding domain via their AB, CC', CD', or EF loops and to the TROP2 binding domain via their BC, C'-C" or FG loops. In certain examples, the binding moieties described herein bind to the TROP2 binding domain via their AB, CC', CD', or EF loops and to the CD3 binding domain via their BC, C'-C" or FG loops.

[0115] 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.

[0116] In some cases, the binding moiety comprises a binding site for a bulk serum protein. In some embodiments, CDRs within the binding moiety provide the binding site for the bulk serum protein. The bulk serum protein is, in some instances, 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, CDRs provide the binding site for the immunoglobulin light chain. The immunoglobulin light chain is, in some instances, an Igκ free light chain or an Igλ free light chain.

[0117] In additional embodiments, the binding moiety is any type of polypeptide. For example, in certain cases, the binding moiety is a natural peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered bulk serum protein. In some examples, 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 a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) (such as those of camelid-derived nanobodies). In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, AFFIMERS®, affitins, alphabodies, avimers, DARPINS®, fynomers, Kunitz domain peptides, or monobodies.

[0118] The binding moieties described herein are contemplated to comprise at least one cleavable linker. In one aspect, the cleavable linker comprises a polypeptide having an amino acid sequence that is specifically recognized and cleaved. In some cases, the binding moieties described herein comprise a protease-cleavable linker that is specifically recognized and cleaved. In some embodiments, the protease-cleavable linker is specifically recognized by a matrix metalloprotease (MMP), such as MMP9. 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 specifically recognized by MMP11.

[0119] Proteases are proteins that, in some cases, cleave proteins in an amino acid 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, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, and HIV-1. Proteases, 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 plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26). [Table 1] TIFF2026502002000003.tif108151 Proteases are known to be secreted by some diseased cells and tissues, such as tumor or cancer cells, creating a protease-rich or protease-enriched microenvironment. In some cases, 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 cells, and other cells. In some cases, proteases, such as proteases that target amino acid sequences found in microbial peptides, are present in a subject's blood. This feature allows targeted therapeutic agents, such as antigen-binding proteins, to have additional specificity. This is because T cells do not bind to antigen-binding proteins outside of the protease-rich microenvironment of the targeted cell or tissue. Other non-limiting examples of linkers that can be utilized in the constructs described herein are listed in the Sequence Listing below.

[0120] Incorporation into chimeric antigen receptors (CARs) In certain instances, the TROP2-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 the TROP2-binding proteins described herein, e.g., containing anti-TROP2 single-domain antibodies. In one embodiment, a CAR containing a TROP2-binding protein described herein is linked via a hinge region to a transmembrane domain and further 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 an amino acid sequence encoding an intracellular signaling domain, e.g., 4-1BB and / or CD3 zeta. Exemplary sequences of ProCAR comprising a TROP2-binding domain are provided in SEQ ID NOs: 1-57, or in a sequence that is at least about 75% to 100% (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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0121] The conditionally active receptors described herein comprise at least one binding moiety comprising a non-CDR loop. In one embodiment, the binding moiety masks binding of the TROP2-binding domain until activation. The cleavable linker comprises, for example, a protease cleavage site or a pH-dependent cleavage site. In certain cases, the cleavable linker is cleaved only in the tumor microenvironment. Thus, a binding moiety linked to a cleavable linker and further attached to a TROP2-binding domain maintains the TROP2-binding domain in an inactive state in the circulation, in some instances, until the cleavable linker is cleaved in the tumor microenvironment. In some embodiments, the binding moiety binds to the TROP2-binding domain. In some embodiments, the non-CDR loop provides a binding site for the moiety to bind to the TROP2-binding domain. In some embodiments, the binding moiety masks binding of the TROP2-binding domain to its target antigen, for example, through steric occlusion or specific intramolecular interactions (e.g., interactions within different domains of a polypeptide comprising the binding moiety). In some embodiments, the binding moiety further comprises a complementarity determining region (CDR).

[0122] In some cases, 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 corresponding to the conditionally active multispecific TROP2-targeting proteins of this disclosure.

[0123] In some embodiments, the conditionally active receptor comprises a TROP2-binding domain (aTarget1), a cleavable linker, and a binding moiety (aTarget2). The TROP2-binding domain has specificity for a first target (TROP2), 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 TROP2-binding domain to its target. Upon cleavage at the cleavable linker, the binding moiety can be released, allowing binding of the TROP2-binding domain.

[0124] In some embodiments, the inactive receptor (inactive ProCAR) comprises a TROP2 binding domain (anti-tumor targeting sdAb or scFv) linked to a binding moiety (anti-targeting 2 sdAb) via a linker containing a protease cleavage site. The binding moiety contains a masking peptide / site inserted within one or more non-CDR loops, such that the binding moiety binds to and inhibits the TROP2 antigen-binding domain. In some embodiments, the binding moiety has specificity for a given target, as described in more detail elsewhere herein. The receptor also comprises a transmembrane domain and an intracellular signaling domain. The receptor is provided in a T cell (CAR-T). Upon exposure to 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 now 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.

[0125] In some embodiments, the cleavable linker of the binding moiety comprises a protease cleavable site similar to those described above for the conditionally active multispecific proteins comprising a TROP2-binding domain of the present disclosure. For example, in some embodiments, the cleavable linker comprises an amino acid sequence selected from the linker sequences set forth in the Sequence Listing.

[0126] Transmembrane domain The conditionally active chimeric antigen receptors, T cell receptor fusion proteins, and T cell receptors of the present disclosure comprise a transmembrane domain for insertion into a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the TROP2-binding domain and the intracellular domain. In some embodiments, the transmembrane domain is interposed between the TROP2-binding domain and the costimulatory domain.

[0127] Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. As a non-limiting example, the TM sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 551) can be used. Additional non-limiting examples of suitable TM sequences include: a) CD8 beta-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).

[0128] 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 interposed between the TROP2-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 CD8-derived hinge region).

[0129] The hinge region can 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.

[0130] Suitable spacers can be readily selected and can be any of a number of suitable lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0131] Exemplary spacers include glycine polymers (G) n , glycine-serine polymers [e.g., (GS) n (SEQ ID NO: 514), (GSGGS) n (SEQ ID NO: 524) and (GGGS) n (SEQ ID NO:516); where n is an integer of at least 1], glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly 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 (SEQ ID NO:525), GGSGG (SEQ ID NO:526), ​​GSGSG (SEQ ID NO:527), GSGGG (SEQ ID NO:528), GGGSG (SEQ ID NO:529), GSSSG (SEQ ID NO:530), and the like.

[0132] The amino acid sequences of immunoglobulin hinge regions are known in the art; see, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT; CPPC; CPEPKSCDTPPPCPR; (see, e.g., Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT; KSCDKTHTCP; KCCVDCP; KYGPPCP; EPKSCDKTHTCPPCP; human IgG1 hinge; ERKCCVECPPCP; human IgG2 hinge; ELKTPLGDTTHTCPRCP; human IgG3 hinge; SPNMVPHAHHAQ; human IgG4 hinge), and the like.

[0133] 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 can be substituted with Tyr so that the hinge region comprises the sequence EPKSCDKTYTCPPCP. See, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891.

[0134] 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:) or a variant thereof.

[0135] 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 TROP2, 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.

[0136] In some embodiments, the conditionally active chimeric antigen receptor further 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 and no more than 20, no more than 10, or no more than 5 modifications thereto.

[0137] 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 at least one and no more than 20 modifications thereto.

[0138] In one aspect, the present disclosure provides cells (e.g., T cells) engineered to express a CAR. In one aspect, the cells are transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cells (e.g., T cells) are 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 cells may stably express the CAR. In another embodiment, the cells (e.g., T cells) are transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cells may transiently express the CAR.

[0139] Conditionally active T cell receptor fusion proteins In one embodiment, the present disclosure provides conditionally active T cell receptor fusion proteins. As used herein, "T cell receptor (TCR) fusion proteins" or "TFPs" include recombinant polypeptides derived from various polypeptides that comprise a TCR, which recombinant polypeptides are generally i) capable of binding to surface antigens on target cells, and ii) capable of interacting with other polypeptide components of an intact TCR complex, typically when co-located within or on the surface of a T cell.

[0140] The conditionally active TFP comprises a binding moiety, a TROP2-binding domain, and a T cell receptor subunit. In some embodiments, the T cell receptor subunit further comprises at least a portion of a T cell receptor extracellular domain, at least a portion of a transmembrane domain, and at least a portion of a T cell receptor intracellular domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a functional fragment thereof, or an amino acid sequence thereof having at least one, two, or three modifications thereto and no more than 20, no more than 10, or no more than 5 modifications thereto.

[0141] In some embodiments, the T cell receptor intracellular domain comprises a stimulatory domain. The stimulatory domain is derived from a T cell receptor subunit, including, but not limited to, a beta subunit, an alpha 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 a portion thereof, including, but not limited to, the following: CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc Gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CDS, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one, two, or three and no more than 20, no more than 10, or no more than 5 modifications thereto.

[0142] In some embodiments, the conditionally active TFP further 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 and no more than 20, no more than 10, or no more than 5 modifications thereto.

[0143] In some embodiments, the TROP2 binding domain is linked to the T cell receptor extracellular domain by a linker sequence. In some instances, the encoded linker sequence is (G4S) n(where n=1-4) (SEQ ID NO: 531). In some cases, the coding linker sequence includes a long linker (LL) sequence. In some cases, the long coding linker sequence includes (G4S) n (where n=2-4) (SEQ ID NO: 532). In some cases, the coding linker sequence includes a short linker (SL) sequence. In some cases, the short coding linker sequence includes (G4S) n (where n=1 to 3) (SEQ ID NO: 533).

[0144] In one aspect, the present disclosure provides cells (e.g., T cells) engineered to express a conditionally active T cell receptor fusion protein (TFP). In one aspect, the cells are transformed with the conditionally active TFP, and the conditionally active TFP is expressed on the cell surface. In some embodiments, the cells (e.g., T cells) are 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 cells may stably express the conditionally active TFP. In another embodiment, the cells (e.g., T cells) are transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the conditionally active TFP. In some such embodiments, the cells may transiently express the conditionally active TFP.

[0145] 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 binds to a T cell receptor subunit, including, but not limited to, an alpha subunit, a beta subunit, or a combination thereof.

[0146] In some embodiments, the binding moiety can or does mask binding of the 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 a 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 the T cell receptor to its target, e.g., through steric occlusion, specific intermolecular interactions, etc.

[0147] In one aspect, the present disclosure provides cells (e.g., T cells) engineered to express a conditionally active T cell receptor (TCR). In one aspect, the cells are transformed with the conditionally active TCR, and the conditionally active TCR is expressed on the cell surface. In some embodiments, the cells (e.g., T cells) are 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 cells may stably express the conditionally active TCR. In another embodiment, the cells (e.g., T cells) are transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the conditionally active TCR. In some such embodiments, the cells may transiently express the conditionally active TCR.

[0148] 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.

[0149] 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. CRL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL1.4), and the like. No. CRL1573), HLHepG2 cells, HuT-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), etc.

[0150] In some cases, the cells are not immortalized cell lines, but rather cells obtained from an individual (e.g., primary culture cells). For example, in some cases, 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.

[0151] Recent studies have used CAR constructs to induce the activity of natural killer (NK) cells, 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 induce immune responses. Because NK cells do not require HLA matching, they can be used as allogeneic effector cells (Harmanson & Kaufman, 2015). Additionally, therapeutically useful peripheral blood NK cells (PB-NK) can be isolated from donors by simple blood sampling. Useful CAR constructs may contain elements similar to those used to generate CAR-T cells.

[0152] Thus, in some embodiments, the present disclosure provides cells, including NK cells, that comprise 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.

[0153] As discussed above in the context of conditionally active TROP2-binding proteins (e.g., TROP2 ProTriTAC), in some embodiments, the conditionally active chimeric antigen receptors described herein have an improved therapeutic index compared to chimeric antigen receptors that contain the same TROP2-binding domain as the conditionally active variant but are constitutively active rather than conditionally active. For example, in some embodiments, TROP2 ProCAR has a higher therapeutic index than TROP2 CAR. In some embodiments, the increase 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. In some embodiments, the increase in therapeutic index results from conjugation of the TROP2-binding domain to the binding moiety in the presence of a non-CDR loop and a cleavable linker.

[0154] Methods for producing cells containing conditionally active receptors The present disclosure provides methods for producing (making) 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 using an expression vector or RNA (e.g., in vitro transcribed RNA) containing a nucleotide sequence encoding the conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor of the present disclosure. Genetic modification can be performed in vivo, in vitro, or ex vivo. The cells can be, for example, immune cells (e.g., T lymphocytes or NK cells), stem cells, or progenitor cells.

[0155] In some cases, the genetic modification is performed ex vivo, for example, by obtaining T lymphocytes, stem cells, or NK cells from an individual, and genetically modifying the cells obtained from the individual to express a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor of the present disclosure.

[0156] T cell source In some embodiments, the T cell source is obtained from a subject. As used throughout this disclosure, the term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can 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., allogeneic donor-derived CAR T cells), natural killer cells (e.g., donor-derived natural killer cells), peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, many T cell lines available in the art can be used. In certain embodiments of the present disclosure, T cells can be obtained from a blood unit drawn from a subject using a 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. Apheresis products typically contain lymphocytes, such as 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. In one embodiment of the present disclosure, cells are washed with phosphate-buffered saline (PBS). In another embodiment, the wash solution may be calcium-free, magnesium-free, or free of many, if not all, divalent cations. An initial activation step in the absence of calcium may result in enhanced activation. As will be readily understood by those skilled in the art, washing steps can be accomplished by methods known to those skilled in the art, such as 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 the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer.Alternatively, the undesired components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0157] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, e.g., DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In one embodiment, the time is approximately 30 minutes. In another embodiment, the time ranges from 30 minutes to 36 hours or more (and all integer values ​​therein). In another embodiment, the time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the incubation time is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. Longer incubation times can be used to separate 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 patients. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time allowed for T cells to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described in more detail herein), subpopulations of T cells can be preferentially selected or unselected at the beginning of culture or at other times during the process. Also, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected or unselected at the beginning of culture or at other desired times. Multiple rounds of selection can also be used in the context of the present disclosure.In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. The "unselected" cells may also be subjected to further rounds of selection.

[0158] Enrichment of T cell populations by negative selection can be achieved by combining antibodies against surface markers unique to the negatively selected cells. One method is negative magnetic immunoadhesion or flow cytometric cell sorting and / or selection using a cocktail of monoclonal antibodies 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 certain embodiments, it may be desirable to enrich or positively select regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25-conjugated beads or other similar selection methods.

[0159] 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 of screening for cell expression may be determined, for example, by the methods described in PCT Publication No. WO2013 / 126712.

[0160] To isolate a desired cell population by positive or negative selection, the surface (e.g., particles, e.g., beads) and cell concentration 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., increase the cell concentration) to ensure maximum contact between the cells and the 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 another embodiment, a concentration of greater than 100 million cells / ml is used. In another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In yet other embodiments, concentrations of 125 million or 150 million cells / ml may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest (e.g., CD28-negative T cells) or cells from samples containing a large number of tumor cells (e.g., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of higher cell concentrations allows for more efficient selection of CD8+ T cells, which typically exhibit weaker CD28 expression.

[0161] In another embodiment, it may be desirable to use a lower cell concentration. By significantly diluting the mixture of T cells and a surface (e.g., particles, e.g., beads), interactions between the particles and the cells are minimized. This selects for cells that express high amounts of the desired antigen that binds to the particles. For example, CD4+ T cells express high levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In one embodiment, the cell concentration used is 5 x 10e6 / ml. In other embodiments, the concentration used can be approximately 1 x 105 / ml to 1 x 106 / ml and any integer value therebetween. In other embodiments, the cells can be incubated on a rotator at various speeds for various lengths of time at 2-10°C or at room temperature.

[0162] Alternatively, stimulatory T cells may be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing step provides a more homogeneous product by removing granulocytes and some monocytes within the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While numerous freezing solutions and parameters are known in the art and are useful in this case, one method involves using 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, such as Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can also be used, or immediate uncontrolled freezing at -20°C or in liquid nitrogen is also possible. In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for 1 hour before activation using the methods of the present disclosure.

[0163] It is also contemplated within the context of the present disclosure that a blood sample or apheresis product may be collected from a subject prior to the time when the expanded cells described herein may be needed. Thus, a cellular source of cells to be expanded can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for numerous diseases or conditions (e.g., those described herein) that would benefit from T cell therapy. In one embodiment, a blood sample or apheresis is collected from a generally healthy subject. In a specific embodiment, a blood sample or apheresis is collected from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and later used. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a particular disease described herein and prior to any treatment. In another embodiment, cells are isolated from a blood sample or apheresis from a subject prior to a number of relevant therapies, including but not limited to, treatment with therapeutic agents such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

[0164] In another embodiment of the present disclosure, T cells are obtained from a patient immediately after a treatment that leaves functional T cells in the subject patient. In this regard, it has been observed that the quality of T cells collected immediately after certain cancer treatments, particularly treatments with drugs that damage the immune system, during the period when patients would normally recover from the treatment, may be optimal or improved in terms of their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for promoting engraftment and in vivo expansion. Thus, in the context of the present disclosure, it is contemplated to collect blood cells, including T cells, dendritic cells, or other hematopoietic cells, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization using GM-CSF) and conditioning regimens may be used to create a condition in the subject that promotes repopulation, recirculation, regeneration, and / or proliferation of specific cell types, particularly within a defined time frame after treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0165] T cell activation and proliferation T cells can generally be activated and expanded using methods described, for example, in 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 A1.

[0166] Generally, T cells of the present disclosure are expanded by contact with a surface bearing an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, T cell populations can be stimulated as described herein, for example, by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Costimulation of accessory molecules on the T cell surface can be achieved using a ligand that binds to the accessory molecule. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. Anti-CD3 antibody and anti-CD28 antibody are used to stimulate the proliferation of either CD4+ or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which may be used as well as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999).

[0167] In certain embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by different protocols. For example, the substance providing each signal can be in solution or bound to a surface. If surface-bound, the substances can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one substance can be bound to a surface and the other substance can be in solution. In one embodiment, the substance providing the costimulatory signal is bound to a cell surface, and the substance providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both substances can be in solution. In one embodiment, the substances can be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding substance that binds to the substance. In this regard, see, for example, U.S. Patent Application Publication Nos. US 20040101519 A1 and US 20060034810 A1 regarding artificial antigen presenting cells (aAPCs) contemplated for use in T cell activation and expansion in the present disclosure.

[0168] In one embodiment, the two substances are immobilized on beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). For example, the substance providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the substance providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, with both substances being co-immobilized on the same bead with equivalent molecular weights. In one embodiment, a 1:1 ratio of each antibody bound to beads for CD4+ T cell proliferation and T cell growth is used. In specific embodiments of the present disclosure, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell proliferation is observed compared to the proliferation observed when a 1:1 ratio is used. In one specific embodiment, an increase of about 1-fold to about 3-fold is observed compared to the proliferation observed when a 1:1 ratio is used. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100 (and all integer values ​​therebetween). In one embodiment of the 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 a specific embodiment of the disclosure, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In one specific 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 another 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 another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

[0169] To stimulate T cells or other target cells, particle-to-cell ratios of 1:500 to 500:1 (and any integer value therebetween) can be used. As can be readily understood by one of ordinary skill in the art, the particle-to-cell ratio can 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 particular embodiments, cell-to-particle ratios in the range of 1:100 to 100:1, and in more specific embodiments, ratios of 1:9 to 9:1 (and any integer value therebetween) can also 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, but specific values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 (particles:T cells). In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In one specific embodiment, the particle-to-cell ratio is 1:5. In yet other 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 then additional particles are added to the cells every day or every other day for up to 10 days, resulting in a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In one specific 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 every day or every other day, resulting in a final 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 every day or every other day, resulting in a final 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 appreciate that various other ratios may also be suitable for use in the present disclosure.In particular, the ratio will vary depending on particle size and cell size and type.

[0170] In yet other embodiments of the present disclosure, cells, e.g., T cells, are combined with agent-coated beads, then the beads and cells are separated, and then the cells are cultured. In another embodiment, the agent-coated beads and cells are not separated prior to culture, but are cultured together. In another embodiment, the beads and cells are first concentrated by applying a force, e.g., a magnetic force, to increase ligation of cell surface markers, thereby inducing cell stimulation.

[0171] For example, cell surface proteins can be ligated by contacting T cells with anti-CD3 and anti-CD28 conjugated paramagnetic beads (3x28 beads). 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 (containing no divalent cations such as calcium and magnesium). Again, one of skill in the art will readily appreciate that any cell concentration can be used. For example, the target cells may be very low in the sample, constituting as little as 0.01% of the sample, or the entire sample (i.e., 100%) may comprise the target cells of interest. Thus, any cell number is within the scope of the present 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 about 2 billion cells / ml is used. In one embodiment, a concentration of greater than 100 million cells / ml is used. In another embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In yet other embodiments, a concentration of 125 or 150 million cells / ml may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest (e.g., CD28-negative T cells). In certain embodiments, such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of higher cell concentrations allows for more efficient selection of CD8+ T cells, which normally exhibit weaker CD28 expression.

[0172] 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 therebetween). 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. Several stimulation cycles may be desirable, 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., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for growth and survival, including: serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, TNF-α, or any other additive for cell growth 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-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, X-Vivo 20, and Optimizer. The media are 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 defined combination of hormones and / or cytokines in amounts sufficient for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and are not included in the culture 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 + 5% CO2).

[0173] T cells subjected to different stimulation times can exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product contains a larger population of helper T cells (TH, CD4+) than cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors generates a population of T cells consisting primarily of TH cells until about day 8-9, while from about day 8-9 onwards, the T cell population includes an increasingly larger population of TC cells.

[0174] Modification of TROP2-binding proteins The TROP2-binding proteins described herein, including TROP2-binding domains (e.g., the TROP2-binding sdAbs of the present disclosure) and TROP2-targeting multispecific proteins (e.g., the TROP2-targeting trispecific or pro-trispecific proteins described herein), encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not encoded by the genetic code, (ii) the mature polypeptide is fused to another compound, such as polyethylene glycol, or (iii) additional amino acids, such as a leader sequence or secretory sequence or an amino acid sequence for protein purification, are fused to the protein.

[0175] 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, e.g., arginylation, and ubiquitination.

[0176] The TROP2-binding proteins described herein may be modified at any site, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Specific common peptide modifications useful for modifying TROP2-binding proteins include glycosylation, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, covalent protection of amino or carboxyl groups, or both, in polypeptides, and ADP-ribosylation.

[0177] In some embodiments, derivatives of the TROP2 binding proteins described herein include immunoreactive modulator derivatives and antigen-binding molecules that contain one or more modifications.

[0178] In some embodiments, the TROP2-binding proteins of the present disclosure are monovalent or multivalent (bivalent, trivalent, etc.). As used herein, the term "valency" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds 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 multiple target binding sites (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).

[0179] In some embodiments, the TROP2-binding proteins are 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 Fc-fused TROP2-binding proteins. In some embodiments, the Fc-fused TROP2-binding proteins of the present disclosure have an extended half-life compared to an otherwise identical TROP2-binding protein. In some embodiments, the Fc-fused TROP2-binding proteins of the present disclosure contain, inter alia, substitutions, mutations, and / or modifications of one or more additional amino acid residues, e.g., in the Fc region, that confer favorable properties to the binding protein, including, but not limited to, altered pharmacokinetics and extended serum half-life.

[0180] In some embodiments, such Fc-fusion TROP2-binding proteins have an extended half-life in mammals (e.g., humans) of greater than 5 days (i.e., more 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. Extended half-life can, in some cases, result in higher serum titers, thereby reducing the frequency of administration of the TROP2-binding protein and / or reducing the administered antibody concentration. In some examples, the in vivo binding and serum half-life of human FcRn high-affinity-binding polypeptides to human FcRn are assayed in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides containing mutant Fc regions.

[0181] In some cases, TROP2 binding proteins are differentially modified during or after production, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting groups / protecting 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.

[0182] The present disclosure also encompasses various post-translational modifications of TROP2-binding proteins, including, for example, N- or O-linked glycans, N- or C-terminal processing, chemical modifications to the amino acid backbone, chemical modifications of N- or O-linked glycans, and the addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. Additionally, TROP2-binding proteins are sometimes modified with a detectable label, such as an enzyme label, a fluorescent label, a radioisotope label, or an affinity label, to enable detection and isolation of modulators.

[0183] Polynucleotides encoding TROP2-binding proteins Also provided in some embodiments are polynucleotide molecules encoding the TROP2-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.

[0184] Polynucleotide molecules are constructed by known methods, for example, by combining genes encoding single-domain TROP2-binding proteins or genes encoding various domains of TROP2-binding proteins containing multiple domains. In some embodiments, the genes encoding the domains are separated by peptide linkers, or in other embodiments, directly linked by peptide bonds to form a single gene construct operably linked to a suitable promoter and, optionally, a suitable transcription terminator, which is expressed in bacteria or other suitable expression systems, such as CHO cells. Depending on the vector system and host used, a number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. The promoter is selected so that it drives the expression of the polynucleotide in the respective host cells.

[0185] In some embodiments, the polynucleotide encoding the TROP2-binding protein described herein is inserted into a vector, preferably an expression vector, representing a further embodiment. The 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.

[0186] A variety of expression vector / host systems can be utilized to contain and express polynucleotides encoding the described TROP2-binding protein polypeptides. Examples of expression vectors include pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) for expression in Escherichia coli (E. coli) or pcDNA5 (Invitrogen) for expression in mammalian cells. Thus, in some embodiments, the TROP2-binding proteins described herein can be produced and isolated by introducing a vector encoding the protein into host cells and culturing the host cells under conditions in which the protein domains are expressed, and can be further purified, if desired.

[0187] Pharmaceutical Composition Additionally, in some embodiments, pharmaceutical compositions are provided comprising an anti-TROP2 binding protein described herein, a vector containing a polynucleotide encoding a TROP2 binding protein polypeptide, or a host cell transformed with this vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is non-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 (e.g., 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 also contain auxiliary agents, such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial attack can be ensured by incorporating various antibacterial and antifungal agents. Another embodiment provides one or more of the TROP2 binding proteins packaged in lyophilized form or in an aqueous medium.

[0188] In some embodiments of the pharmaceutical composition, the TROP2-binding protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, liquid crystal, liposome, quantum dot, superparamagnetic nanoparticle, dendrimer, or nanorod. In other embodiments of the pharmaceutical composition, the TROP2-binding protein is bound to a liposome. In some cases, the TROP2-binding protein is bound to the surface of the liposome. In some cases, the TROP2-binding protein is encapsulated within the shell of the liposome. In some cases, the liposome is a cationic liposome.

[0189] The TROP2 binding proteins described herein are intended for use as pharmaceuticals. Administration can be accomplished by a variety of methods, including 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 contained in the pharmaceutical composition. The administration regimen will be determined by the attending physician and other clinical factors. The dosage for a given patient will depend on numerous factors, including the patient's size, body surface area, age, sex, the particular compound being administered, the time and route of administration, the type of treatment, general health, and other concomitantly administered drugs. An "effective amount" refers to an amount of active ingredient sufficient to affect the course and severity of the disease, resulting in the reduction or amelioration of such a condition, and can be determined using known methods.

[0190] In some embodiments, a TROP2-binding protein of the present disclosure is administered once weekly at a dose of up to 10 mg / kg. In some instances, 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 dose 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 to about 5 μg / kg, about 2 μg / kg, or about 3 μg / kg. / kg to about 10 μg / kg, about 7 μg / kg to about 15 μg / kg, about 12 μg / kg to about 25 μg / kg, about 20 μg / kg to about 50 μg / kg, about 20 μg / kg to about 60 μg / kg, about 35 μg / kg to about 70 μg / kg, about 45 μg / kg to about 80 μg / kg, about 65 μg / kg to about 90 μg / kg, about 85 μg / kg to about 0.1 mg / kg, about 0.095 mg / kg to about 10 mg / kg, and about 20 μg / kg to 540 μg / kg. In some cases, the dosage is about 0.1 mg / kg to about 0.2 mg / kg, about 0.25 mg / kg to about 0.5 mg / kg, about 0.45 mg / kg to about 1 mg / kg, about 0.75 mg / kg to about 3 mg / kg, about 2.5 mg / kg to about 4 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 6 mg / kg, about 5.5 mg / kg to about 7 mg / kg, about 6.5 mg / kg to about 8 mg / kg, about 7.5 mg / kg to about 9 mg / kg, or about 8.5 mg / kg to about 10 mg / kg.In some embodiments, the TROP2 binding proteins of the present disclosure are administered at concentrations of 1 ng / kg, 2 ng / kg, 5 ng / kg, 10 ng / kg, 20 ng / kg, 30 ng / kg, 40 ng / kg, 50 ng / kg, 60 ng / kg, 60 ng / kg, 70 ng / kg, 80 ng / kg, 90 ng / kg, 100 ng / kg, 200 ng / kg, 300 ng / kg, 400 ng / kg, 500 ng / kg, 600 ng / kg, 700ng / kg, 800ng / kg, 900ng / kg, 1μg / kg, 2μg / kg, 5μg / kg, 10μg / kg, 12μg / kg, 15μg / kg, 20μg / kg, 22.5μg / kg, 25μg / kg, 30μg / kg, 40μg / kg, 50μg / kg, 60μg / kg, 70μg / kg, 80μg / kg, 90μg / kg, 100μg / kg, 130μg / kg, 150μg / kg, 180μg / k g, 200μg / kg, 225μg / kg, 250μg / kg, 280μg / kg, 300μg / kg, 350μg / kg, 370μg / kg, 400μg / kg, 430μg / kg, 460μg / kg, 50 0μg / kg, 540μg / kg, 590μg / kg, 600μg / kg, 630μg / kg, 670μg / kg, 700μg / kg, 730μg / kg, 780μg / kg, 800μg / kg, 840μg / kg The drug is administered once weekly at a dose of 900 μg / kg, 950 μg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, or 10 mg / kg. In some embodiments, the administration frequency is less than about daily, every other day, less than once daily, twice weekly, weekly, once every seven days, once every two weeks, once every three weeks, once every four weeks, or once monthly. In some cases, the administration frequency is weekly. In some cases, the administration frequency is weekly and the dosage is up to 10 mg / kg. In some cases, the administration period is from about 1 day to about 4 weeks or longer.

[0191] Therapeutic methods and tumor growth inhibitory properties In certain embodiments, methods are provided for treating a condition associated with malignant cells expressing TROP2 in a subject in need thereof, comprising administering to the subject an effective amount of a TROP2-binding domain or a multispecific protein (including a conditionally active multispecific protein) comprising a TROP2-binding domain of the present disclosure, or a CAR or ProCAR comprising a TROP2-binding protein described herein, or a pharmaceutical composition comprising the same. In some embodiments, the condition is cancer.

[0192] In another aspect, the present disclosure provides a method for inhibiting tumor growth or progression in a subject having malignant cells expressing TROP2, comprising administering to a subject in need thereof an effective amount of a TROP2-binding domain or a multispecific protein comprising a TROP2-binding domain of the present disclosure, or a CAR comprising a TROP2-binding protein 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 TROP2 in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of a TROP2-binding domain or a multispecific protein comprising a TROP2-binding domain of the present disclosure, or a pharmaceutical composition comprising the same. In another aspect, the present disclosure provides a method for inducing tumor regression in a subject having malignant cells expressing TROP2, comprising administering to a subject in need thereof an effective amount of a TROP2-binding domain or a multispecific protein comprising a TROP2-binding domain of the present disclosure, 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 therapeutic agent, such as an antibody. In some embodiments, the second therapeutic agent is a cytokine, TNFα (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 therapy, a TLR (Toll-like receptor) agonist (e.g., TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine.

[0193] In certain embodiments, the TROP2-binding proteins of the present disclosure reduce in vivo tumor cell proliferation when administered to a subject bearing tumor cells that express TROP2. Measurement of tumor cell proliferation reduction can be performed by a number of different methods known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison with a control subject, measurement by imaging techniques (e.g., CT or MRI) with or without isotopes or luminescent molecules (e.g., luciferase) to enhance analysis, etc. In certain embodiments, administration of the TROP2-binding proteins of the present disclosure results in a reduction in in vivo tumor cell proliferation of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control antigen-binding agent, where a reduction in tumor growth of about 100% indicates a complete response or disappearance of the tumor. In still other embodiments, administration of a TROP2-binding protein of the present disclosure results in about a 50-100%, about 75-100%, or about 90-100% reduction in in vivo tumor cell proliferation compared to a control antigen-binding substance. In another embodiment, administration of a TROP2-binding protein of the present disclosure results in about a 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% reduction in in vivo tumor cell proliferation compared to a control antigen-binding substance. In some embodiments, administration of a TROP2-binding protein of the present disclosure results in a complete reduction in in vivo tumor cell proliferation, e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after the initial administration. In some embodiments, the reduction in tumor cell proliferation, e.g., reduction in in vivo tumor cell proliferation, lasts for 1 hour, 2 hours, 5 hours, 10 hours, 23 hours, 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 30 days, 35 days, 40 days, 45 days, 50 days or more.

[0194] In some embodiments, active drugs and prodrugs containing the same TROP2-binding protein of the present disclosure reduce tumor cell proliferation in vivo comparably when administered in equivalent molar amounts to subjects with tumor cells that express TROP2.

[0195] In some embodiments, the TROP2 binding proteins of the present disclosure are administered to treat a neoplastic condition. In some embodiments, the neoplastic condition is benign or malignant, and is a solid tumor or other hematological tumor, and in some embodiments is selected from the group including, but not limited to, adrenal gland tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytic tumor, autonomic ganglionic tumor, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastic disorders, bone cancer (adamantinoma, aneurysmal bone cyst, osteochondroma, osteosarcoma), brain and spinal cord cancer, metastatic brain tumor, breast cancer including triple-negative breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophilic 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, fibrous dysplasia, gallbladder and bile duct cancer, gastric cancer, gastrointestinal tract, gestational trophoblast Sexual disorders, germ cell tumors, glandular disorders, head and neck cancer, hypothalamus, intestinal cancer, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma / benign lipomatous tumors, liposarcoma / malignant lipomatous tumors, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), macrophage disorders, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplasia syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumors, childhood cancer, peripheral nerve sheath tumors, pheochromocytoma, pituitary tumors, prostate cancer, posterior uveal melanoma, rare blood disorders, renal metastases, rhabdoid tumors, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, stromal diseases, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastases and uterine cancer (cervical cancer, endometrial cancer and leiomyoma).

[0196] In certain embodiments, the TROP2-binding proteins of the present disclosure are used as frontline therapy and are administered to subjects who have not previously received treatment for a cancerous condition. In other embodiments, the TROP2-binding proteins of the present disclosure are used to treat subjects who have previously received treatment (with a TROP2-binding protein of the present disclosure or another anti-cancer agent) and who have relapsed or been determined to be refractory to the previous treatment. In some embodiments, the TROP2-binding proteins of the present disclosure are used to treat subjects with recurrent tumors.

[0197] In some embodiments, the TROP2-binding proteins described herein, including the multispecific proteins, CARs, or ProCARs described herein, are administered to treat a wide range of cancers with TROP2 expression and prevalence, including, but not limited to, colorectal cancer, prostate cancer, neuroendocrine cancer, thyroid cancer, lung cancer (both non-small cell and small cell lung cancer), gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, biliary tract and gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, or any combination thereof.

[0198] In some embodiments, the TROP2-binding proteins of the present disclosure are administered to treat proliferative disorders, including solid tumors, including, but not limited to, adrenal, liver, kidney, bladder, breast, stomach, ovary, cervical, uterine, esophageal, colorectal, prostate, pancreatic, lung (both small cell and non-small cell), thyroid, carcinoma, sarcoma, glioblastoma, various head and neck tumors, or any combination thereof.

[0199] In some embodiments, the TROP2-binding proteins of the present disclosure are administered to a subject suffering from melanoma. In some embodiments, the TROP2-binding proteins of the present disclosure are used for the diagnosis, monitoring, treatment, or prevention of melanoma. As used herein, the term "melanoma" includes all types of melanoma, including, but not limited to, primary melanoma, malignant melanoma, cutaneous melanoma, extracutaneous melanoma, superficial spreading melanoma, polypoid melanoma, melanoma, melanoepithelioma, melanosarcoma, melanoma 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 nevus and melanoma (FAM-M) syndrome, desmoplastic malignant melanoma, uveal melanoma, or any combination thereof.

[0200] In some embodiments, possible indications for administration of the TROP2-binding proteins of the present disclosure or pharmaceutical compositions comprising same are neoplastic diseases, particularly epithelial cancers / carcinomas, such as breast cancer, colorectal cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the genitourinary tract, such as ovarian cancer, endometrial cancer, cervical 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 TROP2-binding proteins of the present disclosure or pharmaceutical compositions comprising same is indicated for minimal residual disease, such as early-stage solid tumors, advanced solid tumors or metastatic solid tumors characterized by local and non-local recurrence of tumors caused by single-cell survival, or any combination thereof.

[0201] In selected embodiments, the TROP2 binding proteins of the present disclosure are incorporated into a chimeric antigen receptor (CAR), and the TROP2 CAR is administered in a CAR-based therapy effective in treating, for example, cancers such as epithelial cancers / carcinomas, e.g., breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, cancers of the genitourinary tract, e.g., ovarian cancer, endometrial cancer, cervical 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, thyroid cancer, small cell lung cancer, non-small cell lung cancer (e.g., squamous non-small cell lung cancer or squamous small cell lung cancer), large cell neuroendocrine carcinoma (LCNEC), or any combination thereof.

[0202] Chimeric antigen receptors are generally artificially constructed hybrid proteins or polypeptides containing or including the antigen-binding domain of an antibody linked to a signaling domain (e.g., a T cell signaling domain or a T cell activation domain). In some embodiments, CARs comprising the TROP2-binding proteins of the present disclosure have the ability to redirect the specificity and reactivity of sensitized lymphocytes (e.g., T cells) to TROP2-positive target cells in an MHC-unrestricted manner by utilizing the antigen-binding properties of an antibody or its antigen-binding fragment. This MHC-unrestricted antigen recognition confers oncogenic TROP2 to T cells expressing the TROP2 CAR the ability to recognize tumorigenic TROP2 independently of antigen processing, thereby circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the endogenous T cell receptor (TCR) α and β chains.

[0203] In some embodiments, the disclosed TROP2 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 disease recurs more than 2-3 months after initial treatment), or patients who are resistant to platinum-based agents (e.g., carboplatin, cisplatin, oxaliplatin) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, the disclosed TROP2 CAR therapy is effective in treating ovarian cancer, including ovarian serous carcinoma and ovarian papillary serous carcinoma.

[0204] In another embodiment, the TROP2-binding protein, TROP2 CAR, or TROP2-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 onset of the disease. In some cases, the disorder has been treated, the initial tumor mass has disappeared, shrunk, or otherwise improved, and the patient is therefore asymptomatic or in remission. At such a time, regardless of whether there are little or no signs of disease using standard diagnostic procedures, a pharmaceutically effective amount of the TROP2-binding protein, TROP2 CAR, or TROP2-sensitized lymphocytes of the present disclosure, or any combination thereof, is administered to the subject one or more times. In some embodiments, the TROP2-binding protein, TROP2 CAR, or TROP2-sensitized lymphocytes of the present disclosure, or any combination thereof, is administered periodically 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 the patient's response and clinical and diagnostic parameters.

[0205] In yet another embodiment, the disclosed TROP2-binding proteins, TROP2 CARs, or TROP2-sensitized lymphocytes, or any combination thereof, are used prophylactically or as adjuvant therapy to prevent or reduce the likelihood of tumor metastasis after tumor debulking treatment. As used herein, "tumor debulking treatment" refers to any treatment, technique, or method that removes, reduces, treats, or ameliorates tumors or tumor growth. Exemplary tumor debulking treatments include, but are not limited to, surgery, radiation therapy (i.e., beam irradiation), chemotherapy, immunotherapy, or ablation. In some embodiments, the disclosed TROP2-binding proteins, TROP2 CARs, or TROP2-sensitized lymphocytes, or any combination thereof, are administered at the appropriate time as indicated by clinical, diagnostic, or theranostic treatment to reduce tumor metastasis. In some embodiments, the administration regimen includes appropriate diagnostic or monitoring techniques that allow for modification of the administration regimen.

[0206] Still other embodiments of the present disclosure include administering a TROP2-binding protein, TROP2 CAR, or TROP2-sensitized lymphocyte, or any combination thereof, of the present disclosure to a subject who is asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, a TROP2-binding protein, TROP2 CAR, or TROP2-sensitized lymphocyte, or any combination thereof, of the present disclosure, is used in a prophylactic sense and administered to patients who have been examined or tested and have one or more notable risk factors (e.g., genomic abnormalities, family history, in vivo or in vitro test results, etc.) but have not developed a tumor. In such cases, one of skill in the art would be able to determine an effective dosing regimen by empirical observation or accepted clinical practice.

[0207] In some embodiments of the methods described herein, the TROP2-binding proteins or compositions described herein are administered in combination with a therapeutic agent (also referred to herein as an additional therapeutic agent) for a particular disease, disorder, or condition. Such agents include therapies including, but 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, ultraviolet light, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, the TROP2-binding proteins described herein are administered in combination with antidiarrheals, antiemetics, analgesics, opioids, and / or nonsteroidal anti-inflammatory agents. In some embodiments, the TROP2-binding proteins described herein are administered in combination with an anti-cancer agent. Non-limiting examples of anti-cancer agents that may be used in various embodiments of the present invention, including the pharmaceutical compositions and 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; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; blu Sulfane; Cactinomycin; Calsterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cilolemycin; Cisplatin; Cladribine; Crisnatol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine 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; Etoprine; Fadrozole hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine phosphate; Fluorouracil; Flulocitabine; Fosquidone; Fostriecin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Ilmofosine; Inter 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; ibroplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melenesin acetate Trol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindomide; Mitocalcine; Mitochromin; Mitogillin; Mitomarcin; Mitomycin; Mitospar; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicama Isin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofurin; Ribopurin; Rogletimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Surofenur; Tallysomycin; Tecogalan sodium; Tegafur; Teloxantrone hydrochloride; Temoporfin; Teniposide;Teloxylon; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestron acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tuburozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; binepidine sulfate; vinglisinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other examples of anti-cancer drugs include, but are not limited to, 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecipenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsal formation protein-1; antiandrogens for prostate cancer; antiestrogens; antitumor agents; antisense oligonucleotides; aphidicolin glycinate; apoptotic gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; Arginine deaminase; Asulaculin; Atamestane; Atlimustine; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azasetron; Azatoxins; Azatyrosine; Baccatin III derivatives; Balanol; Batimastat; BCR / ABL antagonists; Benzochlorins; Benzoylstaurosporines; Beta-lactam derivatives; Beta-arretin; Betaclamycin B; Betulinic acid; bFGF inhibitors; Bicalutamide; Bisantrene; Bisaziridinylspermine; Bisnafide; Bistraten A; Bizelesin; Breflate; Bropirimine; Budotitanium; Buthionine sulfoximine; Calcipotriol; Calphostin C; Camptothecin derivatives; Canarypox IL-2; Capecitabine; Carboxamido-amino-triazoles;Carboxyamidotriazole; CaRest M3; CARN 700; Cartilage-derived inhibitor; Carzelesin; Casein kinase inhibitor (ICOS); Castanospermine; Cecropin B; Cetrorelix; Chlorin; Chloroquinoxaline sulfonamide; Cicaprost; cis-Porphyrin; Cladribine; Clomiphene analogs; Clotrimazole; Collismycin A; Collismycin B; Combretastatin A4; Combretastatin analogs; Conagenin; Crambescidin 816; Crisnatol; Cryptophycin 8; Cryptophycin A derivatives; Curacin A; Cyclopentan Laquinone; Cycloplatin; Sipemycin; Cytarabine ocphosphate; Cytolytic factors; Cytostatin; Dacliximab; Decitabine; Dehydrodidemnin B; Deslorelin; Dexamethasone; Dexphosphamide; Dexrazoxane; Dexverapamil; Diazicon; Didemnin B; Didox; Diethylnorspermine; Dihydro-5-azacytidine; Dihydrotaxol; 9-; Dioxamycin; Diphenylspiromustine; Docetaxel; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Dronabinol ol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emiteflu; epirubicin; epristeride; estramustine analogues; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunornithine hydrochloride; forfenimex; formest Tan; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridone; imiquimod; immunostimulating peptides; insulin-like growth factor-I receptor inhibitors; interferon agonists; interferon;Interleukins; Iobenguane; Iododoxorubicin; Ipomeanol, 4-; Ilopract; Irsogladine; Isobengazole; Isohomohalichondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprorelid + estrogen + progesterone; Leuprorelin; Levamisole; Liarozole; Linear polyamine analogs; Lipophilic disaccharide peptides tides; lipophilic platinum compounds; lisoclinamide 7; lobaplatin; lombricin; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitors (e.g., but not limited to, lovastatin, pravastatin, fluvastatin, statins, simvastatin, and atorvastatin); loxoribine; lurtotecan; lutetium texaphyrin; lisofylline; lytic peptides; maytansine; mannostatin A; marimastat; massoprocol; maspin; matrilysin inhibitors; matrix metalloproteinases Inhibitors; Menogaril; Melbarone; Meterelin; Methioninase; Metoclopramide; MIF inhibitors; Mifepristone; Miltefosine; Millimostim; Mismatched double-stranded RNA; Mitoguazone; Mitolactol; Mitomycin analogs; Mitonafide; Mitotoxin fibroblast growth factor-saporin; Mitoxantrone; Mofalotene; Molgramostim; Monoclonal antibodies; Human chorionic gonadotropin; Monophosphoryl lipid A + Myobacterium cell wall sk; Mopidamol; Multidrug resistance gene inhibitors; Multiple tumor suppressor 1-based therapy ;Mustard anticancer agents;Mycaperoxide B;Mycobacterial cell wall extract;Myriaporone;N-acetyldinaline;N-substituted benzamides;Nafarelin;Nagressip;Naloxone + pentazocine;Napavidin;Naphterpine;Nartograstim;Nedaplatin;Nemorubicin;Neridronic acid;Neutral endopeptidase;Nilutamide;Nisamycin;Nitric oxide modulators;Nitroxide antioxidants;Nitrulline;O6-benzylguanine;Octreotide;Oxenone;Oligonucleotides;Onapristone;Ondansetron;Ondansetron;Oracin; oral cytokine inducers; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomyphen; parabactin; pazelliptin; pegaspargase; ;Perdecin;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron;Perphosphamide;Perillyl alcohol;Phenazinomycin;Phenylacetic acid;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;Propylbisacridone;Prostaglandin J2;Proteasome inhibitors Inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Microalgae protein kinase C inhibitors; Protein tyrosine phosphatase 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; Rohitucine; Romurtide; Roquinimex; Rubidinone B1; Ruboxil; Safingol; Santopine; SarCNU; Sarcophytol A; Sargramostim; Sdi1 mimetic; Semustine; Senescence-derived inhibitor 1; Sense oligonucleotide; Signal transduction inhibitor; Signal transduction modulator; Single-chain antigen-binding protein; Sizofiran; Sobuzoxane; Borocaptate sodium; Sodium phenylacetate; Sorbetol; Somatomedin-binding protein; Sone Lumin; Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Spongestatin 1; Squalamine; Stem cell inhibitors; Stem cell division inhibitors; Stipiamid; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonists; Sladista; Suramin; Swainsonine; Synthetic glycosaminoglycans; Talimustine; Tamoxifen methiodide; Tauromustine; Tazarotene; Tecogalan sodium; Tegafur; Terlapyrylium; Telomerase inhibitors; Temoporfin; Temozolomide; Teniposide;Tetrachlorodecaoxide;Tetrazomine;Taliblastin;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrin thyroid-stimulating hormone;Tin ethyl etiopurpurin;Tirapazamine;Titanocene dichloride;Topsentin;Toremifene;Totipotent stem cell factor;Translation inhibitors;Tretinoin;Triacetyluridine;Triciribine;Trimetrexate;Triptorelin;Tropise Tolon; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector systems; erythrocyte gene therapy; veraresol; veramine; verudin; verteporfin; vinorelbine; vinxartin; 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 employing thalidomide and topoisomerase inhibitors. In some embodiments, the TROP2 binding proteins of the present disclosure are used in combination with gemcitabine. In some embodiments, the TROP2 binding proteins described herein are administered before, during, or after surgery.

[0208] Methods for detecting TROP2 expression and diagnosing TROP2-associated cancers According to another embodiment of the present disclosure, there is provided a kit for detecting TROP2 expression in vitro or in vivo. The kit includes the TROP2-binding protein (e.g., a TROP2-binding protein containing a labeled anti-TROP2 single-domain antibody or its antigen-binding fragment) 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.

[0209] In some cases, TROP2 expression is detected in a biological sample. The sample can be any sample, including, but not limited to, tissue from a biopsy, autopsy, or pathology specimen. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0210] In one embodiment, a method is provided for determining whether a subject has cancer by contacting a sample from the subject with an anti-TROP2 single domain antibody disclosed herein and detecting binding of the single domain antibody to the sample. Enhanced binding of the antibody to the sample compared to binding of the antibody to a control sample identifies the subject as having cancer.

[0211] In another embodiment, there is provided a method for confirming the diagnosis of cancer in a subject by contacting a sample from a subject diagnosed with cancer with an anti-TROP2 single domain antibody disclosed herein and detecting binding of the antibody to the sample. Enhanced binding of the antibody to the sample compared to binding of the antibody to a control sample confirms the diagnosis of cancer in the subject.

[0212] In some embodiments of the disclosed method, the TROP2 single domain antibody is directly labeled. In some embodiments, the method further includes contacting the sample with a second antibody that specifically binds to the anti-TROP2 single domain antibody and detecting binding of the second antibody. Enhanced binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects cancer in the subject or confirms the diagnosis of cancer in the subject. In some cases, the cancer is neuroendocrine cancer, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple-negative breast cancer, or ovarian cancer (e.g., epithelial ovarian cancer), or any other type of cancer that expresses TROP2. In some embodiments, the control sample is a sample from a subject without cancer. In certain embodiments, the sample is a blood or tissue sample.

[0213] In some examples, the antibody that binds (e.g., specifically binds) TROP2 is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) TROP2 (first antibody) is unlabeled, and a second antibody or other molecule that can bind to the antibody that specifically binds TROP2 is labeled. The second antibody is selected so that it can specifically bind to a particular species and class of the first antibody. For example, if the first antibody is llama IgG, the second antibody can be an anti-llama IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for the antibody or second antibody are described above and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Non-limiting exemplary luminescent materials include luminol, non-limiting exemplary magnetic materials include gadolinium, and non-limiting exemplary radioactive labels include fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. 125 I, 131 I, 35 S or 3 Contains H.

[0214] In another embodiment, TROP2 can be assayed in a biological sample by a competitive immunoassay using a TROP2 standard labeled with a detectable substance and an unlabeled antibody that specifically binds to TROP2. In this assay, the biological sample, the labeled TROP2 standard, and the antibody that specifically binds to TROP2 are combined, and the amount of labeled TROP2 standard bound to the unlabeled antibody is measured. The amount of TROP2 in the biological sample is inversely proportional to the amount of labeled TROP2 standard bound to the antibody that specifically binds to TROP2.

[0215] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, an antibody that specifically binds to TROP2 can be used to detect TROP2 production in cells in cell culture. In another embodiment, the antibody can be used to detect the amount of TROP2 in a biological sample, such as a tissue sample or a blood or serum sample. In some examples, the TROP2 is cell-surface TROP2. In other examples, the TROP2 is soluble TROP2 (e.g., TROP2 in a cell culture supernatant or in a body fluid sample such as a blood or serum sample).

[0216] In one embodiment, a kit for detecting TROP2 in a biological sample, such as a blood or tissue sample, is provided. For example, a biopsy may be performed to obtain a tissue sample for histological examination to confirm a diagnosis of cancer in a subject. Alternatively, a blood sample may be taken to detect the presence of a soluble TROP2 protein or fragment. Kits for detecting polypeptides typically include a single-domain antibody according to the present disclosure that specifically binds to TROP2. In some embodiments, the kit includes an antibody fragment, such as an scFv fragment, a VH domain, or a Fab. In another embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic label).

[0217] In one embodiment, the kit includes instructional materials disclosing methods for using antibodies that bind to TROP2. The instructional materials can be written in electronic form (e.g., a computer diskette or compact disc), visual (e.g., a video file), or provided via an electronic network (e.g., the Internet, World Wide Web, an intranet, or other network). The kits can include additional components to facilitate the particular application for which the kit is intended. Thus, for example, the kits 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 secondary antibody, etc.). The kits can additionally include buffers and other reagents routinely used in carrying out the particular method. Such kits and suitable contents are well known to those of skill in the art.

[0218] In one embodiment, the diagnostic kit comprises an immunoassay. While the details of the immunoassay may vary depending on the particular format used, methods for detecting TROP2 in a biological sample generally involve contacting the biological sample with an antibody that specifically reacts with a TROP2 polypeptide under immunologically reactive conditions. The antibody specifically binds under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0219] Methods for determining the presence or absence of cell surface markers are well known in the art. For example, antibodies can 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. Antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS employs more advanced detection levels, such as multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels, to separate or sort cells. See U.S. Patent No. 5,061,620. Any single-domain antibody that binds to TROP2 disclosed herein can be used in these assays. Thus, the antibodies can be used in conventional immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation.

[0220] Specific Definitions The terminology used herein is for descriptive purposes only and is not intended to be limiting. As used herein, the singular forms "a," "an," "an," "the ...

[0221] The terms "about" or "approximately" refer to 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" may conventionally mean within one or more standard deviations for a given value. In this application and claims, where a particular value is described, unless otherwise indicated, the word "about" should be interpreted to mean within an acceptable error range for that particular value.

[0222] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to a situation characterized by the supervision (e.g., full-time or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, nursing assistant, or hospice worker).

[0223] The term "antibody" typically refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains bound by covalent disulfide bonds and non-covalent interactions. Human light chains contain a variable domain (VL) and a constant domain (CL), where the constant domain can be readily classified as kappa or lambda based on amino acid sequence and locus. Each heavy chain contains one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD, contains three domains designated CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length (generally about 10 to about 60 amino acids in IgG) segment rich in proline and cysteine. The variable domains of both the light and heavy chains are linked to the constant domains by a "J" region of about 12 or more amino acids, with the heavy chain also having a "D" region of about 10 additional amino acids. Each class of antibody further contains interchain and intrachain disulfide bonds formed by pairs of cysteine ​​residues. Two types of natural disulfide bridges or bonds exist in immunoglobulin molecules: interchain and intrachain disulfide bonds. The location and number of interchain disulfide bonds vary depending on the immunoglobulin class and species. Interchain disulfide bonds are located on the surface of the immunoglobulin, are solvent accessible, and are usually relatively easily reduced. The human IgG1 isotype has 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 attachment. As is well known, the cysteine-rich IgG1 hinge region of the heavy chain is generally considered to consist of three parts: the upper hinge, the core hinge, and the lower hinge. Those skilled in the art will understand that the IgG1 hinge region contains intra-heavy chain cysteines that comprise inter-chain disulfide bonds (two heavy chains / heavy chains, two heavy chains / light chains) that confer 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 by Kabat et al., infra).

[0224] The term "antibody" as used herein 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 (including muteins and variants thereof), 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 Fv (sdFv), Fd fragments (consisting of a VH domain and a CH1 domain), linear antibodies, single domain antibodies, e.g., for example nanobodies, or single variable domain antibodies comprising only one variable domain, such as sdAbs (VH, VL or VHH domains), "rIgG" ("half antibodies"), diabodies, single chain diabodies, tandem diabodies (Tandabs), tandem di-scFvs, tandem tri-scFvs, "minibodies" (which in some cases are exemplified by the following structures: (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 comprising a domain with a binding site for preferential association with or binding to the TROP2 protein. Furthermore, unless context dictates otherwise, this term further includes all antibody classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The heavy chain constant domains that correspond to the different antibody classes are typically represented by the corresponding lowercase Greek letters alpha, delta, epsilon, gamma, and nu, respectively.The light chains of antibodies from 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 their constant domains. In some embodiments, the TROP2-binding protein comprises a heavy chain-only antibody, such as a VH or VHH domain. In some cases, the TROP2-binding protein comprises a heavy chain-only antibody that is an engineered human VH domain. In some instances, the engineered human VH domain is produced by panning a phage display library. In some embodiments, the TROP2-binding protein comprises a VHH. As used herein, "VHH" refers to a single-chain antibody binding domain lacking a light chain. In some cases, the VHH is derived from a type of antibody found in camelids or cartilaginous fish that naturally lacks light chains, or is a synthetic, deimmunized VHH that can be constructed accordingly. Each heavy chain comprises a variable region encoded by a V exon, a D exon, and a J exon. In some cases, the VHH is a naturally occurring VHH, such as 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, vicuna, 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, a huacaya alpaca or a Suri alpaca).

[0225] As used herein, the term "variable region" or "variable domain" refers to the fact that certain portions of the variable domain differ significantly in sequence among antibodies and are responsible for the binding and specificity of each individual antibody to its respective antigen. However, the variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). Each naturally occurring heavy-chain and light-chain variable domain contains four FR regions, which are primarily in a β-sheet configuration and are connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in 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 directly involved in antibody binding to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.The assignment of amino acids to each domain, framework region, and CDR, in some embodiments, unless otherwise indicated, conforms to one of the numbering schemes provided by: Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co. or AbM (Oxford Molecular / MSI Pharmacopeia). In some embodiments of the present disclosure, the TROP2-binding protein is a heavy chain-only antibody, e.g., a VH or VHH domain, comprising three CDRs. Such heavy chain-only antibodies, in some embodiments, bind to TROP2 as a monomer and do not depend on dimerization with the VL (variable light chain) region for optimal binding affinity.

[0226] "Kabat variable domain residue numbering" or "Kabat 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). When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the 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). Kabat residue numbering may be determined for a given antibody by aligning homologous regions of the antibody sequence with the "standard" Kabat numbering sequence. The CDRs of this disclosure are not necessarily intended to correspond to the Kabat numbering convention.

[0227] 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 that corresponds to the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences.

[0228] As used herein, "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. A single antigen can have multiple epitopes. Thus, an antibody can bind to different regions on the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include sugar moieties, phosphoryl groups, or sulfonyl groups on the antigen.

[0229] 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, determined after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, without taking into account any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved by various methods within the skill of the art, for example, using publicly available computer software programs such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared.

[0230] As used herein, "elimination half-life" is used in its ordinary sense as set forth in Goodman and Gillman, The Pharmaceutical Basis of Therapeutics 21-25 (eds. Alfred Goodman Gilman, Louis S. Goodman and Alfred Gilman, 6th ed. 1980). Briefly, the term is intended 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 the drug concentration usually does not approach the concentration required for saturation of the elimination process. The rate of an exponential process is determined by its rate constant k (which represents the fractional change per unit time) or its half-life t 1 / 2 (the time required for 50% of the process to be completed). The units of these two constants are hr-1 and hr, respectively. The first-order rate constant and the half-life of a reaction are simply related (k × t 1 / 2 =0.693) and can be interconverted accordingly. First-order elimination kinetics involves the loss of a constant fraction of the drug per unit time, so that a plot of the logarithm of the drug concentration against time is a straight line at all times after the initial distribution phase (i.e., after drug absorption and distribution are complete). From such a graph, the half-life of the drug elimination can be accurately determined.

[0231] As used herein, the term "binding affinity" refers to the affinity of the proteins described in this disclosure for their binding targets, and is used to refer to the "K D When two or more proteins are shown to have comparable binding affinities for their binding targets, the K D Values ​​are within ±2-fold of each other. When two or more proteins are shown to have comparable binding affinity to a single binding target, the K for each protein's binding to that single binding target is DValues ​​are within ±2-fold of each other. When a protein is shown to bind to two or more targets with comparable binding affinity, the K for that protein's binding to those two or more targets is D The values ​​are within ±2-fold of each other. Generally, the higher the K D In some embodiments, the "K D " is measured by surface plasmon resonance assay or radiolabeled antigen binding assay (RIA) using a BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, "on-rate" or "rate of association" or "association rate" or "k", and "off-rate" or "rate of dissociation" or "dissociation rate" or "koff" are also measured by surface plasmon resonance technology using a BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). In additional embodiments, "K D"," "kon," and "koff" are measured using the OCTET® system (Pall Life Sciences). In an exemplary method for measuring binding affinity using the OCTET® system, a ligand (e.g., biotinylated human or cynomolgus TROP2) is immobilized on the surface of an OCTET® streptavidin capillary sensor chip. The streptavidin chip is then activated with approximately 20-50 μg / ml of human or cynomolgus TROP2 protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocking agent. For binding kinetic measurements, the TROP2-binding protein variant is introduced at a concentration ranging from approximately 10 ng / mL to approximately 100 μg / mL, approximately 50 ng / mL to approximately 5 μg / mL, or approximately 2 ng / mL to approximately 20 μg / mL. In some embodiments, the TROP2-binding single domain protein is used at a concentration ranging from about 2 ng / mL to about 20 μg / mL. Complete dissociation is observed in the negative control (assay buffer) containing no binding protein. The kinetic parameters of the binding reaction are then determined using an appropriate tool, such as ForteBio software.

[0232] In some embodiments, "treatment" or "treat" or "treated" as used herein refers to therapeutic treatment, the purpose of which is to delay (alleviate) an undesirable physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical result. For purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder, or disease; stabilization (i.e., not worsening) of the condition, disorder, or disease state; delay in the onset or progression of the condition, disorder, or disease; improvement in the condition of the condition, disorder, or disease; and remission (partial or complete) (whether detectable or undetectable) or improvement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without undue side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, "treatment" or "treating" or "treated" refers to prophylactic treatment, the purpose of which is to delay the onset of or reduce the severity of an undesirable physiological condition, disorder, or disease, for example, in a person who is predisposed to the disease (e.g., an individual who has genetic markers for a disease such as breast cancer).

[0233] As used herein, "TriTAC," "TROP2-targeting TriTAC," or "TROP2-targeting trispecific protein" refers to a trispecific binding protein that is not conditionally activated and comprises a binding moiety specific for a bulk serum protein, a first target antigen-binding domain, and a second target antigen-binding domain, wherein at least one of the first target antigen-binding domain and the second target antigen-binding domain comprises a TROP2 binding protein described herein, and at least one of the first target antigen-binding domain and the second target antigen-binding domain comprises a domain that binds to CD3, such as human CD3.

[0234] As used herein, "ProTriTAC" or "TROP2-targeting pro-trispecific protein" refers to a conditionally activated trispecific binding protein comprising (i) a cleavable linker (e.g., comprising the amino acid sequences set forth in SEQ ID NOS: 497-543) and (ii) a binding moiety specific for a bulk serum protein and also comprising a masking moiety (e.g., comprising the amino acid sequence set forth in SEQ ID NOS: 549) that prevents binding of the first target antigen-binding domain or the second target antigen-binding domain to its target, wherein at least one of the first target antigen-binding domain and the second target antigen-binding domain comprises a TROP2-binding protein described herein. The ProTriTAC protein of the present disclosure can, in some cases, be activated from the masked state to the active state by cleavage of the cleavable linker in a protease-rich environment, such as the tumor microenvironment, to form an active drug. The active drugs provided herein, in some cases, comprise a TROP2-binding domain of the present disclosure and a CD3-binding domain of the present disclosure. An example of an active agent is provided in SEQ ID NO:229-264, or in an amino acid sequence that is at least about 75% to 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:229-264, for example, an amino acid sequence that is about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:229-264.

[0235] As used herein, "non-cleavable prodrug" 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 SEQ ID NO: 696). An example of an active drug is provided in SEQ ID NOs: 229-264, or in an amino acid sequence that is at least about 75% to 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 229-264, e.g., an amino acid sequence that is about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0236] In non-beta sandwich scaffolds (e.g., DARPIN®, AFFIMER®, affibodies), "non-CDR loop" refers to a region that is (1) suitable for sequence randomization to allow for engineered specificity for a second antigen, and (2) distal to the primary specificity-determining region typically used on the scaffold to allow 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 shown below: [Table 2] As used herein, "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that confers antigen specificity to a cell (e.g., a T cell). 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. As used herein, "ProCAR" refers to a conditionally activatable CAR that contains the TROP2-binding domain of the present disclosure.

[0237] Example The present application may be better understood by reference to the following non-limiting examples, which are provided as illustrative embodiments of the present application. The following examples are presented to more fully illustrate the embodiments and should not be construed in any way as limiting the broad scope of the present application.

[0238] Example 1: Screening of phage display libraries to identify TROP2-binding domains Llamas were immunized with purified TROP2 dimer protein expressed in 293 cells. A phage display library for heavy chain variable antibody domain expression was constructed from circulating B cells isolated from immunized llamas (van der Linden et al., 2000, J Immunol Methods 240:185-195). Llama anti-TROP2 protein was expressed in Escherichia coli (E. coli), periplasmic extracts were prepared, and phage clones were screened for binding to the TROP2 dimer by colorimetric ELISA. Fifty-two unique heavy chain-only sequences (SEQ ID NOS: 1-52) were identified that generated signals in ELISA screening compared with controls containing human or cynomolgus monkey TROP2 protein (Table 3). The CDR1, CDR2, and CDR3 sequences for these heavy chain variable domains are SEQ ID NOS: 58-109, 115-166, and 172-223, respectively. [Table 3] TIFF2026502002000006.tif92156 The numbers in Table 1 represent absorbance measurements for the colorimetric ELISA.

[0239] Example 2: Incorporation of a heavy chain-only single domain antibody binding to TROP2 into a fusion protein and T cell dependent cytotoxicity assay The anti-TROP2 antibody sequence was cloned into a DNA construct for expression of a recombinant fusion protein (SEQ ID NOs: 229-258). The coding sequence of the fusion protein contained a signal peptide for secretory cell expression, a stub mimicking a cleaved form of a conditionally active T cell engager (SEQ ID NO: 495), a humanized anti-CD3 antibody scFv fragment (SEQ ID NO: 494), one of the anti-TROP2 antibody variable domains (SEQ ID NOs: 1-52), and a six-histidine repeat sequence (SEQ ID NO: 496). A linker sequence was inserted at the junction between the antibody domains (SEQ ID NO: 497). These anti-CD3 / anti-TROP2 fusion protein constructs were transfected into Expi293 cells (Life Technologies). The amount of fusion protein in the conditioned medium from transfected Expi293 cells was quantified using an Octet instrument in the presence of either Protein A or an anti-6xHis chip, using a fusion protein of a molecular weight similar to the anti-CD3 / anti-TROP2 protein as a standard.

[0240] Conditioned medium was tested in a T cell-dependent cytotoxicity assay (TDCC) (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, target cells (either luciferase-labeled H292 cells or luciferase-labeled HT1376 cells, both of which express TROP2) were combined with purified human T cells and a titration of anti-CD3 / anti-TROP2 fusion protein. If the fusion protein induces T cells to kill target cells, the signal in the luciferase assay performed 48 hours after the start of the experiment should decrease. Figures 1-12 show graphs of TDCC viability results. EC50 values ​​from the TDCC assay are shown in Table 4. The most potent molecules exhibited EC50 values ​​of 37.4 pM for H292 cells and 152 pM for H1376 cells. The negative control for the TDCC assay was anti-GFP / anti-albumin / anti-CD3 protein, which did not induce T cells to kill H292 cells except for slight activity at the highest concentration tested (e.g., Figures 5 and 11). [Table 4] Example 3: Humanization of TROP2-binding antibodies and T-cell-dependent cytotoxicity assay Four of the llama anti-TROP2 antibody sequences (SEQ ID NOS: 16, 41, 43, and 52) were humanized by grafting their CDR sequences onto human germline sequences (SEQ ID NOS: 53-57), while retaining some llama framework sequences to ensure the antibodies did not lose activity. These humanized sequences, along with their parental constructs, were cloned into expression constructs for expression as anti-CD3 / anti-TROP2 fusion proteins (SEQ ID NOS: 259-263) in Expi293 cells, as described above. The amount of anti-CD3 / anti-TROP2 fusion protein in the conditioned medium was quantified as described above. Culture supernatants containing the anti-CD3 / anti-TROP2 fusion proteins were used in TDCC assays as described above, using only the TROP2-expressing luciferase-labeled cell lines H292, HT1376, or HCC70. The results of the TDCC assays are plotted in Figures 13-18, and the EC50 values ​​for inducible T cell killing are shown in Table 3. Potent inducible T cell killing was observed with both the llama TROP2 antibody and the humanized TROP2 antibody.

[0241] The humanized conjugate 2TRH79B was used to produce a fusion protein containing an anti-ALB domain containing a non-CDR loop mask that blocks CD3 binding, linked to the anti-CD3 and anti-TROP2 2TRH79B conjugate by a cleavable linker (SEQ ID NO: 498). The 2TRH79B conjugate and its parent construct were purified and quantified. The anti-CD3 / anti-TROP2 fusion protein was used in a TDCC assay using three different luciferase-labeled TROP2-expressing cell lines: HCC70, HPAF-II, or CAL27. The results of the TDCC assay are plotted in Figures 19-21, and the EC50 values ​​for induced T cell killing are shown in Table 5. [Table 5] The humanized conjugate 2TRH79B is a sequence variant of the 2TRH79 conjugate. A DNA construct was created containing an anti-ALB domain sequence containing a non-CDR loop mask that blocks CD3 binding, linked by a cleavable linker (L040) to the anti-CD3 and anti-TROP2 2TRH79B conjugate sequence (SEQ ID NO: 498). This fusion construct was transfected into Expi293 cells, and conditioned medium was collected several days later. The anti-ALB:anti-CD3:anti-TROP2 2TRH79B fusion protein was purified by Protein A chromatography using standard binding and elution conditions. DNA constructs containing an anti-CD3 domain linked by a GGGGSGGGS linker to one of three anti-TROP2 sequences (i.e., the llama anti-TROP2 conjugate sequence 2TRL79, and the humanized anti-TROP2 conjugate sequence 2TRH79 and 2TRH79B) were also transfected into Expi293 cells for protein expression. Five days after transfection, conditioned medium was collected and the anti-CD3:anti-TROP2 sequences were purified by immobilized metal affinity chromatography (IMAC) using standard methods. The anti-ALB:anti-CD3:anti-TROP2 2TRH79B fusion protein and anti-CD3:anti-TROP2 sequences were tested in TDCC assays using three different luciferase-labeled TROP2-expressing cell lines: HCC70, HPAF-II, or CAL27, in the presence of 15 mg / ml human serum albumin. The results of the TDCC assays are plotted in Figures 19-21, and the EC50 values ​​for inducible T cell killing are shown in Table 4. Among the anti-CD3:anti-TROP2 proteins containing the humanized anti-TROP2 domain, the 2TRH79B protein was more potent, exhibiting only 2-3-fold lower efficacy than proteins containing the llama anti-TROP2 domain. Comparing the EC50 values ​​for the anti-CD3:anti-TROP2 2TRH79B protein and the anti-ALB:anti-CD3:anti-TROP2 protein (Table 6), it can be seen that the anti-ALB:anti-CD3:anti-TROP protein (2TRH79B L040 ProTriTAC) produced killing with 36- to 173-fold reduced efficacy compared to the anti-CD3:anti-TROP2 protein (2TRH79B).This indicates that the presence of the anti-ALB domain containing the non-CDR loop mask that binds to the anti-CD3 domain reduces TDCC activity. This difference may be even greater if the cell line used in this assay has protease activity that partially activates the anti-ALB:anti-CD3:anti-TROP protein. [Table 6] Example 4: Demonstration of improved tolerability in mice provided by exemplary anti-CD3 / anti-TROP2 fusion proteins All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Harpoon Therapeutics (Protocol No. HAR-001-2019). Animals were purchased from Jackson Laboratory and housed in a pathogen-free animal facility located at Harpoon Therapeutics in accordance with IACUC guidelines. All studies were performed in 6-, 7-, or 11-week-old NSG™ (NOD-SCID IL2R gamma null) female mice (n = 5–10 per group). Mice were age-matched within each experiment.

[0242] NSG™ mice were subcutaneously implanted with a mixture of Trop2-expressing human tumor cells, HCC70 (10E6), CAL27 (5E6), or HPAFII (10E6), in a 1:2 E:T ratio with activated and expanded human T cells (5E6, 2.5E6, or 5E6, respectively) into the right flank (day 0). In HCC70 xenograft experiments (experiments) 1 and 2, treatment was initiated 7 days after tumor establishment (mean 184 mm3 (HCC70 experiment 1) or 155 mm3 (HCC70 experiment 2)). In CAL27 xenografts, treatment was initiated on day 4, after tumor establishment (mean 120 mm3). In HPAFII xenografts, treatment was initiated on day 4, after tumor establishment (mean 128 mm3). Mice were administered repeated intraperitoneal doses (qd × 14) of negative control, non-Trop2-targeting anti-GFP TriTAC, anti-Trop2 2TRL79 (llama conjugate) ProTriTAC linker 040, anti-Trop2 2TRH79 (humanized conjugate) ProTriTAC linker 040, or anti-Trop2 2TRH79B (humanized conjugate) ProTriTAC linker 040. Tumor growth was monitored at least twice weekly as indicated. The mean tumor volumes shown were calculated from measurements taken on the final day of each xenograft model study. Statistics represent RM one-way ANOVA with Dunnett's post-hoc test, where all groups were compared to the negative control, anti-GFP TriTAC. Results from each admix-treated xenograft rodent study are plotted in Figures 22-26, and statistics for each xenograft model study are shown in Table 7. [Table 7] Example 5: Equivalent potent antitumor activity achieved by equivalent molar concentrations of prodrug and active drug All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Harpoon Therapeutics (Protocol No. HAR-001-2019). Animals were purchased from Jackson Laboratory and then housed in a pathogen-free animal facility located at Harpoon Therapeutics in accordance with IACUC guidelines. All studies were performed in 6-, 7-, or 11-week-old NSG™ (NOD-SCID IL2R gamma null) female mice (n = 5–10 per group). Mice were age-matched for each experiment. A mixture of Trop2-expressing HCC70 cells (10E6) and activated, expanded human T cells (5E6) at an E:T ratio of 2.5 million:5 million was subcutaneously implanted into the right flank of NSG™ mice (day 0). In HCC70 xenograft experiments (Experiments 1 and 2), treatment was initiated seven days after tumor establishment (day 7) [average 184 mm3 (HCC70 Experiment 1) or 155 mm3 (HCC70 Experiment 2)]. Mice were administered repeated intraperitoneal doses (qd × 14) of the control dose (300 μg / kg), prodrug (30 μg / kg, 300 μg / kg, or 3000 μg / kg), or active drug (22.5 μg / kg or 225 μg / kg), respectively. Tumor growth was monitored at least twice weekly, as indicated. The mean tumor volume shown was calculated from measurements taken on the final day of each xenograft model study. Statistics represent RM one-way ANOVA with Dunnett's post-hoc test, where all groups were compared to the negative control anti-GFP TriTAC. Results from the admix-treated xenograft rodent study are plotted in Figure 27. The results showed that equivalent molar concentrations of the prodrug and active drug exhibited equally potent antitumor activity (Figure 27).

[0243] Example 6: Measurement of binding activity and species cross-reactivity of TroP2 ProTriTAC linker 40 molecules as assessed by biolayer interferometry Biolayer interferometry (BLI) is a well-established analytical method used to measure the kinetics of specific biomolecular interactions. One molecule, the target ligand, is tagged or modified to enable specific capture and presentation to a second molecule (analyte) in solution. In some applications, the ligand can be expressed as a fusion protein with histidine repeats, an antibody Fc constant domain, or conjugated to a small molecule such as biotin. During the loading step, the ligand is captured on a glass fiber biosensor chip that has been chemically derivatized with a substance capable of binding to the tagged ligand with high affinity (i.e., in the above example, metal-chelating nitrilotriacetic acid, an anti-Fc monoclonal antibody, or streptavidin). Using an instrument such as the Octet RED96 (Sartorius), white light is shone onto the biosensor chip and reflected from two surfaces: the biocompatible surface containing the immobilized ligand and the reference layer. While the reflection distance to the reference layer is constant, the reflection distance from the surface-immobilized ligand changes depending on the binding or dissociation of the analyte, resulting in interference of the light waves and a change in the measured amplitude over time. Binding sensorgrams generated from serial dilutions of known analyte concentrations are then globally fit to a one-to-one binding model. This global fit yields the binding dissociation constant (K D The association rate constant (k) and dissociation rate constant (k) are determined, which are used to calculate the affinity of the antibody. Because the streptavidin-biotin interaction is known to be one of the highest biomolecular affinities identified, and to minimize the confounding contribution of other ligands / surface chemistries, all ligands in this study were biotinylated.

[0244] To better characterize and aid in the identification of relevant toxic species, we evaluated the binding kinetics and affinity of TroP2 ProTriTAC linker 40 molecules (2TRH79B) for tumor-associated calcium signaling factor 2 (Trop2). Biotinylated derivatives of target ligands from human and non-human primate (NHP) species (cynomolgus monkeys) were prepared. Accordingly, binding of 2TRH79B and 2TRH79 to biotinylated target ligands was assessed by biolayer interferometry (BLI) in the absence or presence of calcium. Figures 28A-28B show that 2TRH79B binds with comparable affinity to human (Figure 28A) and cynomolgus monkey (cyno) Trop2 (Figure 28B) in the absence or presence of calcium (47-51 nM or 50 nM, respectively). Table 8 summarizes findings from the binding kinetics studies. [Table 8] The binding kinetics and affinity of a related construct, a second TroP2 ProTriTAC linker 40 molecule (2TRH79), were also assessed by BLI to human and cynomolgus Trop2 in the absence or presence of calcium, and the results were similar: 2TRH79 bound to human and cynomolgus Trop2 with comparable affinity (168–186 nM or 122–179 nM, respectively) in the absence or presence of calcium (data not shown).

[0245] Example 7: One-Month Dose Escalation / Maximum Tolerated Dose Study In this dose escalation / maximum tolerated dose study, TroP2 ProTriTAC linker 40 molecules (2TRH79B) were used for intravenous (slow bolus) injection in cynomolgus monkeys.

[0246] The primary objective of this Good Laboratory Practice (GLP)-compliant, ascending-dose study was to evaluate the potential toxicity and systemic exposure of TroP2 ProTriTAC when administered to cynomolgus monkeys by intravenous (iv) slow-bolus injection at 20, 60, 180, and 540 μg / kg on days 1, 8, 15, and 22, respectively. Figure 29 shows the experimental design. The toxicokinetic (TK) properties of TroP2 ProTriTAC were also measured. Study endpoints included mortality, clinical observations (cageside daily and detailed weekly after dosing), body weight, qualitative food intake, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), bioanalytical and TK parameters, cytokine analysis, organ weights, and gross and microscopic examinations.

[0247] TroP2 ProTriTAC was well tolerated up to 540 μg / kg, the highest dose tested. PK was assessed by capture with a biotinylated anti-idiotypic Ab recognizing the αCD3 domain and detection with a sulfo-tagged anti-idiotypic Ab raised against the αALB domain. Figure 30 demonstrates that TroP2 ProTriTAC exhibits favorable pharmacokinetics, half-life, and systemic accumulation. Hematological changes included mild to moderate decreases in neutrophil counts at all dose levels, as well as transient decreases in lymphocyte and basophil counts during the third week of the study. There were no notable findings in plasma cytokines (IFNγ, IL-1β, IL-2, IL-6, IL-10, IL-8, TNF-α) throughout the study; no notable findings from coagulation, clinical chemistry, or urinalysis parameters; and no notable histopathological findings based on organ weights and gross examination. [Table 9] TIFF2026502002000013.tif242170TIFF2026502002000014.tif243170TIFF2026502002000015.tif242170TIFF202650200200 0016.tif245170TIFF2026502002000017.tif249170TIFF2026502002000018.tif249170TIFF2026502002000019.tif249170TIF F2026502002000020.tif248170TIFF2026502002000021.tif250170TIFF2026502002000022.tif250170TIFF2026502002000023 .tif246170TIFF2026502002000024.tif246170TIFF2026502002000025.tif245169TIFF2026502002000026.tif243170TIFF202 6502002000027.tif247170TIFF2026502002000028.tif249169TIFF2026502002000029.tif249170TIFF2026502002000030.ti f240170TIFF2026502002000031.tif251170TIFF2026502002000032.tif246170TIFF2026502002000033.tif248170TIFF202650 2002000034.tif238170TIFF2026502002000035.tif244170TIFF2026502002000036.tif244170TIFF2026502002000037.tif244170TIFF2026502002000038.tif186170While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious 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 present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in the practice of the present disclosure.It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A TROP2-binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228.

2. The TROP2-binding domain of claim 1, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58 to 114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115 to 171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172 to 228.

3. The TROP2-binding domain according to claim 1 or 2, comprising an amino acid sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57.

4. The TROP2-binding domain according to any one of claims 1 to 3, wherein the TROP2-binding domain is part of a multispecific protein.

5. The TROP2-binding domain of claim 4 , wherein the multispecific protein further comprises a CD3-binding domain.

6. The TROP2 binding domain of claim 5 , wherein the multispecific protein comprises an active drug form.

7. The TROP2-binding domain according to any one of claims 4 to 6, wherein the multispecific protein further comprises a bulk serum protein-binding domain.

8. The TROP2 binding domain of claim 7 , wherein the bulk serum protein comprises a serum albumin protein.

9. The TROP2-binding domain according to claim 8 , wherein the serum albumin protein comprises human serum albumin protein.

10. The TROP2-binding domain of any one of claims 7 to 9, wherein the bulk serum protein-binding domain comprises a sequence that is at least 75% identical to SEQ ID NO: 493 or 549.

11. The TROP2-binding domain according to any one of claims 5 to 9, wherein the CD3-binding domain comprises a sequence that is at least 75% identical to SEQ ID NO:

494.

12. The TROP2-binding domain according to any one of claims 4 to 11, wherein the multispecific protein comprises a sequence that is at least about 75% identical to the sequence set forth in SEQ ID NOs: 229 to 264.

13. The TROP2-binding domain according to any one of claims 7 to 9, wherein the bulk serum protein-binding domain is a binding moiety comprising a linker and a masking moiety, and the masking moiety masks the binding of the TROP2-binding domain or the CD3-binding domain to its respective target.

14. The TROP2-binding domain of any one of claims 7 to 9 and 13, wherein the multispecific protein comprises a non-cleavable prodrug form.

15. The TROP2-binding domain according to claim 13 or 14, wherein the masking portion comprises a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560, or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560.

16. The TROP2-binding domain according to any one of claims 13 to 15, wherein the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497 to 545, or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497 to 545.

17. The TROP2-binding domain of any one of claims 13 to 16, wherein the bulk serum protein-binding domain comprises a sequence that is at least 75% identical to SEQ ID NO: 493 or 549.

18. The TROP2-binding domain according to any one of claims 5 to 9 and 13 to 17, wherein the CD3-binding domain comprises a sequence that is at least 75% identical to the sequence set forth in SEQ ID NO:

494.

19. The TROP2-binding domain of any one of claims 7 to 9 and 13 to 18, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

20. The TROP2-binding domain of any one of claims 7 to 9 and 13 to 19, wherein the multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

21. The TROP2-binding domain of any one of claims 7 to 9 and 13 to 20, wherein the multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

22. 7. The TROP2-binding domain of claim 6, wherein the active agent comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

23. The TROP2-binding domain according to any one of claims 1 to 3, wherein the TROP2-binding domain is a part of a chimeric antigen receptor (CAR) or a conditionally activatable chimeric antigen receptor (ProCAR), and the CAR further comprises at least one of a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

24. 24. The TROP2-binding domain of claim 23, wherein the TROP2-binding domain is a part of ProCAR, and the ProCAR further comprises (a) a binding moiety comprising a non-CDR loop and a cleavable linker, (b) a transmembrane domain, and (c) an intracellular signaling domain, and the binding moiety masks binding of the TROP2-binding domain to its target.

25. 25. The TROP2 binding domain of claim 24, wherein the binding portion further comprises one or more complementarity determining regions (CDRs).

26. 26. The TROP2 binding domain of claim 25, wherein the non-CDR loops provide a binding moiety specific for a bulk serum protein.

27. 27. The TROP2 binding domain of claim 26, wherein the bulk serum protein comprises at least one of serum albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, fibrinogen, or pentameric IgM.

28. 28. The TROP2 binding domain of claim 27, wherein the bulk serum protein comprises serum albumin.

29. The TROP2-binding domain of claim 28, wherein the serum albumin is human serum albumin.

30. The TROP2-binding domain according to any one of claims 23 to 29, wherein 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), whose amino acid sequence has at least one and not more than 20 modifications thereto.

31. 31. The TROP2 binding domain of claim 30, wherein said at least one and no more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to ligand binding to the encoded T cell receptor fusion protein.

32. The TROP2-binding domain according to any one of claims 23 to 31, wherein the CAR or ProCAR further comprises a transmembrane domain, and the transmembrane domain is 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 and not more than 20 modifications thereto.

33. The TROP2-binding domain according to any one of claims 23 to 32, wherein the CAR or ProCAR further comprises an intracellular signaling domain, and the intracellular signaling domain is derived from CD3 epsilon, CD3 gamma, CD3 delta, CD3 alpha, CD3 beta, or a combination thereof.

34. The TROP2 binding domain of claim 33, wherein the intracellular signaling domain is derived from CD3 epsilon.

35. A method for treating or ameliorating a proliferative disease or a neoplastic disease, comprising administering the TROP2-binding domain according to any one of claims 1 to 34 or a pharmaceutical composition comprising the same to a subject in need of such treatment or amelioration.

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

37. A conditionally active TROP2 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 a TROP2 binding domain, the TROP2 binding domain comprising complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114; the conditionally active TROP2-binding protein, wherein R2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228, wherein the non-CDR loops are capable of binding to the TROP2-binding domain or the second target antigen-binding domain, and the binding moiety may or does mask binding of the TROP2-binding domain or the second target antigen-binding domain to its target.

38. 38. The conditionally active TROP2 binding protein of claim 37, wherein the binding moiety comprises a masking moiety, and the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560, or a sequence containing one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560.

39. 39. The conditionally active TROP2 binding protein of claim 37 or 38, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543, or a sequence containing one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 497-543.

40. 40. The conditionally active TROP2 binding protein of any one of claims 37 to 39, wherein the binding portion comprises a sequence that is at least 75% identical to SEQ ID NO:

493.

41. 41. The conditionally active TROP2 binding protein of any one of claims 37 to 40, wherein the second target antigen-binding domain (T2) comprises a CD3-binding domain.

42. 42. The conditionally active TROP2 binding protein of claim 41, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to SEQ ID NO:

494.

43. A method for treating or ameliorating a proliferative disease or a neoplastic disease in a subject in need thereof, comprising administering to the subject a conditionally active TROP2-binding protein according to any one of claims 37 to 42 or a pharmaceutical composition comprising the same.

44. 44. The method of claim 43, wherein the subject is a human.

45. A method for conferring anti-tumor immunity to a subject in need thereof, comprising administering to the subject the conditionally active TROP2-binding protein according to any one of claims 37 to 42 or a pharmaceutical composition comprising the same.

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

47. The TROP2-binding domain according to any one of claims 1 to 34, wherein the binding domain is a humanized antibody or an antigen-binding fragment thereof.

48. The TROP2-binding domain according to any one of claims 1 to 34 and 47, wherein the binding domain is a single-domain antibody, a VHH domain, an scFv, a VH domain, a VL domain, Fab, F(ab')2, Fab', a non-Ig domain, a ligand, a knottin, or a small molecule substance.

49. 49. The TROP2 binding domain of claim 48, wherein the binding domain comprises a single domain antibody.

50. The binding domain binds to TROP2 with a binding affinity (K Ｄ The TROP2-binding domain according to any one of claims 1 to 3 and 47 to 49, which binds to the TROP2-binding domain via the nucleotide sequence (SEQ ID NO: 1).

51. 51. The TROP2 binding domain of any one of claims 1 to 3 and 47 to 50, wherein the binding domain binds to human TROP2, mouse TROP2, cynomolgus monkey TROP2, or a combination thereof.

52. A multispecific protein comprising the TROP2-binding domain according to any one of claims 1 to 3 and 47 to 51.

53. 53. The multispecific protein of claim 52, wherein the TROP2 binding domain further comprises a CD3 binding domain (anti-CD3 domain).

54. 54. The multispecific protein of claim 53, wherein the anti-TROP2 domain and the anti-CD3 domain are in an anti-TROP2:anti-CD3 orientation.

55. 54. The multispecific protein of claim 53, wherein the anti-TROP2 domain and the anti-CD3 domain are in an anti-CD3:anti-TROP2 orientation.

56. 56. The multispecific protein according to any one of claims 52 to 55, comprising a TROP2-binding domain (anti-TROP2 domain), a CD3-binding domain (anti-CD3 domain), and an albumin-binding domain (anti-ALB domain) according to any one of claims 1 to 3 and 47 to 51.

57. 57. The multispecific protein of any one of claims 52 to 56, wherein the anti-CD3 domain comprises the amino acid set forth in SEQ ID NO:

494.

58. 58. The multispecific protein of any one of claims 56 to 57, wherein the anti-ALB domain comprises the amino acid sequence set forth in SEQ ID NO:

493.

59. 59. The multispecific protein of any one of claims 56 to 58, wherein the anti-TROP2 domain, the anti-CD3 domain and the anti-ALB domain are present in the orientation anti-CD3:anti-ALB:anti-TROP2.

60. 59. The multispecific protein of any one of claims 56 to 58, wherein the anti-TROP2 domain, the anti-CD3 domain and the anti-ALB domain are present in the orientation anti-TROP2:anti-ALB:anti-CD3.

61. 59. The multispecific protein of any one of claims 56 to 58, wherein the anti-TROP2 domain, the anti-CD3 domain and the anti-ALB domain are present in the orientation anti-ALB:anti-TROP2:anti-CD3.

62. 59. The multispecific protein of any one of claims 56 to 58, wherein the anti-TROP2 domain, the anti-CD3 domain and the anti-ALB domain are present in the orientation anti-CD3:anti-TROP2:anti-ALB.

63. 59. The multispecific protein of any one of claims 56 to 58, wherein the anti-TROP2 domain, the anti-CD3 domain and the anti-ALB domain are present in the orientation anti-ALB:anti-CD3:anti-TROP2.

64. 59. The multispecific protein of any one of claims 56 to 58, wherein the anti-TROP2 domain, the anti-CD3 domain and the anti-ALB domain are present in the orientation anti-TROP2:anti-CD3:anti-ALB.

65. A multivalent protein comprising a sequence set forth in any one of SEQ ID NOs: 229-264.

66. An active drug comprising a sequence set forth in any one of SEQ ID NOs: 229-264.

67. An active drug comprising a sequence set forth in any one of SEQ ID NOs: 1-57.

68. 66. A pharmaceutical composition comprising: (i) (a) a TROP2-binding domain according to any one of claims 1 to 34 and 47 to 51; (i) (b) a conditionally active TROP2-binding protein according to any one of claims 37 to 42; (i) (c) a multispecific protein according to any one of claims 52 to 64; (i) (d) a multivalent protein according to claim 65; or (i) (e) an active drug according to claim 66 or 67; and (ii) a pharmaceutically acceptable carrier.

69. A method for producing a TROP2-binding domain, comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the TROP2-binding domain according to any one of claims 1 to 3 and 47 to 51 under conditions that allow expression of the TROP2-binding domain, and recovering and purifying the produced protein from the culture.

70. 65. A method for producing a multispecific protein, comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding domains of the multispecific TROP2 binding protein of any one of claims 52 to 64 under conditions allowing expression of the multispecific protein, and recovering and purifying the produced protein from the culture.

71. A method for treating or ameliorating a proliferative disease or a neoplastic disease in a subject in need thereof, comprising administering to the subject a TROP2-binding domain described in any one of claims 1 to 34 and 47 to 51 or a pharmaceutical composition described in claim 68.

72. A method for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a multispecific protein according to any one of claims 52 to 64, a multivalent protein according to claim 65, an active drug according to claim 66 or 67, or a pharmaceutical composition according to claim 68.

73. 73. The method of claim 71 or 72, wherein the subject is a human.

74. 74. The method of claim 73, wherein the method further comprises administering an agent, wherein the agent is a biological therapeutic, a cytokine, 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 therapy, a TLR (toll-like receptor) agonist, or a tumor vaccine.

75. The method of any one of claims 71 to 74, wherein the TROP2-binding domain selectively binds to tumor cells that express TROP2.

76. 76. The method of any one of claims 71 to 75, wherein the neoplastic disease comprises a solid tumor disease.

77. 77. The method of claim 76, wherein the solid tumor disease is metastatic.

78. 78. The method of any one of claims 71 to 77, wherein the neoplastic disease comprises at least one of oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof.

79. 47. The method of any one of claims 43 to 46, wherein the method further comprises administering a substance, wherein the substance is a biological therapeutic, a cytokine, 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 therapy, a TLR (toll-like receptor) agonist or a tumor vaccine.

80. 80. The method of claim 79, wherein the TROP2 binding domain selectively binds to tumor cells that express TROP2.

81. 81. The method of claim 79 or 80, wherein the neoplastic disease comprises a solid tumor disease.

82. 82. The method of claim 81, wherein the solid tumor disease is metastatic.

83. 83. The method of any one of claims 79 to 82, wherein the neoplastic disease is at least one of colorectal cancer, oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof.

84. A method for producing a conditionally active TROP2-binding protein, comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of the conditionally active TROP2-binding protein according to any one of claims 37 to 42 under conditions that allow expression of the conditionally active TROP2-binding protein, and recovering and purifying the produced protein from the culture.

85. 66. A method for producing a multivalent protein, comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of the multivalent protein of claim 65 under conditions that allow expression of the multivalent protein, and recovering and purifying the produced protein from the culture.

86. A method for producing an active drug, comprising culturing a host transformed or transfected with a vector containing one or more nucleic acid sequences encoding a domain of an active drug described in claim 66 or 67 under conditions that allow expression of the active drug, and recovering and purifying the produced drug from the culture.

87. A cell comprising the CAR according to any one of claims 23 and 30 to 33.

88. A cell comprising the ProCAR according to any one of claims 23 to 33.

89. 89. The cell of claim 87 or 88, wherein the cell is a T cell or an NK cell.

90. A method for producing CAR or ProCAR, comprising transfecting the cell according to any one of claims 87 to 89 with a vector or RNA comprising a nucleotide sequence encoding CAR or ProCAR.

91. 43. The conditionally active TROP2 binding protein of any one of claims 37 to 42, wherein the protein comprises a sequence that is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

92. 43. The conditionally active TROP2 binding protein of any one of claims 37 to 42, wherein the protein comprises a sequence that is at least about 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

93. 43. The conditionally active TROP2 binding protein of any one of claims 37 to 42, wherein the protein comprises a sequence that is at least about 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

94. 94. The conditionally active TROP2 binding protein of any one of claims 37 to 42 and 91 to 93, wherein the conditionally active TROP2 binding protein has a greater therapeutic index compared to a TROP2 binding protein that does not include the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active TROP2 binding protein.

95. 95. The conditionally active TROP2 binding protein of claim 94, wherein the conditionally active TROP2 binding protein has a therapeutic index that is at least about 5 to about 100 times greater than the therapeutic index of a TROP2 binding protein that does not include the binding moiety (M) or the cleavable linker (L) but is otherwise identical to the conditionally active TROP2 binding protein.

96. 96. A pharmaceutical composition comprising: (i) (a) the conditionally active TROP2 binding protein of any one of claims 91 to 95; and (ii) a pharmaceutically acceptable carrier.

97. 97. A method for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 96.

98. A method for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a conditionally active TROP2 binding protein described in claim 94 or 95 or a pharmaceutical composition described in claim 96.

99. 99. The method of claim 97 or 98, wherein the subject is a human.

100. 100. The method of any one of claims 97-99, wherein the neoplastic disease comprises at least one of oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof.

101. 1. A method for increasing the therapeutic index of a TROP2 binding domain, the method comprising conjugating the TROP2 binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein: the non-CDR loops comprise binding sites specific for the TROP2 binding domain, - the TROP2 binding domain is masked from binding to its target by the binding moiety; - the method wherein the TROP2 binding domain binds to its target upon cleavage of the cleavable linker.

102. 102. The method of claim 101, wherein the TROP2-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: 58-114 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228.

103. 103. The method of claim 101 or 102, wherein the TROP2 binding domain conjugated to the binding moiety is part of a conditionally active multispecific protein, wherein the conditionally active multispecific protein further comprises a CD3 binding domain.

104. 104. The method of any one of claims 101 to 103, wherein the binding moiety comprises a sequence that is at least about 75% identical to SEQ ID NO:

493.

105. The method of claim 103 or 104, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO:

494.

106. 106. The method of any one of claims 101 to 105, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497 to 543 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497 to 543.

107. The method of any one of claims 101 to 106, wherein the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57.

108. 108. The method of any one of claims 101 to 107, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

109. 108. The method of any one of claims 101 to 107, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

110. 108. The method of any one of claims 101 to 107, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

111. 111. The method of any one of claims 101 to 110, wherein the TROP2 binding domain conjugated to the binding moiety is part of a conditionally active chimeric antigen receptor, wherein the conditionally active chimeric antigen receptor further comprises at least one of a transmembrane domain, an intracellular signaling domain, and a costimulatory domain.

112. 112. The method of claim 111, wherein the TROP2-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: 58-114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228.

113. 113. The method of claim 111 or 112, wherein the binding moiety comprises a sequence that is at least about 75% identical to SEQ ID NO:

493.

114. The method of any one of claims 111 to 113, wherein the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57.

115. The method of any one of claims 111 to 113, wherein the TROP2 binding domain comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57.

116. A method of increasing the therapeutic index of a TROP2 binding protein comprising a first target antigen-binding domain and a second target antigen-binding domain, wherein at least one of the first and second target antigen-binding domains comprises a TROP2 binding domain, the method comprising conjugating the first or second target antigen-binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein: the non-CDR loops comprise binding sites specific for the first or second target antigen-binding domain; - at least one of the first or second target antigen-binding domains is masked from binding to its target by a binding moiety; wherein the masked first or second target antigen-binding domain binds to its target upon cleavage of the cleavable linker.

117. 117. The method of claim 116, wherein the TROP2-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: 58-114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228.

118. 118. The method of claim 116 or 117, wherein the non-CDR loop comprises a binding site specific for the TROP2 binding domain.

119. 118. The method of claim 116 or 117, wherein at least one of the first or second target antigen binding domains comprises a CD3 binding domain.

120. 120. The method of claim 119, wherein the non-CDR loop comprises a binding site specific for the CD3 binding domain.

121. The method of claim 120, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO:

494.

122. 122. The method of any one of claims 116 to 121, wherein the binding moiety comprises a sequence that is at least about 75% identical to SEQ ID NO:

493.

123. The method of any one of claims 116 to 122, wherein the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57.

124. 124. The method of any one of claims 116 to 123, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497 to 543, or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497 to 543.

125. 125. The method of any one of claims 116 to 124, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

126. 125. The method of any one of claims 116 to 124, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

127. 125. The method of any one of claims 116 to 124, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

128. A method for increasing the therapeutic index of a TROP2 binding protein comprising a TROP2 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.

129. 129. The method of claim 128, wherein the TROP2-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: 58-114 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114, CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171, and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence containing one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228.

130. The method of claim 128 or 129, wherein the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO:

494.

131. 131. The method of any one of claims 128-130, wherein the binding moiety comprises a sequence that is at least about 75% identical to SEQ ID NO:

493.

132. The method of any one of claims 128 to 131, wherein the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 57.

133. 133. The method of any one of claims 128-132, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543, or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497-543.

134. 134. The method of any one of claims 128-133, wherein the TROP2-binding domain is part of a conditionally active multispecific protein, and wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

135. 134. The method of any one of claims 128 to 133, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

136. 134. The method of any one of claims 128-133, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

137. 1. A conditionally active TROP2-targeting multispecific protein comprising a TROP2-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 TROP2-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: 58-114 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 58-114; CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 115-171; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 172-228.

138. 138. The conditionally active TROP2-targeting multispecific protein of claim 137, wherein the albumin binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO:

493.

139. 139. The conditionally active TROP2-targeting multispecific protein of claim 137 or 138, wherein the TROP2-binding domain comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57.

140. 140. The conditionally active TROP2-targeting multispecific protein of any one of claims 137 to 139, wherein the cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497 to 543, or a sequence comprising one or more substitutions within a sequence selected from the group consisting of SEQ ID NOs: 497 to 543.

141. 141. The conditionally active TROP2-targeting multispecific protein of any one of claims 137 to 140, wherein the conditionally active multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

142. 141. The conditionally active TROP2-targeting multispecific protein of any one of claims 137 to 140, wherein the conditionally active multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

143. 141. The conditionally active TROP2-targeting multispecific protein of any one of claims 137 to 140, wherein the conditionally active multispecific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229 to 264.

144. 144. A pharmaceutical composition comprising the conditionally active TROP2-targeting multispecific protein of any one of claims 137-143.

145. 145. The pharmaceutical composition of claim 144, further comprising a pharmaceutically acceptable carrier.

146. 144. A method for producing a conditionally active TROP2-targeted multispecific protein, comprising culturing a host transformed or transfected with a vector comprising one or more nucleic acid sequences encoding domains of the conditionally active TROP2-targeted multispecific protein of any one of claims 137 to 143 under conditions allowing expression of the conditionally active TROP2-targeted multispecific protein, and recovering and purifying the produced protein from the culture.

147. 144. A method for treating or ameliorating a proliferative or neoplastic disease in a subject in need thereof, comprising administering to the subject a conditionally active TROP2-targeted multispecific protein of any one of claims 137-143 or a pharmaceutical composition of claim 144 or 145.

148. 148. The method of claim 147, wherein the neoplastic disease comprises a solid tumor disease.

149. 149. The method of claim 148, wherein the solid tumor disease is metastatic.

150. 150. The method of any one of claims 147-149, wherein the neoplastic disease comprises at least one of oral cancer, colorectal cancer, head and neck cancer, prostate cancer, liver cancer, cervical cancer, nasopharyngeal cancer, thyroid cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, bladder cancer, gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, nasal NK / T cell lymphoma, glioma, glioblastoma, osteosarcoma, pituitary adenoma, or any combination thereof.

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