Trop2-targeting trispecific proteins for the

Abstract

Provided herein are TROP2 binding proteins, pharmaceutical compositions comprising such proteins or fragments thereof, as well as nucleic acids, recombinant expression vectors, and host cells for making such TROP2 binding proteins. Also disclosed are methods of preventing and / or treating diseases, conditions, and disorders using the disclosed TROP2 binding proteins.

Description

[0001] Cross-references

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

[0003] Incorporated by Reference

[0004] 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 Art

[0005] TROP2 is a protein encoded by the TACSTD2 gene in humans. This antigen is a member of a family of at least two type I membrane proteins. It transduces intracellular calcium signals and acts as a cell surface receptor. Studies have shown that aberrant TROP2 protein overexpression is associated with several cancers, including colorectal, pancreatic, gastric, oral squamous cell carcinoma, ovarian, and breast cancer. Cancers with high TROP2 expression are associated with increased disease recurrence and drug resistance, making it a poor prognostic factor for survival.

[0006] There is a need for more treatment options that allow physicians to select therapeutics with optimal side effect profiles for individual patients.The present disclosure provides novel polypeptide and protein therapeutics useful in methods of treatment, particularly methods of treating conditions associated with aberrant expression of TROP2. Summary of the Invention

[0007] Provided herein are TROP2 binding domains comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises 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. In some embodiments, the amino acid sequence is at least about 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57. 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 pharmaceutical form. In some embodiments, the multispecific protein further comprises a bulk serum protein binding domain. In some embodiments, the bulk serum protein comprises serum albumin. In some embodiments, the serum albumin comprises human serum albumin. In some embodiments, the bulk serum protein binding domain comprises a sequence that is at least 75% identical to SEQ ID NO: 493 or 549. In some embodiments, the CD3 binding domain comprises a sequence that is at least 75% identical to SEQ ID NO: 494. In some embodiments, the multispecific protein comprises a sequence that is at least about 75% identical to a 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 is capable of masking binding of the TROP2 binding domain or the CD3 binding domain to its respective target. In some embodiments, the multispecific protein comprises a noncleavable prodrug form. In some embodiments, the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOs: 550 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 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 in a sequence selected from the group consisting of SEQ ID NOs: 497-543.In some embodiments, the large volume serum protein binding domain comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NO: 493. In some embodiments, wherein the CD3 binding domain comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NO: 494. In some embodiments, wherein the multispecific protein comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NO: 229-264. In some embodiments, the multispecific protein comprises a sequence that is at least 85% identical to a sequence selected from SEQ ID NO: 229-264. In some embodiments, the active drug comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NO: 229-264.

[0008] Provided herein is 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 or a pharmaceutical composition comprising the same according to the description herein. In some embodiments, the subject is a human.

[0009] 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; (b) the TROP2 binding domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 115-171; and the CDR3 comprises a sequence selected from SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 172-228, wherein the binding moiety is capable of masking the binding of the TROP2 binding domain to its target.

[0010] In some embodiments, the TROP2 binding domain comprises a sequence that is at least about 75% identical to a sequence selected from SEQ ID NOs: 1-57.

[0011] 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, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228, wherein the non-CDR loop is capable of binding the TROP2 binding domain or the second target antigen binding domain, and wherein the binding moiety is capable of masking the binding of the TROP2 binding domain or the second target antigen binding domain to its target. In some embodiments, wherein the binding moiety comprises a masking moiety, and wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NOs: 550 or 558-560, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560. In some embodiments, 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 relative to a sequence selected from the group consisting of SEQ ID NOs: 497-543. In some embodiments, wherein the binding moiety comprises a sequence that is at least 75% identical to 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 that is at least 75% identical to SEQ ID NO: 494.

[0012] Provided herein is a method for treating or ameliorating a proliferative disease or a neoplastic disease, comprising administering to the subject a conditionally active chimeric antigen receptor described herein or a pharmaceutical composition comprising the same. In some embodiments, the subject is a human.

[0013] Provided herein is 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 or a pharmaceutical composition comprising the same according to the description herein. In some embodiments, the subject is a human.

[0014] In some embodiments, the domain is a humanized antibody or antigen binding fragment thereof. In some embodiments, the binding domain is a single domain antibody, a VHH domain, a scFv, a VH domain, a VL domain, a Fab, a F(ab')2, a Fab', a non-Ig domain, a ligand, a knottin, or a small molecule entity. In some embodiments, the binding domain comprises the 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. D ) binds to TROP2. In some embodiments, the binding domain binds to human TROP2, mouse TROP2, cynomolgus monkey TROP2, or a combination thereof.

[0015] Provided herein is a multispecific protein comprising a TROP2 binding domain, wherein the TROP2 binding domain is according to described herein. In some embodiments, it comprises a TROP2 binding domain (anti-TROP2 domain) and a CD3 binding domain (anti-CD3 domain) according to described herein. In some embodiments, the anti-TROP2 domain and the anti-CD3 domain are in an anti-TROP2:anti-CD3 orientation. In some embodiments, the anti-TROP2 domain and the anti-CD3 domain are in an anti-CD3:anti-TROP2 orientation. In some embodiments, it comprises a TROP2 binding domain (anti-TROP2 domain), the CD3 domain (anti-CD3 domain), and an albumin binding domain (anti-ALB domain) according to described herein. In some embodiments, the anti-CD3 domain comprises an amino acid sequence as set forth in SEQ ID NO: 494. In some embodiments, the anti-ALB domain comprises an amino acid sequence as set forth in SEQ ID NO: 493. In some embodiments, the anti-TROP2 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3:anti-ALB:anti-TROP2 orientation. In some embodiments, the anti-TROP2 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-TROP2:anti-ALB:anti-CD3 orientation. In some embodiments, the anti-TROP2 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB:anti-TROP2:anti-CD3 orientation. In some embodiments, the anti-TROP2 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-CD3:anti-TROP2:anti-ALB orientation. In some embodiments, the anti-TROP2 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-ALB:anti-CD3:anti-TROP2 orientation. In some embodiments, the anti-TROP2 domain, the anti-CD3 domain, and the anti-ALB domain are in an anti-TROP2:anti-CD3:anti-ALB orientation.

[0016] Provided herein is a multivalent protein comprising a sequence as set forth in any one of SEQ ID NOS: 229-264.

[0017] Provided herein is an active pharmaceutical comprising a sequence as set forth in any one of SEQ ID NOS: 229-264.

[0018] Provided herein is an active pharmaceutical comprising a sequence as set forth in any one of SEQ ID NOS: 1-57.

[0019] Provided herein is a pharmaceutical composition 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 pharmaceutical described herein, and (ii) a pharmaceutically acceptable carrier.

[0020] Provided herein is a method of producing a TROP2 binding domain described herein, the method comprising culturing a host transformed or transfected with a vector comprising 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.

[0021] Provided herein is a method of producing a multispecific protein 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 the multispecific TROP2 binding protein under conditions that allow expression of the multispecific protein, and recovering and purifying the produced protein from the culture.

[0022] Provided herein is 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 herein or a pharmaceutical composition described herein.

[0023] Provided herein is 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 multispecific protein according to described herein, a multivalent protein according to described herein, an active pharmaceutical according to described herein, or a pharmaceutical composition according to described herein. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering a combination of a certain agent and a TROP2 binding domain according to described herein, a multispecific protein according to described herein, a multivalent protein according to described herein, an active pharmaceutical according to described herein, or a pharmaceutical composition according to 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 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 combination of a certain agent and any one of the conditionally active TROP2 binding proteins or a pharmaceutical composition comprising the same according to 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.

[0024] Provided herein is a method of producing a conditionally active TROP2 binding protein according to 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 according to described herein under conditions that allow expression of the conditionally active TROP2 binding protein, and recovering and purifying the produced protein from the culture.

[0025] Provided herein is a method of producing a multivalent protein according to 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 multivalent protein according to described herein under conditions that allow expression of the multivalent protein, and recovering and purifying the produced protein from the culture.

[0026] Provided herein is a method of producing an active pharmaceutical according to 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 an active pharmaceutical according to described herein under conditions that allow expression of the active pharmaceutical, and recovering and purifying the produced protein from the culture.

[0027] Provided herein is a conditionally active TROP2 binding protein, wherein the conditionally active TROP2 binding protein has a higher therapeutic index compared to a TROP2 binding protein that does not comprise (a) a binding moiety but is otherwise identical to the conditionally active TROP2 binding protein. In some embodiments, the therapeutic index of the conditionally active TROP2 binding protein is at least about 5-fold to about 100-fold higher than the therapeutic index of a TROP2 binding protein that does not comprise (a) a 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, 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: 229-264.

[0028] Provided herein is a pharmaceutical composition comprising: (i) a conditionally active TROP2 binding protein according to described herein and (ii) a pharmaceutically acceptable carrier.

[0029] Provided herein is 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 chimeric antigen receptor according to described herein or a pharmaceutical composition according to described herein.

[0030] Provided herein is 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 described herein or a pharmaceutical composition according to described herein. In some embodiments, the subject is a human. In some embodiments, the neoplastic disease is 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.

[0031] Provided herein is a method of improving 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 loop comprises a binding site specific for the TROP2 binding domain, wherein the TROP2 binding domain is masked from binding its target by the binding moiety, wherein the TROP2 binding domain binds its target upon cleavage of the cleavable linker. In some embodiments, the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 115-171; and the CDR3 comprises a sequence selected from SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 172-228. In some embodiments, the TROP2 binding domain conjugated to the binding moiety is part of a conditionally active multi-specific protein, wherein the conditionally active multi-specific protein further comprises a CD3 binding domain. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to SEQ ID NO: 493. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 494. In some embodiments, the cleavable linker comprises a sequence selected from SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 497-543. In some embodiments, the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NOs: 1-57. In some embodiments, the conditionally active multi-specific protein comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NOs: 229-264. In some embodiments, the conditionally active multi-specific protein comprises a sequence that is at least 85% identical to a sequence selected from SEQ ID NOs: 229-264. In some embodiments, the conditionally active multi-specific protein comprises a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 229-264.In some embodiments, the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to 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.

[0032] Provided herein is a method of improving 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 target antigen binding domain and the second target antigen binding domain comprises a TROP2 binding domain, the method comprising conjugating the first target antigen binding domain or the second target antigen binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, wherein the non-CDR loop comprises a binding site specific for the first target antigen binding domain or the second target antigen binding domain, wherein at least one of the first target antigen binding domain and the second target antigen binding domain is masked by the binding moiety from binding its target, and wherein the masked first target antigen binding domain and the masked second target antigen binding domain bind their targets upon cleavage of the cleavable linker. In some embodiments, the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in 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 target antigen binding domain or the second target antigen binding domain comprises a CD3 binding domain. In some embodiments, the non-CDR loop comprises a binding site specific for the CD3 binding domain. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 494. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to 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 cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions in 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 multi-specific 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 multi-specific 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 method of improving 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. In some embodiments, the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the CD3 binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 494. In some embodiments, the binding moiety comprises a sequence that is at least about 75% identical to 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 cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 497-543. In some embodiments, the conditionally active multi-specific 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 multi-specific 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 multi-specific protein comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

[0034] Provided herein is a TROP2-targeted conditionally active multispecific protein comprising: a TROP2 binding domain, a CD3 binding domain, 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, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228. In some embodiments, the albumin binding domain comprises a sequence that is at least about 75% identical to SEQ ID NO: 494. 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 cleavable linker comprises a sequence selected from the group consisting of SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions in 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.

[0035] Provided herein is a pharmaceutical composition comprising a TROP2-targeted conditionally active multispecific protein described herein. In some embodiments, it further comprises a pharmaceutically acceptable carrier.

[0036] Provided herein is a method of producing a TROP2-targeted conditionally active multispecific protein described herein, the method comprising culturing a host cell transformed or transfected with a vector comprising one or more nucleic acid sequences encoding a domain of a TROP2-targeted conditionally active multispecific protein described herein under conditions that allow expression of the TROP2-targeted conditionally active multispecific protein described herein, and recovering and purifying the produced protein from the culture.

[0037] Provided herein is 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-targeting conditionally active multispecific protein described herein or a pharmaceutical composition according to 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 is 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 DESCRIPTION OF DRAWINGS

[0038] 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 application will be obtained by reference to the following detailed description that sets forth illustrative

[0039] Figure 1 Results from TDCC assays using H292 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 3TRL3, 2TRL92, 3TRL77, 2TRL4, and 2TRL76 are provided.

[0040] Figure 2 Results from TDCC assays using H292 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 3TRL3, 2TRL92, 3TRL77, 2TRL4, and 2TRL76 are provided.

[0041] Figure 3 Results from TDCC assays using H292 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 3TRL56, 3TRL87, 2TRL33, 2TRL1, and 2TRL5 are provided.

[0042] Figure 4 Results from TDCC assays using H292 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 2TRL8, 2TRL27, 2TRL46, 2TRL69, and 2TRL94 are provided.

[0043] Figure 5Results from a TDCC assay using H292 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL79, 2TRL81, 2TRL18, 3TRL27, and a GFP negative control are presented.

[0044] Figure 6 Results from a TDCC assay using H292 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL28, 3TRL39, 3TRL82, 2TRL68, 2TRL64, and 3TRL53 are presented.

[0045] Figure 7 Results from a TDCC assay using H1376 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL3, 2TRL92, 3TRL77, 2TRL4, and 2TRL76 are presented.

[0046] Figure 8 Results from a TDCC assay using H1376 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL16, 2TRL29, 3TRL58, 2TRL31, and 2TRL70 are presented.

[0047] Figure 9 Results from a TDCC assay using H1376 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 3TRL56, 3TRL87, 2TRL33, 2TRL1, and 2TRL5 are presented.

[0048] Figure 10 Results from a TDCC assay using H1376 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL8, 2TRL27, 2TRL46, 2TRL69, and 2TRL94 are presented.

[0049] Figure 11 Results from a TDCC assay using H1376 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL79, 2TRL81, 2TRL18, 3TRL27, and a GFP negative control are presented.

[0050] Figure 12 Results from a TDCC assay using H1376 cells and anti-CD3 / anti-TROP2 fusion proteins containing the llama anti-TROP2 sequences 2TRL28, 3TRL39, 3TRL82, 2TRL68, 2TRL64, and 3TRL53 are presented.

[0051] Figure 13 Results from TDCC assays using H292 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 3TRL53, 3TRH53, 2TRL76, and 2TRH76 are provided.

[0052] Figure 14 Results from TDCC assays using H292 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRL81, and 2TRH81 are provided.

[0053] Figure 15 Results from TDCC assays using HT1376 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 3TRL53, 3TRH53, 2TRL76, and 2TRH76 are provided.

[0054] Figure 16 Results from TDCC assays using HT1376 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRL81, and 2TRH81 are provided.

[0055] Figure 17 Results from TDCC assays using HCC70 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 3TRL53, 3TRH53, 2TRL76, and 2TRH76 are provided.

[0056] Figure 18 Results from TDCC assays using HCC70 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRL81, and 2TRH81 are provided.

[0057] Figure 19 Results from TDCC assays using HCC70 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequences 2TRL79, 2TRH79, 2TRH79B, and 2TRH79B L040 ProTriTAC are provided.

[0058] Figure 20Results from TDCC assays using HPAF-II cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama and humanized anti-TROP2 sequences 2TRL79, 2TRH79, 2TRH79B, and 2TRH79B L040 ProTriTAC are provided.

[0059] Figure 21 Results from TDCC assays using CAL27 cells and anti-CD3 / anti-TROP2 fusion proteins comprising humanized anti-TROP2 sequences 2TRH79B and 2TRH79B L040 ProTriTAC are provided.

[0060] Figure 22 Results from a combination therapy xenograft rodent study using HCC70 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequence 2TRL79 ProTriTAC are shown.

[0061] Figure 23 Results from a combination therapy xenograft rodent study using HCC70 cells and anti-CD3 / anti-TROP2 fusion proteins comprising llama anti-TROP2 sequence 2TRL79 ProTriTAC are shown.

[0062] Figure 24 Results from a combination therapy xenograft rodent study using HCC70 cells and anti-CD3 / anti-TROP2 fusion proteins comprising humanized anti-TROP2 sequence 2TRH79 ProTriTAC are shown.

[0063] Figure 25 Results from a combination therapy xenograft rodent study using CAL27 cells and anti-CD3 / anti-TROP2 fusion proteins comprising humanized anti-TROP2 sequence 2TRH79B ProTriTAC are shown.

[0064] Figure 26 Results from a combination therapy xenograft rodent study using HPAF-II cells and anti-CD3 / anti-TROP2 fusion proteins comprising humanized anti-TROP2 sequence 2TRH79B ProTriTAC are shown.

[0065] Figure 27 Prodrugs and active drugs were shown to exhibit comparable strong anti-tumor activity in HCC70 cells.

[0066] Figures 28A-28B Anti-CD3 / anti-TROP2 fusion proteins comprising humanized 2TRH79B were shown to exhibit comparable strong anti-tumor activity in HCC70 cells in the absence or presence of calcium (47-51 nM or 50 nM, respectively) to human (Figure 28A ) and cynomolgus monkey (cyno) Trop2 ( Figure 28B ) considerable affinity binding.

[0067] Figure 29 The experimental design of the one-month dose-escalation / maximum tolerated dose study is shown.

[0068] Figure 30 The anti-CD3 / anti-TROP2 fusion protein containing humanized 2TRH79B was shown to exhibit favorable pharmacokinetics, half-life, and systemic accumulation. DETAILED DESCRIPTION

[0069] Described herein are trispecific proteins targeting TROP2, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for preparing such proteins. Also provided are methods for preventing and / or treating diseases, conditions, and disorders using the disclosed trispecific proteins targeting TROP2. The trispecific proteins targeting TROP2 are capable of specifically binding to TROP2 and CD3 and have a half-life extension domain, such as a domain that binds to human albumin (ALB).

[0070] TROP2 binding protein

[0071] Proteins that bind to TROP2, pharmaceutical compositions thereof, and nucleic acids, recombinant expression vectors, and host cells for preparing such proteins are described herein. Methods of preventing and / or treating diseases, conditions, and disorders using the disclosed TROP2 binding proteins are also provided. In some embodiments, the TROP2 binding protein is part of a multispecific (e.g., trispecific) protein that comprises a TROP2 binding domain as described herein.

[0072] Trophoblast cell surface antigen (Trop2), or tumor-associated calcium signal transducer 2 (TACSTD2), or epithelial glycoprotein 1 (EGP-1), or pancreatic cancer marker protein GA733-1, or GP50, or membrane fraction 1 surface marker 1 (M1S1), is a ubiquitously expressed 35 kDa type I transmembrane glycoprotein with four N-linked glycosylation sites and composed of 323 amino acids encoded by the TACSTD2 gene. It is a member of the tumor-associated calcium signal transducer (TACSTD) family and is structurally related to the epithelial cell adhesion molecule (EpCAM). Trop-2 comprises a large extracellular domain, a transmembrane domain, and an intracellular tail. The crystal structure of the TROP2 extracellular domain reveals a compact subunit composed of three domains: the N-terminal (ND), the type 1 thyroglobulin (TY), and the C-terminal (CD) domain. The TROP2 extracellular domain is capable of dimerization.

[0073] TROP2 has similar claudin-interaction capabilities as its paralog EpCAM, both of which are involved in signal transduction initiated by extracellular domain proteolytic cleavage. Trop-2 plays a vital role in embryonic development, placental tissue formation, embryonic implantation, stem cell proliferation, and organ development. TROP2 is a stem / progenitor cell marker and low basal expression levels of TROP2 are found on the surface of multiple normal epithelial tissues including skin and oral mucosa. TROP2 overexpression is observed in many types of malignant epithelial tumors, such as 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., breast 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 cancer, extrahepatic cholangiocarcinoma (hilar cholangiocarcinoma), oral squamous cell carcinoma, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, laryngeal squamous cell carcinoma, liver fluke associated cholagiocarcinoma, lung adenocarcinoma, hepatocellular carcinoma, cervical squamous cell carcinoma, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma. Trop2 is also highly expressed in non-epithelial origin tumors, such as melanoma, nasal NK / T cell lymphoma, glioma and glioblastoma, and osteosarcoma. TROP2 overexpression is also associated with pituitary adenomas. Despite frequent overexpression of Trop2 in the tumorigenic process, genetic analyses have shown that point mutations and copy number variations of the TACSTD2 gene are quite rare in human tumors. TROP2 overexpression is associated with a worse prognosis for survival and drug resistance.

[0074] Exemplary protein sequences for TROP2 are provided 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 provided 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 provided 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 provided in Refseq XP_005543292.2. In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein encoded by a nucleic acid as provided in RefSeq NM_002353. In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein encoded by a nucleic acid as provided in RefSeq NM_0200047. In some embodiments, a TROP2 binding protein of the present disclosure binds to a TROP2 protein comprising an amino acid sequence as set forth in SEQ ID NO: 546 or 547.

[0075] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL (SEQ ID NO: 546).

[0076] HTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL (SEQ ID NO: 547).

[0077] In some embodiments, the TROP2 binding domain binds to the extracellular domain of a mature TROP2 protein. In some embodiments, the TROP2 binding domain binds to the transmembrane domain of a mature TROP2 protein. In some embodiments, the TROP2 binding domain binds to the intracellular tail of a mature TROP2 protein.

[0078] In some embodiments, the TROP2 binding domain binds to a protein comprising a sequence that is truncated 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 examples, the TROP2 binding domains disclosed herein recognize an epitope within TROP2, as in certain cases the TROP2 binding protein interacts with one or more amino acids found within a domain of human TROP2. The epitope bound by the antibody can consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of TROP2. Alternatively, the epitope can consist of multiple non-contiguous amino acids (or sequences of amino acids) located within a domain of TROP2.

[0079] In some embodiments, the TROP2 binding domains disclosed herein recognize full-length TROP2. In certain examples, the TROP2 binding domains disclosed herein recognize an epitope within TROP2, as in certain cases the TROP2 binding protein interacts with one or more amino acids found within a domain of human TROP2. The epitope bound by the antibody can consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within a domain of TROP2. Alternatively, the epitope can consist of multiple non-contiguous amino acids (or sequences of amino acids) located within a domain of TROP2.

[0080] In some embodiments, the TROP2 binding proteins of the present disclosure bind to full-length TROP2 protein or fragments thereof, such as fragments within full-length TROP2 protein that comprise an epitope, as described above. In some cases, the fragments comprising an epitope include antigenic or immunogenic fragments of TROP2 protein and derivatives thereof. In some embodiments, the fragments comprising an antigenic epitope, including antigenic or immunogenic fragments, are 12 amino acids or more, such as 20 amino acids or more, 50 or 100 amino acids or more. In some embodiments, the TROP2 fragments comprise 95% or more of the full protein length, 90% or more, 75% or 50% or 25% or 10% or more of the full protein length. In some embodiments, the fragments of TROP2 comprising an epitope, including antigenic or immunogenic fragments, are capable of eliciting a relevant immune response in a patient. In some embodiments, the derivatives of TROP2 include variants of the sequence in which one or more (e.g., 1-20, such as 15 amino acids, or up to 20% of the total number of amino acids in the protein, such as up to 10% or 5% or 1%) deletions, insertions, or substitutions have been made to the TROP2 sequence provided in SEQ ID NO: 546 or 547.

[0081] In some embodiments, the substitutions include conservative substitutions. In some examples, the derivatives and variants have substantially the same biological function as the protein from which they are derived. For example, in some cases, the derivatives and variants of TROP2 have comparable antigenicity or immunogenicity to the protein from which they are derived, have ligand binding activity or active receptor complex formation ability of the protein from which they are derived, or preferably both, and have the same tissue distribution as TROP2.

[0082] In some embodiments, the TROP2 binding protein specifically binds to TROP2 with the same or better affinity as a reference TROP2 binding protein, in such embodiments, the TROP2 binding protein comprises an affinity matured TROP2 binding molecule and is derived from a TROP2 binding parent molecule comprising one or more amino acid mutations (e.g., stabilizing mutations, destabilizing mutations) relative to the TROP2 binding parent molecule. In some embodiments, the affinity matured TROP2 binding molecule has better stability relative to a selected destabilizing agent than a reference TROP2 binding parent molecule. In some embodiments, the affinity matured TROP2 binding molecule is identified in a process comprising panning one or more pre-candidate TROP2 binding molecules expressed in a phage display library derived from one or more TROP2 binding parent molecules against a TROP2 protein, such as a human TROP2 protein. In some embodiments, the pre-candidate TROP2 binding molecule comprises an amino acid substitution in a variable region, CDR, or framework residue relative to the parent molecule.

[0083] As used herein, "phage display" refers to a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of 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 those sequences that bind to a target molecule 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, randomized peptides and small proteins by fusion to gene III or gene VIII of filamentous phage. See, e.g., Wells and Lowman, Curr. Opin. Struct. Biol, 3:355-362 (1992), and references cited therein. In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein, such that the phage particle displays one copy of the fusion protein or none. Affinity interactions are reduced relative to multivalent phage, so selection is based on intrinsic 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).

[0084] In some embodiments, panning includes using varying binding times and concentrations to identify TROP2 binding molecules with increased or decreased binding rates from pre-candidate TROP2 binding molecules. In some embodiments, panning includes using varying wash times to identify TROP2 binding molecules with increased or decreased dissociation rates from pre-candidate TROP2 molecules. In some embodiments, panning includes using both varying binding times and varying wash times. In some embodiments, one or more stabilizing mutations are combined, for example by shuffling to create a second round combinatorial library from such mutants and performing a second round of panning, to increase the stability of the affinity matured TROP2 binding molecules, followed by binding selection.

[0085] In some embodiments, the affinity matured TROP2 binding molecules have equal or better affinity for TROP2 protein (e.g., human TROP2 protein) than the TROP2 binding parent molecule, but have reduced cross-reactivity with selected substances such as ligands, proteins, antigens, etc. that are not the TROP2 epitope to which the TROP2 binding parent molecule is specific or designed to be specific, or in some embodiments, have increased cross-reactivity. With respect to the latter, in some embodiments, the affinity matured TROP2 binding molecules are more successfully tested in animal models if they react with human TROP2 and the corresponding target in the animal model, e.g., mouse TROP2 or 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, 10 nM or less, and to cyno 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 molecules identified after one round of panning bind to human TROP2 with an affinity of about 5 nM or less, such as 1 nM or less, and to cyno TROP2 with an affinity of about 7.5 nM or less, such as 1 nM or less. In some embodiments, the 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 cyno TROP2 with an affinity of about 3.5 nM or less.

[0086] In some embodiments, the TROP2 binding protein comprises an antigen-specific binding domain polypeptide that specifically binds to a target, such as a target on a diseased cell or a target on other cells that support a disease state, such as a target on stromal cells that support tumor growth or a target on immune cells that support disease-mediated immunosuppression. In some examples, the antigen-specific binding domain comprises an antibody, a single chain antibody, a Fab, a Fv, a T cell receptor binding domain, a ligand binding domain, a receptor binding domain, a domain antibody, a single domain antibody, a minibody, a nanobody, a peptibody, or various other antibody mimetics, such as affitins, alphabodies, atrimers, CTLA4-based molecules, adnectins, anticalins, Kunitz domain-based proteins, avimers, knottins, fynomers, affibodies, affilins, monobodies, and armadillo repeat-based proteins.

[0087] In some embodiments, the TROP2 binding domain is an anti-TROP2 antibody or antigen-binding fragment thereof, or an antibody variant of a 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 an anti-TROP2 antibody or antigen-binding fragment thereof as described herein are contemplated. For example, in certain embodiments, amino acid sequence variants of an anti-TROP2 antibody or antigen-binding fragment thereof as described herein are contemplated to improve its binding affinity and / or other biological properties. Exemplary methods for making amino acid variants include, but are not limited to, introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody or antigen-binding fragment thereof.

[0088] Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., antigen binding. In certain embodiments, variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and framework regions. Examples of such substitutions are described infra. Amino acid substitutions can be introduced into an antibody of interest, or an antigen binding fragment thereof, and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, altered antibody dependent cellular cytotoxicity (ADCC) or improved T cell mediated cellular cytotoxicity (TDCC). Both conservative and non-conservative amino acid substitutions are contemplated for the production of antibody variants.

[0089] In another example of substitutions used to generate variant anti-TROP2 antibodies, or antigen binding fragments thereof, one or more hypervariable region residues of a parent antibody are substituted. Typically, the variants are then selected for an improvement in a desired property as compared to the parent antibody, or antigen binding fragment thereof, e.g., increased affinity, decreased affinity, decreased immunogenicity, increased pH dependence of binding.

[0090] In some embodiments, the TROP2 binding domain is a single domain antibody (sdAb), such as a heavy chain variable domain (VH) specific for TROP2, a variable domain of a llama-derived sdAb (VHH), a peptide, a ligand, or a small molecule entity. In some embodiments, the TROP2 binding domain described herein is any domain that binds to TROP2, including but not limited to a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In certain embodiments, the TROP2 binding domain is a single domain antibody. In other embodiments, the TROP2 binding domain is a peptide. In further embodiments, the TROP2 binding domain is a small molecule.

[0091] In general, it should be noted that the term single domain antibody, as used herein in its broadest sense, is not limited to a particular biological source or a particular method of preparation. A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds that are different from those derived from antibodies. The single domain antibody can be any single domain antibody in the art, or any future single domain antibody. The single domain antibody can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, bovine. For example, in some embodiments, the single domain antibody of the present disclosure is obtained by: (1) by isolating a VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, in particular from a mammalian species, such as from a human, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab", or by expressing a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques to make a protein, polypeptide or other amino acid sequence; (7) by using nucleic acid synthesis techniques known in the art to make a nucleic acid encoding a single domain antibody, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing.

[0092] In one embodiment, the single domain antibody corresponds to a VHH domain of a naturally occurring heavy chain antibody directed against TROP2. As further described herein, such VHH sequences can generally be generated or obtained by suitably immunizing a llama species with TROP2 (i.e., to generate an immune response and / or heavy chain antibodies directed against TROP2), obtaining a suitable biological sample (such as a blood sample, a serum sample or a B cell sample) from said llama, and using any suitable technique known in the art to generate a VHH sequence directed against TROP2 starting from said sample.

[0093] In another embodiment, such naturally occurring VHH domains against TROP2 are obtained from naive libraries of camelid VHH sequences, e.g. by screening such libraries using TROP2 or at least one portion, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, e.g. 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 in WO 00 / 43507.

[0094] In a further embodiment, yet another technique for obtaining VHH sequences against TROP2 involves suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e. to generate an immune response and / or heavy chain antibodies against TROP2), obtaining a suitable biological sample (such as a blood sample, serum sample or B cell sample) from said transgenic mammal and then using any suitable technique known in the art to generate VHH sequences against TROP2 starting from said sample. For example, rats or mice expressing heavy chain antibodies as well as other methods and techniques described in WO 02 / 085945 and WO 04 / 049794 can be used for this purpose.

[0095] In some embodiments, anti-TROP2 single domain antibodies of the present disclosure include single domain antibodies having an amino acid sequence corresponding to an amino acid sequence of a non-human antibody and / or naturally occurring VHH domain, e.g., an American camelid anti-TROP2 antibody, but which have been "humanized," i.e., by replacing one or more amino acid residues in the amino acid sequence of the non-human anti-TROP2 and / or naturally occurring VHH sequence, particularly in the framework sequences, with one or more amino acid residues present at the corresponding position in a VH domain from a conventional 4-chain antibody from a human (e.g., as described above). This can be done in a manner known in the art, which will be readily apparent to one of skill in the art, e.g., based on the further description herein. Moreover, it should be noted that such humanized anti-TROP2 single domain antibodies of the present disclosure are obtained in any suitable manner known per se (i.e., as indicated by points (1)-(8) above), and thus are not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VHH domains as starting material. In some further embodiments, single domain anti-TROP2 antibodies as described herein include single domain antibodies having an amino acid sequence corresponding to an amino acid sequence of a naturally occurring VH domain, but which have been "camelized," i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues present at the corresponding position in a VHH domain of a heavy chain antibody. Such "camelized" substitutions are preferably inserted at amino acid positions forming and / or present at the VH-VL interface, and / or at so-called camelid hallmark residues. See, e.g., WO 94 / 04678, and Davies and Riechmann (1994 and 1996). Preferably, the VH sequence used as starting material or starting point for generating or designing a camelized single domain is a VH sequence from a mammal, more preferably a VH sequence from a human, such as a VH3 sequence. However, it should be noted that in certain embodiments, such camelized anti-TROP2 single domain antibodies of the present disclosure are obtained in any suitable manner known in the art (i.e., as indicated by points (1)-(8) above), and thus are not strictly limited to polypeptides obtained using polypeptides comprising non-human anti-TROP2 antibodies and / or naturally occurring VH domains as starting material. For example, as described further herein, both "humanization" and "camelization" are performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then altering one or more codons in the nucleotide sequence in a manner that the new nucleotide sequence encodes a "humanized" or "camelized" single domain antibody, respectively. This nucleic acid can then be expressed to provide the desired anti-TROP2 single domain antibody of the present disclosure.Alternatively, in other embodiments, the amino acid sequences of the desired humanized or camelized anti-TROP2 single domain antibodies of the present disclosure are designed based on the amino acid sequences of naturally occurring VHH domains or VH domains, respectively, and then synthesized de novo using known peptide synthesis techniques. In some embodiments, the nucleotide sequences encoding the desired humanized or camelized anti-TROP2 single domain antibodies of the present disclosure are designed based on the amino acid sequences or nucleotide sequences of naturally occurring VHH domains or VH domains, respectively, and then synthesized de novo using known nucleic acid synthesis techniques, followed by expression of the resulting nucleic acids using known expression techniques to provide the desired anti-TROP2 single domain antibodies of the present disclosure.

[0096] Other suitable methods and techniques for obtaining the anti-TROP2 single domain antibody nucleic acids of the present disclosure starting from naturally occurring VH sequences or VHH sequences include, for example, combining one or more portions of one or more naturally occurring VH sequences (such as one or more framework (FR) sequences and / or complementarity determining region (CDR) sequences), one or more portions of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide an anti-TROP2 single domain antibody of the present disclosure or a nucleotide sequence or nucleic acid encoding the same.

[0097] In some embodiments, the TROP2 binding domain is an anti-TROP2 specific antibody comprising a heavy chain variable complementarity determining region CDR1, a heavy chain variable CDR2, a heavy chain variable CDR3, a light chain variable CDR1, a light chain variable CDR2, and a light chain variable CDR3. In some embodiments, the TROP2 binding domain comprises any domain that binds to TROP2, including but not limited to a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen binding fragment such as a single domain antibody (sdAb), a Fab, a Fab’, a F(ab)2, and a 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 Fv (dsFv) fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some embodiments, the TROP2 binding domain is a single domain antibody. In some embodiments, the anti-TROP2 single domain antibody comprises heavy chain variable complementarity determining regions (CDRs), CDR1, CDR2, and CDR3.

[0098] In some embodiments, the TROP2 binding domain is a polypeptide comprising an amino acid sequence consisting of four framework regions / sequences (fl-f4) interrupted by three complementarity determining regions / sequences (CDR1, CDR2, and CDR3), as shown in the following formula: fl-r1-f2-r2-f3-r3-f4, where r1, r2, and r3 are CDR1, CDR2, and CDR3, respectively, and fl, f2, f3, and f4 are framework residues. The framework residues of the TROP2 binding proteins of the present disclosure comprise, 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 comprise, 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.

[0099] In some embodiments, the binding proteins described herein comprise a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding proteins have at least 70-95% or more homology to a sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding proteins have at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology to a sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding proteins have at least 70-95% or more identity to a sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof. In some embodiments, the TROP2 binding proteins have at least 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity to a sequence selected from the group consisting of SEQ ID NOs: 1-57, subsequences thereof, and variants thereof.

[0100] In some embodiments, the CDR1 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions as compared to a sequence selected from SEQ ID NOs: 58-114. In some embodiments, the CDR2 comprises a sequence as set forth in any one of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions as compared to a sequence selected from SEQ ID NOs: 115-171. In some embodiments, the CDR3 comprises a sequence as set forth in any one of SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions as compared to a sequence selected from SEQ ID NOs: 172-228.

[0101] In various embodiments, the TROP2 binding domain of the present disclosure is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from SEQ ID NOs: 58-114, 115-171, and 172-228.

[0102] 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 an amino acid sequence set forth in SEQ ID NOs: 58-114.

[0103] 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: 115-171.

[0104] 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: 172-228.

[0105] In some embodiments, the TROP2 binding protein is cross-reactive to 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 bind to human TROP2 with a human K D (h K D ) of about 10-9 M or less. In certain embodiments, the TROP2 binding domains disclosed herein bind to cynomolgus monkey TROP2 with a cynomolgus monkey K D (c K D ) of about 10-9 M or less. In certain embodiments, the TROP2 binding domains disclosed herein bind to mouse TROP2 with a mouse K D (m K D ) of about 10-9 M or less. In certain embodiments, the TROP2 binding domains disclosed herein bind to cynomolgus monkey TROP2 and human TROP2 with cynomolgus monkey K D (c K D ) and human K D (h K D ) of about 10-9 M or less, respectively. In certain embodiments, the TROP2 binding domains disclosed herein bind to cynomolgus monkey TROP2 and human TROP2 with cynomolgus monkey K D (c K D ), mouse K D (m K D ), and human K D (h K D) binds to cynomolgus monkey TROP2, mouse TROP2, and human TROP2. In some embodiments, the TROP2 binding protein binds to human, mouse, and cynomolgus monkey TROP2 with comparable binding affinities (i.e., h K D , m K D , and c K D values differ by no more than ±10%). In some embodiments, the TROP2 binding domains disclosed herein bind Trop2 in the presence of calcium. In some embodiments, the TROP2 binding domains disclosed herein bind Trop2 in the absence of calcium.

[0106] In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 500 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 450 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 400 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 350 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 300 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 250 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 200 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 150 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 100 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 80 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 50 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 40 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 200 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 150 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.001 nM to about 100 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.1 nM to about 90 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.2 nM to about 80 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.3 nM to about 70 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.4 nM to about 50 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.5 nM to about 30 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.6 nM to about 10 nM. In some embodiments, the hKD, mKD, and cKD are in the range from about 0.7 nM to about 8 nM.In some embodiments, the hKD, mKD, and cKDare in the range from about 0.8 nM to about 6 nM. In some embodiments, the hKD, mKD, and cKDare in the range from about 0.9 nM to about 4 nM. In some embodiments, the hKD, mKD, and cKDare in the range from about 1 nM to about 2 nM.

[0107] In some embodiments, the hkon(1 / Ms)e5, ckon(1 / Ms)e5, mkon(1 / Ms)e5range from about 0.001 to about 100, for example, about 0.1-1, about 0.5-0.9. In some embodiments, the hKoff(1 / s), cKoff(1 / s), and mKoff(1 / s) range from about 1 x 10 -2 to 9 x 10 -6 , for example, about 1 x 10 -2 to 9 x 10 -3 , about 4 x 10 -3 to 6 x 10 -3 .

[0108] In some embodiments, any of the foregoing TROP2 binding domains (e.g., anti-TROP2 single domain antibodies of SEQ ID NOs: 1-57) is labeled with an affinity peptide for ease of purification. In some embodiments, the affinity peptide tag is six contiguous histidine residues, also known as 6X-his (SEQ ID NO: 496).

[0109] In certain embodiments, the TROP2 binding domains of the present disclosure preferentially bind membrane-bound TROP2 over soluble TROP2. Membrane-bound TROP2 refers to TROP2 that is present in or on the cell membrane surface of a cell expressing TROP2. Soluble TROP2 refers to TROP2 that is no longer present in or on the cell membrane surface of a cell that is or has been expressing TROP2. In certain cases, soluble TROP2 is present in the blood and / or lymphatic circulation of a subject. In one embodiment, the binding of the TROP2 binding domain to membrane-bound TROP2 is less than 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold than the binding to soluble TROP2. In one embodiment, the antigen binding proteins of the present disclosure preferentially bind membrane-bound TROP2 by a factor of 30-fold than the binding to soluble TROP2. The preferential binding of an antigen binding protein to membrane-bound TROP2 over soluble TROP2 can be readily determined using a binding assay.

[0110] In some embodiments, the TROP2 binding proteins are quite small, and in some embodiments are no larger than 40 kDa, no larger than 30 kDa, no larger than 25 kDa, no larger than 20 kDa, no larger than 15 kDa, or no larger than 10 kDa. In certain cases, the TROP2 binding proteins are 5 kDa or less if they are peptides or small molecule entities.

[0111] In other embodiments, the TROP2 binding proteins described herein comprise a small molecule entity (SME) conjugate to TROP2. SME conjugates are small molecules that are on average about 500 Da to 2000 Da in size, and 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 such as LPETG (SEQ ID NO: 548). To attach the SME conjugate to the TROP2 binding protein comprising the sortase recognition sequence, the protein is incubated with the sortase and the SME conjugate, whereby the sortase attaches the SME conjugate to the recognition sequence. In still other embodiments, the TROP2 binding proteins described herein comprise a knottin peptide for binding to TROP2. Knottins are disulfide-stabilized peptides with a cysteine knot scaffold, and have an average size of about 3.5 kDa. Knottins have been contemplated to bind to certain tumor molecules such as TROP2. In further embodiments, the TROP2 binding proteins described herein comprise a natural TROP2 ligand.

[0112] In some embodiments, the TROP2 binding proteins comprise more than one domain, and are single polypeptide designs with flexible linkages between the domains. This allows for easy production and manufacture of the TROP2 binding proteins, as they can be encoded by a single cDNA molecule that is easily introduced into a vector. In addition, in some embodiments where the TROP2 binding proteins described herein are monomeric single polypeptide chains, there are no chain pairing issues or need for dimerization. It is contemplated that in such embodiments, the TROP2 binding proteins described herein have reduced tendency to aggregate.

[0113] In TROP2 binding proteins comprising more than one domain, the domains are connected by one or more internal connecting linkers. In certain embodiments, the internal linker is "short", i.e., consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the internal linker consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the internal linker is "long", i.e., consists 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, contiguous amino acid residues. With respect to the amino acid composition of the internal linker, a peptide is selected that imparts flexibility to the TROP2 binding protein, does not interfere with the binding domains, and is resistant to protease cleavage. For example, glycine and serine residues often provide protease resistance. Examples of internal linkers suitable for connecting domains in a TROP2 binding protein include, but are not limited to, (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), (GGGGG)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), (GGGGSGGGGSGGGGS) (SEQ ID NO: 523), or (GGGGSGGGS) (SEQ ID NO: 524).

[0114] In some cases where the TROP2 binding protein comprises more than one domain, the domains within the TROP2 binding protein are conjugated using an enzymatic site-specific conjugation method that involves the use of a mammalian or bacterial transglutaminase. Microbial transglutaminases (mTGs) are versatile tools in modern research and biotechnology. The availability of a large number of relatively pure enzymes, ease of use, and lack of regulation by calcium and guanine-5'-triphosphate (GTP) make mTGs the primary cross-linking enzyme used in the food industry and biotechnology. Currently, mTGs are used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. See, e.g., Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem. 25, 5, 855-862.

[0115] In some examples, a TROP2 binding protein comprising more than one domain is provided, wherein one of the domains comprises an acceptor glutamine in the constant region, which can then be conjugated to another domain through a lysine-based linker (e.g., any primary amine chain that is a substrate for TGase, e.g., comprising an alkyl amine, oxoamine), where the conjugation only occurs on one or more acceptor glutamine residues present in the targeted moiety outside of the antigen binding site (e.g., outside of the variable region, in the constant region). Thus, the conjugation does not occur on 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 a lysine-based linker in the presence of TGase.

[0116] In some embodiments where one or more domains within the TROP2 binding protein are directly linked, a hybrid vector is prepared in which the DNA encoding the directly linked domains are themselves directly linked to each other. In some embodiments where a linker is used, a hybrid vector is prepared in which the DNA encoding one domain is linked to DNA encoding one end of the linker moiety, and the DNA encoding the other domain is linked to the other end of the linker moiety.

[0117] In some embodiments, the TROP2 binding protein 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 of a camelid-derived single domain antibody (VHH). In other embodiments, the TROP2 binding protein is a non-Ig binding domain, i.e., an antibody mimetic, such as an anticalin, an affilin, an affibody molecule, affitins, alphabodies, avimers, fynomers, kunitz domain peptides, and monobodies. In further embodiments, the TROP2 binding protein is a ligand or peptide that binds to or associates with TROP2. In still further embodiments, the TROP2 binding protein is a knottin. In still further embodiments, the binding domain for TROP2 is a small molecule entity.

[0118] In certain embodiments, a TROP2 binding protein according to the present disclosure can be incorporated into a trispecific protein targeting TROP2. In some embodiments, the trispecific protein comprises a CD3 binding domain, a half-life extension domain, and a TROP2 binding domain, according to the present disclosure. In some embodiments, the TROP2 binding trispecific protein comprises a trispecific antibody.

[0119] Multispecific TROP2 targeting proteins, such as trispecific proteins targeting TROP2 (also referred to herein as TROP2 targeting TriTAC proteins or molecules)

[0120] 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.

[0121] In further embodiments, the multispecific protein further comprises a domain that specifically binds to a T cell receptor (TCR). In some embodiments, the multispecific protein further comprises a domain that specifically binds to an alpha chain of a TCR. In some embodiments, the multispecific protein further comprises a domain that specifically binds to a beta chain of a TCR.

[0122] In certain embodiments, the CD3 binding domain of the multispecific protein not only exhibits efficient CD3 binding affinity for human CD3, but also shows superior cross-reactivity with the respective cynomolgus monkey CD3 proteins. In some examples, the CD3 binding domain of the multispecific protein cross-reacts with CD3 from cynomolgus monkey. In certain examples, the CD3 binding human: cynomolgus monkey K D (h K D :c K D ) ratio is 20: 1 to 1 :2.

[0123] In some embodiments, the CD3 binding domain of the multispecific protein is any domain that binds to CD3, including but not limited to a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or an antigen-binding fragment of a CD3-binding antibody such as a single domain antibody (sdAb), a Fab, a F(ab')2, and a 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 Fv (dsFv) fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some cases, it is beneficial for the CD3 binding domain to be derived from the same species in which the multispecific protein comprising a single domain serum albumin binding protein described herein will ultimately be used. For example, for human use, it can be beneficial for the CD3 binding domain of a multispecific protein comprising 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 a 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 a sequence that is at least about 75% to 100% identical to SEQ ID NO: 494, such as at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 494.

[0124] In some embodiments, the serum albumin binding domain (also referred to herein as a half-life extension domain) of a multispecific protein comprising a TROP2 binding protein as 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, 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 of a camelid-derived sdAb (VHH), or an antigen binding fragment of a HSA binding antibody, such as a Fab, F(ab’)2, and 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, and a dimer consisting of one heavy chain and one light chain, a peptide, a ligand, or a small molecule entity specific for serum albumin. In certain embodiments, the HSA binding domain is a single domain antibody. In other embodiments, the serum albumin binding domain is a peptide. In further embodiments, the serum albumin binding domain is a small molecule. In some embodiments, the serum albumin binding domain of a multispecific binding protein comprising a single chain variable fragment CD3 binding protein is expected to be fairly small, and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD. In certain examples, the serum albumin binding domain is 5 kD or less if it is a peptide or small molecule entity. Exemplary amino acid sequences of serum albumin binding domains of multispecific (e.g., trispecific) TROP2 targeting TriTAC proteins of the present disclosure are provided as SEQ ID NO: 493 or 566, or a sequence that is at least about 75% to 100% identical to SEQ ID NO: 493 or 566, such as at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 493 or 566.

[0125] The half-life extension domain of the multispecific binding protein comprising a single-chain variable fragment CD3 binding protein as described herein results in a change in the pharmacodynamics and pharmacokinetics of the single-chain variable fragment CD3 binding protein itself. As described above, the half-life extension domain extends the elimination half-life. The half-life extension domain also changes pharmacodynamic properties, including a change in tissue distribution, penetration, and diffusion of the single-chain variable fragment CD3 binding protein. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissue, and enhanced efficacy compared to the protein without the half-life extension domain. In one embodiment, the method of treatment effectively and efficiently utilizes a reduced amount of the multispecific binding protein comprising a single-chain variable fragment CD3 binding protein, resulting in reduced side effects, e.g., reduced cytotoxicity to off-target, e.g., non-tumor cells.

[0126] In addition, in some embodiments, the binding affinity of the half-life extension domain is selected so as to target a particular elimination half-life in a particular multispecific binding protein comprising a TROP2 binding protein as described herein. Thus, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a medium binding affinity. In yet other embodiments, the half-life extension domain has a low or minimal binding affinity. Exemplary binding affinities include a Kd of 10 nM or less (high), 10 nM to 100 nM (medium), and greater than 100 nM (low). D As described above, the binding affinity to serum albumin is determined by known methods, such as surface plasmon resonance (SPR).

[0127] In certain embodiments, a 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 extension domain; and (C) a third domain that is a TROP2-binding protein as described herein. In certain embodiments, the first domain comprises an scFv that specifically binds to CD3. For example, the CD3 is a human CD3 protein. In certain embodiments, the second domain comprises an sdAb that specifically binds to a large volume serum protein. In some cases, the large volume serum protein is an albumin, such as a serum albumin, such as human serum albumin. In some embodiments, domains (A), (B), and (C) are connected by linkers LI and L2 in either of the following orientations: H2N-(A)-LI-(C)-L2-(B)-COOH, H2N-(B)-LI-(A)-L2-(C)-COOH, H2N-(C)-LI-(B)-L2-(A)-COOH, H2N-(C)-LI-(A)-L2-(B)-COOH, H2N-(A)-LI-(B)-(C)-L2-COOH, or H2N-(B)-(C)-(A)-COOH.

[0128] In some embodiments, a TROP2-targeting multispecific protein of the present disclosure comprises an amino acid sequence that is at least about 70% to about 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 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%, to about 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-57 and 229-264.

[0129] conditionally active multispecific TROP2-targeting proteins, such as conditionally active TROP2-targeting tri-specific proteins (also referred to herein as TROP2-targeting ProTriTACs or pro-tri-specific proteins or molecules)

[0130] One embodiment of the present disclosure provides a conditionally active multispecific protein comprising a TROP2-binding domain disclosed herein (e.g., in some embodiments, the present disclosure provides a TROP2-targeting pro-tri-specific / ProTriTAC protein comprising a TROP2-binding domain of the present disclosure).

[0131] In some embodiments, the conditionally active multispecific protein further comprises a domain that specifically binds to CD3 and a binding moiety that specifically binds to a bulky serum protein, such as human serum albumin. In some embodiments, the binding moiety is capable of masking the interaction of the TROP2 binding domain or the CD3 binding domain with its target. In some embodiments, the binding moiety of the present disclosure comprises a masking moiety and a cleavable linker, such as a protease cleavable linker. Exemplary sequences of masking moieties within a binding moiety are provided in SEQ ID NOs: 550 and 558-560, or sequences comprising one or more substitutions relative to a sequence selected from 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 a sequence selected from SEQ ID NOs: 497-543, or a sequence comprising one or more substitutions relative to a sequence selected from 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 a sequence as set forth in SEQ ID NO: 544 or 545, or a sequence comprising one or more substitutions relative to SEQ ID NO: 544 or 545. In some embodiments, the binding moiety comprises a 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 a sequence selected from SEQ ID NO: 549. In some embodiments, the CD3 binding domain of a TROP2 ProTriTAC of the present disclosure comprises a 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.

[0132] In some embodiments, a TROP2-targeting ProTriTAC of the present disclosure comprises, from N-terminus to C-terminus, a binding moiety that is an anti-ALB domain comprising a non-CDR loop having a binding site for a CD3 binding domain (e.g., a CD3 binding domain having SEQ ID NO: 549 or a sequence at least about 75% identical thereto), a cleavable linker, a CD3 binding domain, and an anti-TROP2 binding domain on the C-terminus. In some embodiments, the TROP2 binding domain of the ProTriTAC is at least about 60%, about 61%, at least about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0133] 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 a 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 as set forth in SEQ ID NOs: 1-57 and 229-264, pharmaceutical compositions comprising the same, and methods of using the same to treat diseases such as neoplastic diseases as described herein.

[0134] In some embodiments, a TROP2-targeting ProTriTAC of the present disclosure comprises, as an uncleavable prodrug form, 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 a sequence selected from the group consisting of SEQ ID NOs: 1-57.

[0135] Exemplary sequences of active TROP2-targeting drugs (CT) as described herein are 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 about 100% identical to SEQ ID NOs: 1-57 and 229-264.

[0136] The binding moiety is capable of synergistically expanding the therapeutic window of the conditionally active TROP2-targeting pro-trispecific protein by both steric masking and specific masking. In some embodiments, the binding moiety combines steric masking (e.g., by binding to large volume serum albumin) and specific masking (e.g., by non-CDR loop binding to CDRs of the anti-TROP2 domain or anti-CD3 scFv domain). For example, the binding moiety masks or is capable of masking binding of the TROP2 binding domain (e.g., hiding the TROP2 binding domain and / or preventing premature binding) until the ability to activate and sequence “masking” can be tested using an assay that can utilize the activity of the masking sequence (e.g., a sequence selected from SEQ ID NOs: 550 and 558-560, or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 550 and 558-560) with and without masking. Briefly, to test masking, ProCAR constructs are constructed with or without the masking moiety in, for example, the CC’ loop. T cells are infected with lentivirus made from the constructs to produce CAR-T cells, which are subsequently stained with anti-FLAG antibody and TROP2-Fc, and a fluorescently labeled secondary antibody, and analyzed by flow cytometry. Staining dot plots are generated and compared. In some cases, the masking moiety is a masking peptide / moiety that is inserted into one or more non-CDR loops such that the binding moiety binds and inhibits the TROP2 antigen binding domain until the construct is delivered into the tumor microenvironment. In some cases, modifying the non-CDR loop within the binding moiety does not affect albumin binding. In some cases, a protease-cleavable linker is capable of activating the TROP2-targeting pro-trispecific protein in a single proteolytic event, thereby allowing more efficient conversion of the pro-trispecific molecule in the tumor microenvironment. Further, in some cases, tumor-associated proteolytic activation reveals an active T cell engager that has minimal extra-tumoral activity after activation. In some embodiments, the present disclosure provides a half-life extended T cell engager format (ProTriTAC) comprising the TROP2 binding moieties described herein, which in some cases represents a new and improved method for engineering conditionally active T cell engagers.

[0137] In some embodiments, the half-life of the conditionally active pro-trispecific form of the TROP2 binding domain in the systemic circulation is extended by using a binding moiety as described above, which acts as a safety switch that keeps the pro form of the multispecific protein in an inert state until it reaches the tumor microenvironment, where it is conditionally activated by cleavage of the linker and is able to bind its target antigen. In certain cases, the safety switch has several advantages; some examples 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 the undesired activated protein in the systemic circulation, thereby minimizing the spread of impurities related to chemistry, manufacturing, and control, such as pre-activated drug product, endogenous viruses, host cell proteins, DNA, leachables, antifoam agents, antibiotics, toxins, solvents, heavy metals; (iv) reducing the concentration of the undesired activated protein in the systemic circulation, thereby minimizing the spread of impurities, aggregates, breakdown products, product variants due to oxidation, deamidation, denaturation, loss of C-terminal Lys in MAbs; (v) preventing aberrant activation in the circulation; (vi) reducing toxicity associated with leakage of the activated species from diseased tissues or other pathophysiological conditions such as tumors, autoimmune diseases, inflammation, viral infections, tissue remodeling events (such as myocardial infarction, skin wound healing), or external insults (such as X-ray, CT scan, UV exposure); and (vii) reducing non-specific binding of the conditionally active TROP2 targeting protein. In addition, upon activation of the safety switch, or in other words, upon its cleavage, the conditionally active TROP2 targeting protein is dissociated from the safety switch, thereby providing an extended half-life and thus clearance from the circulation. For example, if the drug is inadvertently activated outside of the tumor environment, or leaks out of the tumor environment after activation, it can be rapidly cleared and is less likely to cause damage to normal tissues, thus reducing toxicity.

[0138] In some embodiments, the conditionally active multispecific TROP2 binding proteins described herein have an improved therapeutic index compared to TROP2 binding proteins that do not have conditionally activity but have constitutive activity. For example, in some embodiments, the TROP2 ProTriTAC has an increased therapeutic index compared to a TROP2 TriTAC. In some embodiments, the increase in therapeutic index is at least about 2-fold to about 1000-fold, such as about 4-fold to about 800-fold, about 6-fold to about 800-fold, about 6-fold to about 600-fold, about 10-fold to about 400-fold, about 20-fold to about 200-fold, about 30-fold to about 150-fold, about 50-fold to about 100-fold. In some embodiments, the increase in therapeutic index is due to the conjugation of the TROP2 binding domain with a binding moiety as described above employing a non-CDR loop and a cleavable linker.

[0139] In some embodiments, the "therapeutic index" (TI) (also known as the "therapeutic window") is a comparison of the minimum amount of a therapeutic agent (e.g., TROP2 TriTAC, TROP2 ProTriTAC, TROP2 CAR, TROP2 ProCAR) that results in a therapeutic effect (e.g., increased survival rate of a patient having a TROP2-expressing cancer treated with the therapeutic agent described above) to the minimum tolerated dose. In some cases, an improvement in the therapeutic index is manifested as an improvement in EC 50 of T cell-mediated killing of cancer cells compared to TROP2 ProTriTAC.

[0140] In some embodiments, the conditionally active TROP2-targeting protein format confers a significantly longer serum half-life to the TROP2-binding domain and reduces the likelihood of its undesired activation in circulation, resulting in a "biologically better" format.

[0141] The binding moiety as described herein comprises at least one non-CDR loop. In some embodiments, the non-CDR loop provides a binding site for the binding of 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, through steric hindrance, through specific intermolecular interactions, or a combination of both.

[0142] In some embodiments, the binding moieties described herein further comprise complementarity determining regions (CDRs), e.g., CDRs specific for binding to 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). The Ig can be of any class or subclass (IgGl, 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 loops make up the “complementarity determining regions” (CDRs) that determine the antigen-binding specificity of the molecule. IgG molecules comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), which are highly variable in sequence and / or involved in antigen recognition and / or form the structurally defined loops, wherein the more conserved regions called framework regions (FRs) are interspersed. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0143] In some embodiments, the binding moieties of the present disclosure are heavy chain-only antibodies. In some embodiments, the variable domain of a heavy chain-only antibody has several beta strands arranged in two folds. The variable domain of a heavy chain-only antibody comprises three hypervariable loops, or complementarity determining regions (CDRs), and framework regions FR1, FR2, FR3, and FR4. The three CDRs (CDR1, CDR2, CDR3) of the variable domain are clustered at one end of the beta barrel. The CDRs are loops connecting the beta strands B-C, C’-C”, and F-G of the immunoglobulin fold, while the bottom loops connecting the beta strands AB, CC’, C”-D, and E-F of the immunoglobulin fold, and the top loop connecting the D-E strands of the immunoglobulin fold are non-CDR loops.

[0144] In some embodiments of the disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of a "non-CDR loop" of a binding moiety described herein, such as a binding moiety that is a heavy chain-only antibody. In some embodiments of the disclosure, at least some of the amino acid residues of constant domain CH1, CH2, or CH3 are part of a "non-CDR loop" of a binding moiety described herein. In some embodiments, the non-CDR loop comprises 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; the DE, BD, GF, A (A1A2)B, and EF loops of an I (intermediate)-set domain of an Ig or Ig-like molecule.

[0145] Within the variable domain, the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are believed to be the scaffolding for the CDRs. However, in some cases, some FR residues play an important role in antigen recognition and binding. Framework residues that affect Ag binding are divided into two categories. The first are FR residues that contact the antigen and are thus part of the binding site, and some of these residues are in sequence proximity to the CDRs. Other residues are residues that are in sequence distance from the CDRs but are in close proximity to them in the 3-D structure of the molecule, e.g., loops in the heavy chain.

[0146] In some embodiments, the non-CDR loops are modified to generate an antigen binding site specific for a large volume 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.

[0147] It is contemplated that various techniques can be used to modify the non-CDR loops, such as site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid that is foreign to the non-CDR loop amino acid sequence, amino acid substitution. In some examples, an antigenic peptide is inserted into a non-CDR loop. In some examples, a non-CDR loop is replaced with an antigenic peptide. In some cases, the modification to generate an antigen binding site is in only one non-CDR loop. In other cases, more than one non-CDR loop is modified. For example, the modification is in any one of the non-CDR loops, i.e., AB, CC', C"D, EF, and D-E. In some cases, the modification is in the DE loop. In other cases, the modification is in all four of the AB, CC', C"D, E-F loops.

[0148] In certain examples, a binding moiety described herein binds to a TROP2 binding domain via its AB, CC, C”D, or EF loop, and to a large volume serum protein such as albumin via its B-C, C’-C”, or F-G loop. In certain examples, the binding moiety binds to a TROP2 binding domain via its AB, CC, C”D, and EF loop, and to a large volume serum protein such as albumin via its BC, C’C”, and FG loop. In certain examples, the binding moiety binds to a TROP2 binding domain via one or more of its AB, CC, C”D, and E-F loop, and to a large volume serum protein such as albumin via one or more of its BC, C’C”, and FG loop. In certain examples, the binding moiety binds to a large volume serum protein such as albumin via its AB, CC, C”D, or EF loop, and to a TROP2 binding domain via its BC, C’C”, or FG loop. In certain examples, the binding moiety binds to a large volume serum protein such as albumin via its AB, CC, C”D, and EF loop, and to a TROP2 binding domain via its BC, C’C”, and FG loop. In certain examples, a binding moiety of the first embodiment binds to a large volume serum protein such as albumin via one or more of its AB, CC, C”D, and E-F loop, and to a TROP2 binding protein via one or more of its BC, C’C”, and FG loop. In certain examples, a binding moiety described herein binds to a CD3 binding domain via its AB, CC, C”D, or EF loop, and to a large volume serum protein such as albumin via its B-C, C’-C”, or F-G loop. In certain examples, a binding moiety described herein binds to a large volume serum protein such as albumin via its AB, CC, C”D, or EF loop, and to a CD3 binding domain via its B-C, C’-C”, or F-G loop. In certain examples, a binding moiety described herein binds to a CD3 binding domain via its AB, CC, C”D, or EF loop, and to a TROP2 binding domain via its B-C, C’-C”, or F-G loop. In certain examples, a binding moiety described herein binds to a TROP2 binding domain via its AB, CC, C”D, or EF loop, and to a CD3 binding domain via its B-C, C’-C”, or F-G loop.

[0149] Large volume serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAbs, scFvs, Fabs, VHHs, fibronectin type III domains, immunoglobulin-like scaffolds (as set forth in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPins, cysteine knot peptides, lipocalins, three-helix bundle scaffolds, protein G-related albumin binding modules, or DNA or RNA aptamer scaffolds.

[0150] In some cases, the binding moiety comprises a binding site for a large volume serum protein. In some embodiments, the CDRs within the binding moiety provide a binding site for a large volume serum protein. In some examples, the large volume serum protein is a globulin, albumin, transferrin, IgGl, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the binding moiety comprises a binding site for an immunoglobulin light chain. In some embodiments, the CDRs provide a binding site for an immunoglobulin light chain. In some examples, the immunoglobulin light chain is an Ig kappa-free light chain or an Ig lambda-free light chain.

[0151] In further embodiments, the binding moiety is any kind of polypeptide. For example, in certain cases, the binding moiety is a natural peptide, a synthetic peptide, or a fibronectin scaffold or an engineered large volume 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, humanized antibodies. In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a soluble TCR fragment, a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived nanobody (VHH). In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, affitins, alphabodies, avimers, fynomers, kunitz domain peptides, and monobodies.

[0152] It is contemplated herein that the binding moieties described herein comprise at least one cleavable linker. In one aspect, the cleavable linker comprises a polypeptide having a sequence that is recognized and cleaved in a sequence-specific manner. In some cases, the binding moieties described herein comprise a protease-cleavable linker that is recognized and cleaved in a sequence-specific manner. In some embodiments, the protease-cleavable linker is recognized in a sequence-specific manner by a matrix metalloproteinase (MMP), such as MMP9. In some cases, the protease-cleavable linker is recognized by MMP9 comprises a polypeptide having the amino acid sequence PR(S / T)(L / I)(S / T). In some cases, the protease-cleavable linker recognized by MMP9 comprises a polypeptide having the amino acid sequence LEATA. In some cases, the protease-cleavable linker is recognized in a sequence-specific manner by MMP11.

[0153] A protease is a protein that cleaves proteins in some cases in a sequence-specific manner. Proteases include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hKl, hKlO, hKl 5, fibrinolysin, collagenase, collagenase IV, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidin, bromelain, calpain, caspases, caspase-3, Mir 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, malarial aspartic proteases, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteinases (MMPs), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enteropeptidase, prostate specific target (PSA, hK3), interleukin-1 beta converting enzyme, thrombin, FAP (FAP-alpha), dipeptidyl peptidases, and dipeptidyl peptidase IV (DPPIV / CD26).

[0154] Table 1: Exemplary proteases and protease recognition sequences

[0155]

[0156]

[0157] Proteases are known to be secreted by certain diseased cells and tissues, such as tumor or cancer cells, thereby creating a protease-rich microenvironment or protease- abundant 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. Protease-secreting cells surrounding a tumor 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 are present in a subject’s blood, such as proteases that target amino acid sequences found in microbial peptides. This feature allows a targeted therapeutic, such as an antigen-binding protein, to have additional specificity because T cells will not be bound by the antigen-binding protein except in the protease-rich microenvironment of the target cell or tissue. Other non-limiting examples of linkers that can be used in the constructs described herein are provided in the sequence listing below.

[0158] Integration into a chimeric antigen receptor (CAR)

[0159] In certain examples, TROP2 binding proteins of the present disclosure can be incorporated into a chimeric antigen receptor (CAR) or ProCAR. Engineered immune effector cells, such as T cells or NK cells, can be used to express a CAR that includes an anti-TROP2 binding protein containing, for example, an anti-TROP2 single domain antibody described herein. In one embodiment, a CAR including a TROP2 binding protein described herein is linked via a hinge region to a transmembrane domain and further linked to a costimulatory domain, for example, a functional signaling domain obtained from OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB. In some embodiments, the CAR further comprises a sequence encoding an intracellular signaling domain such as 4-1BB and / or CD3 zeta. Exemplary sequences of ProCARs comprising a TROP2 binding domain are provided in SEQ ID NOS: 1-57, or a sequence that is at least about 75% to 100% identical, e.g., about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to a sequence selected from SEQ ID NOS: 1-57.

[0160] The conditionally active receptors described herein comprise at least one binding moiety comprising a non-CDR loop. In one aspect, the binding moiety masks binding of the TROP2 binding domain until activation. The cleavable linker, for example, comprises a protease cleavage site or a pH-dependent cleavage site. In certain cases, the cleavable linker is cleaved only in the tumor microenvironment. Thus, in some examples, the binding moiety linked to the cleavable linker and further bound to the TROP2 binding domain maintains the TROP2 binding domain in an inert state in circulation until the cleavable linker is cleaved in the tumor microenvironment. In some embodiments, the binding moiety is bound to the TROP2 binding domain. In some embodiments, the non-CDR loop provides a binding site for the binding of the moiety to the TROP2 binding domain. In some embodiments, the binding moiety masks binding of the TROP2 binding domain to its target antigen, for example, by steric blocking, by specific intramolecular interactions, for example, within different domains of the polypeptide comprising the binding moiety. In some embodiments, the binding moiety further comprises a complementarity determining region (CDR).

[0161] In some cases, as described above in the context of the conditionally active multispecific TROP targeting proteins of the present disclosure, the binding moiety of the CAR or proCAR described herein is a domain derived from an immunoglobulin molecule (Ig molecule).

[0162] 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 further has a modified non-CDR loop that can inhibit binding of the TROP2 binding domain to its target. Upon cleavage at the cleavable linker, the binding moiety can be released, enabling binding of the TROP2 binding domain.

[0163] In some embodiments, the active receptor (inactive ProCAR) comprises a TROP2 binding domain (anti-tumor target sdAb or scFv) linked to a binding moiety (anti-Target 2 sdAb) by a linker comprising a protease cleavage site. The binding moiety comprises a masking peptide / moiety inserted into one or more non-CDR loops, such that the binding moiety binds and inhibits the TROP2 antigen binding domain. In some embodiments, the binding moiety has specificity for a given target, as further described elsewhere herein. The receptor further comprises a transmembrane domain and an intracellular signaling domain. The receptor is provided in a T cell (CAR-T). Upon exposure to a tumor environment, the protease cleavage site is cleaved by a tumor-associated protease, thereby activating the receptor, resulting in an active receptor that does not comprise the binding moiety. The receptor now comprises an active antigen binding domain. When the receptor is internalized by a cell, a new receptor is produced that comprises the binding moiety and is inactive.

[0164] In some embodiments, the cleavable linker of the binding moiety comprises a protease cleavable site similar to that described above with respect to the conditionally active multi-specific proteins comprising a TROP2 binding domain of the disclosure. For example, in some embodiments, the cleavable linker comprises a sequence selected from the linker sequences provided in the sequence listing.

[0165] Transmembrane domain

[0166] The conditionally active chimeric antigen receptors, T cell receptor fusion proteins, and T cell receptors of the disclosure comprise a transmembrane domain for insertion into the membrane of a eukaryotic cell. 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.

[0167] Any transmembrane (TM) domain that provides insertion of the 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. Other non-limiting examples of suitable TM sequences include, but are not limited to: 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).

[0168] Hinge region

[0169] 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 region”), wherein the hinge region is 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 hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0170] The hinge region can have a length of from about 4 amino acids to about 50 amino acids (aa), e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.

[0171] Suitable spacers can be readily selected and can be any of a number of suitable lengths, e.g., 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0172] Exemplary spacers include glycine polymers (G)n, glycine-serine polymers (including, for example, (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 thus can serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more phi-psi space accessible than alanine, and is less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Exemplary spacers comprise an amino acid sequence, 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.

[0173] Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT; CPPC; CPEPKSCDTPPPCPR; (see, e.g., Glaser et al., (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT; KSCDKTHTCP; KCCVDCP; KYGPPCP; EPKSCDKTHTCPPCP; human IgGl hinge; ERKCCVECPPCP; human IgG2 hinge; ELKTPLGDTTHTCPRCP; human IgG3 hinge; SPNMVPHAHHAQ; human IgG4 hinge); and the like.

[0174] In some embodiments, the hinge region comprises the amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4 hinge region. The hinge region can 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 a human IgGl hinge can be substituted with Tyr, such that the hinge region comprises the sequence EPKSCDKTYTCPPCP; see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891.

[0175] 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.

[0176] Conditionally active chimeric antigen receptors

[0177] 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 CARs of the present disclosure comprise multiple domains, including a binding moiety, a target antigen binding domain that binds 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.

[0178] In some embodiments, the conditionally active chimeric antigen receptor further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein including, but not limited to, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and an amino acid sequence thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications.

[0179] In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, 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, a functional fragment thereof, and an amino acid sequence thereof having at least one but not more than 20 modifications.

[0180] In one aspect, the present disclosure provides a cell (e.g., a T cell) engineered to express a CAR. In one aspect, the cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the CAR. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cell can transiently express the CAR.

[0181] Conditionally active T cell receptor fusion proteins

[0182] In one embodiment, the present disclosure provides a conditionally active T cell receptor fusion protein. As used herein, a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from various polypeptides comprising a TCR that is generally capable of i) binding to a surface antigen on a target cell, and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on a T cell.

[0183] 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, a transmembrane domain, and a T cell receptor intracellular domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein including, but not limited to, a TCR a chain, a TCR b 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 but not more than 20, 10, or 5 modifications.

[0184] In some embodiments, the T cell receptor endodomain comprises a stimulatory domain. The stimulatory domain can be 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 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 modifications but not more than 20, 10, or 5 modifications.

[0185] In some embodiments, the conditionally active TFP further comprises a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein, including but not limited to OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one, two, or three modifications but not more than 20, 10, or 5 modifications.

[0186] In some embodiments, the TROP2 binding domain is linked to the T cell receptor ectodomain by a linker sequence. In some cases, the encoded linker sequence comprises (G4S)n, where n = 1 to 4 (SEQ ID NO: 531). In some cases, the encoded linker sequence comprises a long linker (LL) sequence. In some cases, the encoded long linker sequence comprises (G4S)n, where n = 2 to 4 (SEQ ID NO: 532). In some cases, the encoded linker sequence comprises a short linker (SL) sequence. In some cases, the encoded short linker sequence comprises (G4S)n, where n = 1 to 3 (SEQ ID NO: 533).

[0187] In one aspect, the present disclosure provides a cell (e.g., a T cell) engineered to express a conditionally active T cell receptor fusion protein (TFP). In one aspect, the cell is transformed with a conditionally active TFP, and the conditionally active TFP is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding a conditionally active TFP. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the conditionally active TFP. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TFP. In some such embodiments, the cell can transiently express the conditionally active TFP.

[0188] Conditionally active T cell receptor

[0189] 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 receptors of the present disclosure comprise a binding moiety. In some embodiments, the binding moiety is attached to a T cell receptor subunit, including but not limited to an alpha subunit, a beta subunit, or a combination thereof.

[0190] In some embodiments, the binding moiety is capable of masking the binding of the T cell receptor to its target. In some embodiments, the binding moiety binds to the T cell receptor. In some embodiments, a non-CDR loop provides a binding site for the moiety to bind to the T cell receptor. In some embodiments, a non-CDR loop provides a binding site specific for a T cell receptor alpha, a T cell receptor beta, or a combination thereof. In some embodiments, the binding moiety masks the binding of the T cell receptor to its target, e.g., by steric hindrance, by specific intermolecular interactions.

[0191] In one aspect, the present disclosure provides a cell (e.g., a T cell) engineered to express a conditionally active T cell receptor (TCR). In one aspect, the cell is transformed with a conditionally active TCR, and the conditionally active TCR is expressed on the cell surface. In some embodiments, the cell (e.g., a T cell) is transduced with a viral vector encoding a conditionally active TCR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express the conditionally active TCR. In another embodiment, the cell (e.g., a T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a conditionally active TCR. In some such embodiments, the cell can transiently express the conditionally active TCR.

[0192] Cell

[0193] In one embodiment, the disclosure provides a cell comprising a chimeric antigen receptor or conditionally active chimeric antigen receptor, a conditionally active T cell receptor fusion protein, or a conditionally active T cell receptor of the disclosure. The cell can be a mammalian cell.

[0194] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL 9618, CCL 61, CRL 9096), 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. CRL 1721), COS cells, COS-7 cells (ATCC No. CRL 1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL 1573), HL HepG2 cells, HuT-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0195] In some cases, the cell is not an immortalized cell line, but is a cell obtained from an individual (e.g., a primary cell). For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

[0196] In recent studies, CAR constructs have been used to guide natural killer (NK) cell activity, as reviewed by Hermanson & Kaufman (2015, Front Immunol 6: 195) and Carlsten & Childs (2015, Front Immunol 6: 266). Similar to T cells, NK cells can be transfected with CAR expression constructs and used to induce an immune response. Since NK cells do not require HLA matching, they can be used as allogeneic effector cells (Harmanson & Kaufman, 2015). In addition, peripheral blood NK cells (PB-NK) for treatment can be isolated from donors by simple blood draws. The CAR construct used may include elements similar to those used to prepare CAR-T cells.

[0197] Thus, in some embodiments, the present disclosure provides cells comprising NK cells comprising a chimeric antigen receptor, a conditionally active chimeric antigen receptor, a conditionally active T cell receptor fusion protein, or a conditionally active T cell receptor of the present disclosure.

[0198] As discussed above in the context of conditionally active TROP2 binding proteins (e.g., TROP2 ProTriTAC), in some embodiments, compared to comprising the same TROP2 binding domain as the conditionally active variant, but being a constitutively active rather than a conditionally active chimeric antigen receptor, the conditionally active chimeric antigen receptor described herein has an improved therapeutic index. For example, in some embodiments, TROP2 ProCAR has a therapeutic index increased than TROP2 CAR. In some embodiments, the increase in therapeutic index is at least about 2 times to about 1000 times, for example, about 4 times to about 800 times, about 6 times to about 800 times, about 6 times to about 600 times, about 10 times to about 400 times, about 20 times to about 200 times, about 30 times to about 150 times, about 50 times to about 100 times. In some embodiments, the increase in therapeutic index is attributed to the conjugation of the TROP2 binding domain to the binding portion as described above using a non-CDR loop and a cleavable linker.

[0199] Methods for generating cells containing conditionally active receptors

[0200] The present disclosure provides a method of producing a cell comprising a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor. The method generally involves genetically modifying a mammalian cell with an expression vector or RNA (e.g., in vitro transcribed RNA) comprising a nucleotide sequence encoding a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor of the present disclosure. The genetic modification can be performed in vivo, in vitro, or ex vivo. The cell can be, for example, an immune cell (e.g., a T lymphocyte or NK cell), a stem cell, or a progenitor cell.

[0201] In some cases, the genetic modification is performed ex vivo. For example, a T lymphocyte, stem cell, or NK cell is obtained from an individual; and the cell obtained from the individual is genetically modified to express a conditionally active chimeric antigen receptor, T cell receptor fusion protein, or T cell receptor of the present disclosure.

[0202] Sources of T cells

[0203] In some embodiments, the source of T cells is obtained from a subject. The term "subject" as used throughout the present disclosure is intended to include living organisms (e.g., mammals) 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 variety 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 a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell lines available in the art can be used. In certain embodiments of the present disclosure, any technique known to one of skill in the art can be used, such as FICOLL® separation, density gradient centrifugation, or magnetic cell separation. TMIsolation. T cells are obtained from a blood unit collected from a subject. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In one embodiment of the disclosure, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, and can lack magnesium or can lack many, if not all, divalent cations. An initial activation step in the absence of calcium can result in amplified activation. One of ordinary skill in the art will readily appreciate that the washing step can be accomplished by methods known to those of skill in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics CellSaver 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 buffers. Alternatively, the unwanted components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0204] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting mononuclear cells (e.g., by PERCOLL TM gradient centrifugation or by counterflow centrifugal elutriation). Specific T cell subpopulations, 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, CD3+T cells are isolated by positive selection using anti-CD3 conjugated beads (e.g., DYNABEADS® M-450 CD3, ThermoFisher Scientific, Waltham, MA). In another embodiment, CD4+T cells are isolated by negative selection using anti-CD8 conjugated beads (e.g., DYNABEADS® M-450 CD8, ThermoFisher Scientific, Waltham, MA). In another embodiment, CD8+T cells are isolated by negative selection using anti-CD4 conjugated beads (e.g., DYNABEADS® M-450 CD4, ThermoFisher Scientific, Waltham, MA). In another embodiment, CD45RA+T cells are isolated by negative selection using anti-CD45RO conjugated beads (e.g., DYNABEADS® M-450 CD45RO, ThermoFisher Scientific, Waltham, MA). In another embodiment, CD45RO+T cells are isolated by negative selection using anti-CD45RA conjugated beads (e.g., DYNABEADS® M-450 CD45RA, ThermoFisher Scientific, Waltham, MA). T cells are isolated by incubating the M-450 CD3 / CD28 T) together for a period of time sufficient for positive selection of the desired T cells. In one embodiment, the period of time is about 30 minutes. In further embodiments, the period of time ranges from 30 minutes to 36 hours or more and all integral values therebetween. In further embodiments, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the period of time is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. Longer incubation times can be used to isolate T cells in any situation where there are fewer T cells compared to other cell types, such as in the isolation of tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Further, using longer incubation times can increase the efficiency of capturing CD8+ T cells. Thus, by simply shortening or lengthening the time allowed for T cells to bind to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), one can preferentially select for or exclude certain T cell subpopulations at the beginning of the culture or at other points in the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, one can preferentially select for or exclude certain T cell subpopulations at the beginning of the culture or at other desired points in time. Multiple rounds of selection can also be used in the context of the present disclosure. In certain embodiments, it can be desirable to perform the selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

[0205] Enrichment of the T cell population by negative selection can be accomplished with a combination of antibodies to the unique surface markers of the cells being negatively selected. One method is through negative magnetic immunoadherence or flow cytometry cell sorting and / or selection using a cocktail of monoclonal antibodies to cell surface markers present on the cells being negatively selected. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In certain embodiments, it can be desirable to enrich for or positively select for regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

[0206] In one embodiment, a population of T cells can be selected that express 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, such as other cytokines. Methods can be determined to screen for expression by the cells, for example, by the methods described in PCT Publication WO 2013 / 126712.

[0207] To isolate the desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles, such as beads) can be varied. In certain embodiments, it can be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In further embodiments, a concentration of greater than 100 million cells / ml is used. In further embodiments, a cell concentration of 100, 150, 200, 250, 300, 350, 400, 450, or 500 million cells / ml is used. In one embodiment, a cell concentration of 750, 800, 850, 900, 950, or 1 billion cells / ml is used. In further embodiments, a concentration of 1.25 or 1.5 billion cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, the use of high cell concentrations allows for more efficient capture of cells that can express the target antigen of interest weakly (such as CD28 negative T cells) or from samples where many tumor cells are present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells can have therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0208] In another embodiment, it can be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles, such as beads), the interaction between the particles and cells is minimized. This selects for cells that express large amounts of the desired antigen to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells at dilute concentrations. In one embodiment, a concentration of 5 x 10e6 / ml of cells is used. In other embodiments, the concentration used can be about 1 x 10 5 / ml to 1 x 10 6 / ml and any integer value therebetween. In other embodiments, the cells can be incubated on a rotator at varying speeds for varying lengths of time at 2-10 °C or room temperature.

[0209] T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent mononuclear cells from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and would be useful in this context, one approach involves the use of PBS with 20% DMSO and 8% human serum albumin, or a media with 10% Dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% Dextran 40, and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, and then the cells are frozen at a rate of 1 °C per minute to -80 °C and stored in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used as well as uncontrolled snap freezing at -20 °C or in liquid nitrogen. In certain embodiments, the cryopreserved cells are thawed and washed as described herein and allowed to rest at room temperature for 1 hour prior to activation using the methods of the present disclosure.

[0210] It is also contemplated in the context of the present disclosure to collect a blood sample or blood component isolate from a subject at a time period prior to which expanded cells as described herein can be needed. Thus, a source of cells to be expanded can be collected at any necessary time point, and the desired cells, such as T cells, isolated and frozen for later use in a T cell therapy for any number of diseases or conditions that would benefit from a T cell therapy, such as the diseases or conditions described herein. In one embodiment, the blood sample or blood component isolate is taken from a generally healthy subject. In certain embodiments, the blood sample or blood component isolate is taken 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, the T cells can be expanded, frozen and used later. In certain embodiments, the sample is collected from the patient at a short time after diagnosis of a particular disease as described herein, prior to any treatment. In further embodiments, the cells are isolated from a blood sample or blood component isolate of a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, anti-viral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies, or other immune depleting agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

[0211] In further embodiments of the present disclosure, T cells are obtained directly from a patient after a treatment that renders the patient with functional T cells. In this regard, it has been observed that after certain cancer treatments, particularly treatments with agents that compromise the immune system, the quality of the T cells obtained can be optimal or improved shortly after the treatment and during a period in which the patient would normally be recovering from the treatment, as it has the capacity to expand ex vivo. Likewise, after ex vivo manipulation using the methods described herein, these cells can be in a preferred state for engraftment and enhanced expansion in vivo. Thus, it is contemplated in the context of the present disclosure to collect blood cells, including T cells, dendritic cells or other cells of the hematopoietic lineage during this recovery period. Further, in certain embodiments, mobilization (e.g., with GM-CSF) and conditioning regimens can be used to create conditions in a subject in which re-population, re-circulation, regeneration and / or expansion of particular cell types is favored, particularly within a defined time window after a therapy. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0212] Activation and expansion of T cells

[0213] T cells can be activated and expanded generally using methods as described in, e.g., 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 Al.

[0214] Generally, the T cells of the present disclosure can be expanded by contact with a surface to which is attached reagents that stimulate CD3 / TCR complex associated signals and ligands that stimulate costimulatory molecules on the surface of the T cells. In particular, a population of T cells can be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen binding fragment thereof or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) and a calcium ionophore. To costimulate accessory molecules on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate proliferation of the T cells. Anti-CD3 and anti-CD28 antibodies are employed to stimulate proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used as well as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999).

[0215] In certain embodiments, the primary stimulatory signal and the costimulatory signal for a T cell can be provided by different regimens. For example, the agents providing each signal can be in solution or coupled to a surface. When coupled to a surface, the agents can be coupled to the same surface (i.e., an "cis" arrangement) or to different surfaces (i.e., a "trans" arrangement). Alternatively, one agent can be coupled to a surface while the other agent is in solution. In one embodiment, the agent providing the costimulatory signal is bound to the surface of a cell, while the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In one embodiment, the agents 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 agent that will bind to the agent. In this regard, see, e.g., artificial antigen presenting cells (aAPCs) contemplated for use in activating and expanding the T cells of the present disclosure in U.S. Patent Application Publication Nos. US20040101519 Al and US20060034810 Al.

[0216] In one embodiment, the two agents are immobilized on beads (either on the same bead, i.e., "cis," or on different beads, i.e., "trans"). For example, the agent that provides the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent that provides the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and the two agents are co-immobilized on the same bead in equal molecular amounts. In one embodiment, each antibody bound to the bead for CD4+ T cell expansion and T cell growth uses a 1 : 1 ratio. In certain embodiments of the disclosure, a ratio of anti-CD3 antibody:anti-CD28 antibody is used for the bead-bound antibodies such that an increase in T cell expansion is observed compared to expansion observed using a 1 : 1 ratio. In a particular embodiment, an increase of about 1-fold to about 3-fold is observed compared to expansion observed using a 1 : 1 ratio. In one embodiment, the ratio of CD3 antibody:CD28 antibody bound to the bead 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 particle than anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the disclosure, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the bead is greater than 2: 1. In a particular embodiment, the antibodies bound to the bead use a 1 : 100 CD3:CD28 ratio. In another embodiment, the antibodies bound to the bead use a 1 : 75 CD3:CD28 ratio. In a further embodiment, the antibodies bound to the bead use a 1 : 50 CD3:CD28 ratio. In one embodiment, the antibodies bound to the bead use a 1 : 30 CD3:CD28 ratio. In one embodiment, the antibodies bound to the bead use a 1 : 10 CD3:CD28 ratio. In one embodiment, the antibodies bound to the bead use a 1 : 3 CD3:CD28 ratio. In yet another embodiment, the antibodies bound to the bead use a 3 : 1 CD3:CD28 ratio.

[0217] Particle to cell ratios from 1 :500 to 500: 1 and any integer value therebetween can be used to stimulate T cells or other target cells. One of ordinary skill in the art can readily understand that the particle to cell ratio can depend on the size of the particles relative to the target cells. For example, smaller beads can only bind a small number of cells, while larger beads can bind many cells. In certain embodiments, the cell to particle ratio ranges from 1 : 100 to 100: 1 and any integer value therebetween, and in further embodiments, the ratio includes 1 :9 to 9: 1 and any integer value therebetween, can also be used to stimulate T cells. As described above, the ratio of anti-CD3 and anti-CD28 coupled particles to T cells that results in T cell stimulation can vary, but certain values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1, with one preferred ratio being at least 1 : 1 T cells to particles. In one embodiment, a particle to cell ratio of 1 : 1 or less is used. In a particular embodiment, the particle to cell ratio is 1 :5. In further embodiments, the particle to cell ratio can vary depending on the number of days of stimulation. For example, in one embodiment, the particle to cell ratio is 1 : 1 to 10: 1 on the first day, and additional particles are added to the cells every day or every other day for up to 10 days, with a final ratio of 1 : 1 to 1 : 10 (based on cell count on the day of addition). In a particular embodiment, the particle to cell ratio is 1 : 1 on the first day of stimulation, and is adjusted to 1 :5 on the third and fifth days of stimulation. In one embodiment, particles are added every day or every other day until the final ratio on the first day is 1 : 1, and the final ratio on the third and fifth days of stimulation is 1 :5. In one embodiment, the particle to cell ratio is 2: 1 on the first day of stimulation, and is adjusted to 1 : 10 on the third and fifth days of stimulation. In one embodiment, particles are added every day or every other day until the final ratio on the first day is 1 : 1, and the final ratio on the third and fifth days of stimulation is 1 : 10. Those of skill in the art will appreciate that a variety of other ratios can be suitable for the present disclosure. In particular, the ratio will vary depending on the particle size and cell size and type.

[0218] In further embodiments of the present disclosure, the cells, such as T cells, are combined with the agent-coated beads, and then the beads and cells are separated and the cells are subsequently cultured. In alternative embodiments, the agent-coated beads and cells are not separated prior to culture, but are cultured together. In further embodiments, the beads and cells are first concentrated by the application of a force, such as a magnetic force, resulting in an increase in the ligation of cell surface markers, thereby inducing cell stimulation.

[0219] For example, cell surface proteins can be linked by contacting T cells with paramagnetic beads (3×28 beads) to which anti-CD3 and anti-CD28 are attached. 4 to 10 9 T cells) and beads (e.g., a 1:1 ratio M-450CD3 / CD28 T paramagnetic beads) are mixed in a buffer such as PBS (without divalent cations such as calcium and magnesium). Again, one of ordinary skill in the art will readily appreciate that any cell concentration can be used. For example, target cells may be very rare in a sample and comprise only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Therefore, any cell number is within the scope of the present disclosure. In certain embodiments, it may be necessary 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 the particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In one embodiment, greater than 100 million cells / ml is used. In further embodiments, a cell concentration of 10 million, 15 million, 20 million, 25 million, 30 million, 350 million, 40 million, 45 million, or 50 million cells / ml is used. In one embodiment, a cell concentration of 75,000,000, 80,000, 85,000,000, 90,000, 95,000,000 or 100,000,000 cells / ml is used. In a further embodiment, a concentration of 125,000,000 or 150,000,000 cells / ml may be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, using high cell concentrations allows more effective capture of cells that may weakly express the target antigen of interest, such as CD28 negative T cells. Such a cell population may have therapeutic value and needs to be obtained in certain embodiments. For example, using high concentrations of cells allows more effective selection of CD8+ T cells that typically have weaker CD28 expression.

[0220] In one embodiment of the disclosure, the mixture can be cultured for a number of hours (about 3 hours) to about 14 days or any integral hour value therebetween. In one embodiment, the mixture can be cultured for 21 days. In one embodiment of the 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. It can also be desirable to have several stimulation cycles, such that the culture time of the T cells can be 60 days or more. Conditions suitable for T cell culture include an appropriate medium (e.g., basal essential medium or RPMI medium 1640 or X-vivo 15, (Lonza)) that can contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with the addition of amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in an amount sufficient for T cell growth and expansion. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cell cultures to be infused into a subject. The target cells are maintained under conditions required to support growth, e.g., appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0221] T cells that have been exposed to different stimulation times can exhibit different properties. For example, a typical blood or blood component isolated peripheral blood mononuclear cell product has a population of helper T cells (TH, CD4+) that is greater than a population of cytotoxic T cells or suppressor T cells (TC, CD8+). Expansion of T cells by stimulation of CD3 and CD28 receptors ex vivo produces a population of T cells that is primarily composed of TH cells until about day 8-9, and after about day 8-9, the population of T cells includes an increasing population of TC cells.

[0222] TROP2 binding protein modification

[0223] The TROP2 binding proteins described herein, including TROP2 binding domains (e.g., TROP2 binding sdAbs of the present disclosure) and TROP2 targeting multispecific proteins (e.g., TROP2 targeting trispecific or pre-trispecific proteins described herein), include derivatives or analogs in which (i) an amino acid is replaced by an amino acid residue that is not encoded by the genetic code, (ii) a mature polypeptide is fused with another compound, such as a polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader sequence or a secretion sequence or a sequence for purification of the protein.

[0224] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a 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 a phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.

[0225] Modifications are made at any of the positions in the TROP2 binding proteins described herein, including the peptide backbone, the amino acids side chains, and the amino or carboxyl termini. Certain common peptide modifications that can be made to TROP2 binding proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, capping of amino or carboxyl or both amino and carboxyl of polypeptides, and ADP-ribosylation.

[0226] In some embodiments, the derivatives of TROP2 binding proteins as described herein include immunoreactivity modulator derivatives and antigen binding molecules comprising one or more modifications.

[0227] 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 one target molecule or a particular location or site on a target molecule. When an antibody is monovalent, each binding site of the molecule will specifically bind one antigenic location or epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site can specifically bind the same or different molecules (e.g., can bind different ligands or different antigens, or different epitopes or locations on the same antigen).

[0228] In some embodiments, the above-mentioned TROP2 binding proteins are fused to an Fc region from any species, including but not limited to human immunoglobulins, such as human IgGl, human IgG2, human IgG3, 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 otherwise identical TROP2 binding proteins. In some embodiments, the Fc-fused TROP2 binding proteins of the present disclosure comprise, for example, one or more additional amino acid residue substitutions, mutations and / or modifications in the Fc region that result in a binding protein with preferred properties, including but not limited to: altered pharmacokinetics, extended serum half-life, and the like.

[0229] In some embodiments, such Fc-fused TROP2 binding proteins provide an extended half-life in a mammal, such as a human, of longer than 5 days, longer than 10 days, longer than 15 days, longer than 20 days, longer than 25 days, longer than 30 days, longer than 35 days, longer than 40 days, longer than 45 days, longer than 2 months, longer than 3 months, longer than 4 months, or longer than 5 months. In some cases, the extended half-life results in a higher serum titer, thereby reducing the frequency of administration of the TROP2 binding protein, and / or reducing the concentration of antibody to be administered. In some examples, the in vivo binding to human FcRn and serum half-life of a human FcRn high affinity binding polypeptide is determined in a transgenic mouse expressing human FcRn or a transfected human cell line, or in a primate administered a polypeptide with a variant Fc region.

[0230] In some cases, the TROP2 binding proteins are differentially modified during or after production, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be introduced to a TROP2 binding protein by a variety of techniques, including, but not limited to, specific chemical cleavage, by agents such as cyanogen bromide, trypsin, pepsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, and the like.

[0231] Various post-translational modifications of the TROP2 binding proteins encompassed by the present disclosure include, for example, N-linked or O-linked carbohydrate chains, processing at the N- or C-terminus, linkage to chemical moieties to the amino acid backbone, chemical modifications of the N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of expression in prokaryotic host cells. In addition, in some cases, the TROP2 binding proteins are modified with detectable labels such as enzymes, fluorescent, radioactive, or affinity labels to allow detection and isolation of the modulators.

[0232] Polynucleotides encoding TROP2 binding proteins

[0233] In some embodiments, polynucleotide molecules encoding the TROP2 binding proteins described herein are also provided. In some embodiments, the polynucleotide molecules are provided in the form of a DNA construct. In other embodiments, the polynucleotide molecules are provided in the form of a messenger RNA transcript.

[0234] The 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 comprising more than one domain. In some embodiments, the genes encoding the domains are separated by a peptide linker, or in other embodiments, are directly linked by peptide bonds into a single gene construct, which is operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressed in bacteria or other appropriate expression systems, such as CHO cells. Depending on the vector system and host employed, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. The promoters are selected to drive expression of the polynucleotide in the respective host cell.

[0235] In some embodiments, the polynucleotides encoding the TROP2 binding proteins described herein are inserted into a vector, preferably an expression vector, which represents a further embodiment. The recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0236] A variety of expression vector / host systems can be utilized to contain and express polynucleotides encoding the polypeptides of the TROP2 binding proteins. Examples of expression vectors are pSKK for expression in E. coli (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27), or pcDNA5 for expression in mammalian cells (Invitrogen). Thus, in some embodiments, the TROP2 binding proteins as described herein are produced by introducing a vector encoding the proteins as described above into a host cell, and culturing the host cell under conditions permitting expression of the protein domains, which can be isolated and optionally further purified.

[0237] Pharmaceutical compositions

[0238] In some embodiments, also provided are pharmaceutical compositions comprising an anti-TROP2 binding protein described herein, a vector comprising a polynucleotide encoding the TROP2 binding protein or a host cell transformed with the vector, and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is nontoxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated in conventional ways and administered to a subject in a suitable dose. Preferably, the composition is sterile. These compositions can further contain accessories such as preservatives, emulsifying agents and dispersing agents. The action of microorganisms can be prevented by including various antibacterial and antifungal agents. Another embodiment provides one or more of the above TROP2 binding proteins packaged in lyophilized form or in an aqueous medium.

[0239] 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, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the TROP2 binding protein is attached to a liposome. In some cases, the TROP2 binding protein is conjugated 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.

[0240] The TROP2 binding proteins described herein are contemplated for use as a medicament. Administration is achieved through different ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound comprised in the pharmaceutical composition. The administration regimen will be determined by the attending physician, in light of the other clinical factors. The dosage for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, the time and route of administration, the kind of therapy, overall health and other drugs that can be simultaneously administered. An "effective dose" is the amount of active ingredient sufficient to affect the course and severity of the disease, resulting in the reduction or remission of such pathology, and can be determined using known methods.

[0241] In some embodiments, the TROP2 binding proteins of the present disclosure are administered at a dose of up to 10 mg / kg at a frequency of once per week. In some cases, 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 is from about 1 ng / kg to about 10 ng / kg, from about 5 ng / kg to about 15 ng / kg, from about 12 ng / kg to about 20 ng / kg, from about 18 ng / kg to about 30 ng / kg, from about 25 ng / kg to about 50 ng / kg, from about 35 ng / kg to about 60 ng / kg, from about 45 ng / kg to about 70 ng / kg, from about 65 ng / kg to about 85 ng / kg, from about 80 ng / kg to about 1 pg / kg, from about 0.5 pg / kg to about 5 pg / kg, from about 2 pg / kg to about 10 pg / kg, from about 7 pg / kg to about 15 pg / kg, from about 12 pg / kg to about 25 pg / kg, from about 20 pg / kg to about 50 pg / kg, from about 20 pg / kg to about 60 pg / kg, from about 35 pg / kg to about 70 pg / kg, from about 45 pg / kg to about 80 pg / kg, from about 65 pg / kg to about 90 pg / kg, from about 85 pg / kg to about 0.1 mg / kg, from about 0.095 mg / kg to about 10 mg / kg, from about 20 pg / kg to 540 pg / kg. In some cases, the dose is from about 0.1 mg / kg to about 0.2 mg / kg, from about 0.25 mg / kg to about 0.5 mg / kg, from about 0.45 mg / kg to about 1 mg / kg, from about 0.75 mg / kg to about 3 mg / kg, from about 2.5 mg / kg to about 4 mg / kg, from about 3.5 mg / kg to about 5 mg / kg, from about 4.5 mg / kg to about 6 mg / kg, from about 5.5 mg / kg to about 7 mg / kg, from about 6.5 mg / kg to about 8 mg / kg, from about 7.5 mg / kg to about 9 mg / kg, or from about 8.5 mg / kg to about 10 mg / kg.In some embodiments, the TROP2 binding proteins of the present disclosure are administered at a dose 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, 700 ng / kg, 800 ng / kg, 900 ng / kg, 1 pg / kg, 2 pg / kg, 5 pg / kg, 10 pg / kg, 12 pg / kg, 15 pg / kg, 20 pg / kg, 22.5 pg / kg, 25 pg / kg, 30 pg / kg, 40 pg / kg, 50 pg / kg, 60 pg / kg, 70 pg / kg, 80 pg / kg, 90 pg / kg, 100 pg / kg, 130 pg / kg, 150 pg / kg, 180 pg / kg, 200 pg / kg, 225 pg / kg, 250 pg / kg, 280 pg / kg, 300 pg / kg, 350 pg / kg, 370 pg / kg, 400 pg / kg, 430 pg / kg, 460 pg / kg, 500 pg / kg, 540 pg / kg, 590 pg / kg, 600 pg / kg, 630 pg / kg, 670 pg / kg, 700 pg / kg, 730 pg / kg, 780 pg / kg, 800 pg / kg, 840 pg / kg, 900 pg / kg, 950 pg / 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 once a week. In some embodiments, the frequency of administration is about less than once a day, once every other day, less than once a day, twice a week, once a week, 7 times a week, twice a week, three times a week, once every four weeks, or once a month. In some cases, the frequency of administration is once a week. In some cases, the frequency of administration is once a week and the dose is up to 10 mg / kg. In some cases, the duration of administration is from about 1 day to about 4 weeks or more.

[0242] Therapeutic methods and tumor growth reduction properties

[0243] Also provided in certain embodiments are methods of treating a condition associated with TROP2-expressing malignant cells in a subject, comprising administering to a subject in need thereof an effective amount of a TROP2 binding domain or a multi-specific protein comprising a TROP2 binding domain of the present disclosure (including a conditionally active multi-specific protein), 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 a cancer.

[0244] In another aspect, the present disclosure provides a method of inhibiting tumor growth or progression in a subject having TROP2-expressing malignant cells, comprising administering to a subject in need thereof an effective amount of a TROP2 binding domain or a multi-specific 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 of inhibiting metastasis of TROP2-expressing malignant cells in a subject, comprising administering to a subject in need thereof an effective amount of a TROP2 binding domain or a multi-specific 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 of inducing tumor regression in a subject having TROP2-expressing malignant cells, comprising administering to a subject in need thereof an effective amount of a TROP2 binding domain or a multi-specific 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 biologic therapeutic agent, e.g., an antibody. In some embodiments, the second therapeutic agent is a cytokine, TNFa (tumor necrosis factor alpha), a PAP (phosphatidic acid phosphatase) inhibitor, an oncolytic virus, a kinase inhibitor, an IDO (indoleamine-pyrrole 2,3-dioxygenase) inhibitor, a glutaminase GLS1 inhibitor, a CAR (chimeric antigen receptor)-T cell or T cell therapy, a TLR (Toll-like receptor) agonist (e.g., TLR3, TLR4, TLR5, TLR7, TLR9), or a tumor vaccine.

[0245] In certain embodiments, a TROP2 binding protein of the present disclosure reduces the growth of tumor cells in vivo when administered to a subject having tumor cells expressing TROP2. Measurement of reduced tumor cell growth can be determined by a variety of different methods known in the art. Non-limiting examples include direct measurement of tumor size, measurement of excised tumor mass and comparison to control subjects, measurement by imaging techniques (e.g., CT or MRI) that can or can not employ isotopes or light-emitting molecules (e.g., luciferase) to enhance the analysis, and the like. In particular embodiments, administration of a TROP2 binding protein of the present disclosure results in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the growth of tumor cells in vivo as compared to a control antigen binding agent, with about 100% reduction in tumor growth indicating a complete response and tumor disappearance. In further embodiments, administration of a TROP2 binding protein of the present disclosure results in about 50-100%, about 75-100%, or about 90-100% reduction in the growth of tumor cells in vivo as compared to a control antigen binding agent. In further embodiments, administration of a TROP2 binding protein of the present disclosure results in about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% reduction in the growth of tumor cells in vivo as compared to a control antigen binding agent. In some embodiments, administration of a TROP2 binding protein of the present disclosure results in a complete reduction in tumor cell growth, e.g., in vivo tumor cell growth within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 25 days, 30 days of first administration. In some embodiments, the reduction in tumor cell growth (e.g., in vivo tumor cell growth) lasts for more than 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.

[0246] In some embodiments, an active drug and a prodrug containing the same molar equivalent of a TROP2 binding protein of the present disclosure are comparable in their effect to reduce tumor cell growth in vivo when administered to a subject having tumor cells expressing TROP2.

[0247] In some embodiments, a TROP2 binding protein of the present disclosure is administered to treat a neoplastic condition. In some embodiments, the neoplastic condition is benign or malignant; 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-part sarcoma, astrocytic tumor, autonomic ganglia tumor, bladder cancer (squamous cell carcinoma and transitional cell carcinoma), blastocyst cavity disorder, 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, chromophobe renal cell carcinoma cancer, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, epithelial disorder, Ewing's tumor, extraskeletal myxoid chondrosarcoma, fibrous dysplasia of bone, fibrous structure of bone dysplasia, gallbladder and bile duct cancer, gastric cancer, gastrointestinal, gestational trophoblastic disease, germ cell tumor, glandular disorder, head and neck cancer, hypothalamic, intestinal cancer, islet cell tumor, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, liver cancer (hepatoblastoma, hepatocellular carcinoma), lymphoma, lung cancer (small cell carcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma, etc.), macrophage disorder, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, rare hematologic disorder, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, stroma disorder, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastatic cancer, and uterine cancer (cervical cancer, endometrial cancer, and leiomyoma).

[0248] In certain embodiments, a TROP2 binding protein of the present disclosure is used as a first line therapy and is administered to a subject who has not previously been treated for a cancerous condition. In other embodiments, a TROP2 binding protein of the present disclosure is used to treat a subject who has been previously treated (with a TROP2 binding protein of the present disclosure or other anti-cancer agent) and has relapsed or is determined to be refractory to the previous treatment. In some embodiments, a TROP2 binding protein of the present disclosure is used to treat a subject who has a recurrent tumor.

[0249] In some embodiments, the TROP2 binding proteins as described herein, including the multispecific proteins, CARs or ProCARs as described herein, are administered to treat cancers with extensive TROP2 expression and prevalence, including but not limited to colorectal cancer, prostate cancer, neuroendocrine cancer, thyroid cancer, lung cancer (non-small cell lung cancer and small cell lung cancer), gastric cancer, ovarian cancer, endometrial cancer, pancreatic cancer, biliary tract cancer and gallbladder cancer, esophageal cancer, breast cancer, adenocarcinoma, or any combination thereof.

[0250] In some aspects, the TROP2 binding proteins of the present disclosure are administered to treat proliferative disorders including solid tumors, including but not limited to cancer of the adrenal gland, liver, kidney, bladder, breast, stomach, ovary, cervix, uterus, esophagus, colorectal, prostate, pancreas, lung (small cell and non-small cell), thyroid, sarcoma, glioblastoma, and various head and neck tumors, or any combination thereof.

[0251] In some embodiments, the TROP2 binding proteins of the present disclosure are administered to a subject having melanoma. In some embodiments, the TROP2 binding proteins of the present disclosure are used in 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, melanocarcinoma, melanocytoma, melanosarcoma, melanoma in situ, nodular malignant melanoma, lentigo maligna melanoma, malignant lentigo melanoma, mucosal lentigo melanoma, mucosal melanoma, acral-lentiginous melanoma, soft tissue melanoma, ocular melanoma, invasive melanoma, familial atypical mole-melanoma (FAM-M) syndrome, desmoplastic malignant melanoma, uveal melanoma, or any combination thereof.

[0252] In some embodiments, the possible indications for administration of the TROP2 binding proteins of the present disclosure, or pharmaceutical compositions comprising the same, are neoplastic diseases, especially epithelial carcinomas / cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, urogenital tract cancer, e.g., ovarian cancer, endometrial cancer, cervical cancer, and renal cancer, lung cancer, stomach cancer, small bowel 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 the same, are suitable for use in minimal residual disease, such as early stage solid tumors, late stage solid tumors, or metastatic solid tumors, characterized by single cell survival leading to local and non-local recurrence of the tumor, or any combination thereof.

[0253] In selected aspects, 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 to treat a cancer, e.g., an epithelial carcinoma / cancer, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, genitourinary tract cancer, e.g., ovarian cancer, endometrial cancer, cervical cancer, and renal cancer, lung cancer, gastric cancer, small bowel cancer, liver cancer, pancreatic cancer, gall bladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer, small cell lung cancer, non-small cell lung cancer (e.g., squamous cell non-small cell lung cancer or squamous cell small cell lung cancer), large cell neuroendocrine carcinoma (LCNEC), or any combination thereof.

[0254] Chimeric antigen receptors are generally artificially constructed hybrid proteins or polypeptides that contain or comprise an antigen binding domain of an antibody linked to a signaling domain (e.g., a T cell signaling or T cell activation domain). In some embodiments, a CAR comprising a TROP2 binding protein of the present disclosure has the ability to redirect the specificity and reactivity of a sensitized lymphocyte (e.g., a T cell) to TROP2 positive target cells in a non-MHC restricted manner by exploiting the antigen binding properties of an antibody or antigen binding fragment thereof. This non-MHC restricted antigen recognition enables T cells expressing the TROP2 CAR to recognize neoplastic TROP2 independent of antigen processing, thereby circumventing a major mechanism of tumor escape. Moreover, when expressed in T cells, the CAR does not advantageously dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0255] In some embodiments, the disclosed TROP2 binding proteins are administered to a refractory patient (i.e., a patient whose disease has recurred during or shortly after completion of an initial course of therapy); a sensitive patient (i.e., a patient whose recurrence is longer than 2-3 months after primary therapy); or a patient who exhibits resistance to platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel). In another embodiment, the disclosed TROP2 CAR is therapeutically effective to treat ovarian cancer, including ovarian serous carcinoma and ovarian papillary serous carcinoma.

[0256] In another embodiment, the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, are used in maintenance therapy to reduce or eliminate the chance of tumor recurrence after the initial appearance of disease. In some cases, the disease has been treated and the initial tumor mass has been eliminated, reduced, or otherwise ameliorated, so the patient is asymptomatic or in remission. At this point, the subject is administered a pharmaceutically effective amount of the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, one or more times, whether or not there is little or no disease in the disease indication using standard diagnostic procedures. In some embodiments, the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, are administered on a regular schedule, e.g., weekly, biweekly, monthly, every six weeks, every two months, every three months, every six months, or yearly, over a period of time to reduce the likelihood of disease recurrence. Moreover, in some embodiments, such treatment continues for a period of weeks, months, years, or even indefinitely, depending on the patient's response as well as clinical and diagnostic parameters.

[0257] In yet another embodiment, the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, are used in a de-escalation procedure for prophylactic or adjuvant therapy to prevent or reduce the likelihood of tumor metastasis. As used in the present disclosure, "de-escalation procedure" means any procedure, technique, or method that eliminates, reduces, treats, or ameliorates a tumor or tumor proliferation. Exemplary de-escalation procedures include, but are not limited to, surgery, radiation therapy (i.e., beam radiation), chemotherapy, immunotherapy, or ablation. In some embodiments, the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, are administered at appropriate times, as suggested by clinical, diagnostic, or therapeutic procedures, to reduce tumor metastasis. In some embodiments, the dosing regimen is accompanied by appropriate diagnostic or monitoring techniques that allow for modification thereof.

[0258] Other embodiments of the present disclosure include administration of the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, to subjects who are asymptomatic but at risk of developing a proliferative disorder. That is, in some embodiments, the TROP2 binding proteins, TROP2 CARs, or TROP2 sensitized lymphocytes of the present disclosure, or any combination thereof, are used in a prophylactic sense and provided to patients who have been screened or tested and have one or more notable risk factors (e.g., genomic indicators, family history, in vivo or in vitro test results, etc.), but have not yet developed a tumor. In such cases, one of skill in the art will be able to determine an effective dosing regimen by empirical observation or by accepted clinical practice.

[0259] In some embodiments of the methods described herein, a TROP2 binding protein or composition as described herein is administered in combination with an agent for treating a particular disease, disorder, or condition (also referred to herein as an additional therapeutic agent). Such agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutic drugs), hormones (steroids, peptides, etc.), radiation therapy (directed delivery of gamma rays, X-rays, and / or radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, a TROP2 binding protein described herein is administered in combination with an antidiarrheal agent, an antiemetic agent, an analgesic, an opioid, and / or a nonsteroidal anti-inflammatory agent. In some embodiments, a TROP2 binding protein described herein is administered in combination with an anticancer agent. Non-limiting examples of anticancer agents that can be used in various embodiments of the present disclosure, including the pharmaceutical compositions and dosage forms and kits of the present disclosure, include: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; agalsidase beta; agalsidase alfa; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; azacitidine; azactidine; azaribine; baleenstatin; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; bortezomib; bromofosfamide; busulfan; calicheamicin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; chlormethine; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; demethoxyviridin; denileukin diftitox; dexamethasone; dexrazoxane; diaziquone; dihydromaytansine;Mitocarcin; Mitoclopramide; Mitoxetine; Mitomycin; Mitospe; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogamycin; Omaplatin; Oxisulam; Paclitaxel; Pegaspargase; Pelimycin; Pentamidine; Pelimycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Pyroxantrone hydrochloride; Plicamycin; Promectin; Porfimer sodium; Porfimer; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofuranoside; Liboadenosine; Roglulimide; Safingol; Safingol hydrochloride; Semustine; Simtraqin; Spadronate sodium; Sparamycin; Spirogermanium hydrochloride; Spiromustine; Spiro Platinum; streptozotocin; streptozotocin; sulfaquinoxaline; talimycin; tecogalan sodium; tegafur; tiloxantrone hydrochloride; temoporfin; teniposide; tiroxilon; testolactone; thiamidine; thioguanine; thiotepa; thiazolamide nucleoside; tirapazamine; toremifene citrate; triptolon acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronide; triptorelin; tobradazole hydrochloride; uramustine; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinpoxetine sulfate; vinpoxetine sulfate; vinsorbate sulfate; vinorelbine tartrate; vinprodine sulfate; vinblastine sulfate; vorozole; zeniplatin; fenastatin; zorubicin hydrochloride. Other examples of anticancer drugs include, but are not limited to, 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adenosine; adolesin; aldesleukin; ALL-TK antagonists; hexamethylmelamine; aminostine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; anrelix; anti-dorsalizing morphogenetic protein-1 (ADMP-1); protein-1); antiandrogen; prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; glycine aphidicolin; apoptosis gene regulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene;Bisaziridinyl spermine; Bisnafide; Bistratene A; Bisantrene; Breflate; Brostalikmin; Budotitan; Buthionine sulfone imine; Calcipotriol; Camptothecin derivatives; Canarypox IL-2; Capecitabine; Carboxamide-amino-triazole; Carboxyamidotriazole; CaRest M3; CARN 700; Cartilage-derived inhibitor; Carzol; Casekinase inhibitor (ICOS); Castanospermine; Cecropin B; Cetrorelix; Chlorin; Chloquinox; Cicaprost; Cis-porphyrin; Cladribine; Clomifene analog; Clotrimazole; Colemicin A; Colemicin B; Combretastatin A4; Combretastatin analogs; Conagenin; Crambescidin 816; Crestomazone; Cyanophycin A; Cyanophycin A derivative; Curacin A; Cyclopentanoperhydrophenanthrenequinone; Cycloplatam; Debio 14; Cytosine arabinoside octadecyl phosphate; Cytolysin; Cytostatin; Dariximab; Decitabine; Dehydroleucettine B; Deslorelin; Dexamethasone; Dexifosfamide; Dexrazoxane; Dexverapamil; Diaziquone; Didox; Diethylornithine; Dihydro-5-azacytidine; 9-Dihydrotaxol; Dioxamycin; Diphenylspiromustine; Docetaxel; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Dronabinol; Duocarmycin SA; Edetalestil; Edelfoside; Edotrecol; Edrecolomab; Efomycin; Elemene; Eflomithine; Epirubicin; Epithilone; Estramustine analog; Estrogen agonists; Estrogen antagonists; Ethamivan; Etoposide phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Flutamide; Fluasterone; Fludarabine; Fluorodaunorunicin hydrochloride; Fosphanocol; Formestane; Fosquidnonol; Fosspirfen; Gadolinium texaphyrin; Gallium nitrate; Galocitabine; Ganirelix; Gelatinase inhibitor; Gemcitabine; Glutathione inhibitor; Hepsulfam; Heregulin; Hexamethylene bisacetamide; Hypericin; Ibandronate; Idarubicin; Idoxifene; Idomethine; Ilmofosine; Iloperidone; Imidazoacridones; Imiquimod; Immunostimulatory peptides; Insulin-like growth factor-I receptor inhibitor; Interferon agonists; Interferons; Interleukins; Iobenguane; Iodo- doxorubicin; Ipomeanol; Iroplax; Isoalldesrox; Isohomofacundin B; Itasetron; Jasplakinolide;kahalalide F; triacetate discodermolide-N; lanreotide; lenograstim; lentinan sulfate; leponthamide; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen + hydroxyprogesterone caproate; leuprolide; levamisole; liarozole; linear polyamine analogues; lipophilic bisamide peptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lumefantrine; lonomide; losoxantrone; HMG-CoA reductase inhibitors (such as, but not limited to, lovastatin, pravastatin, fluvastatin, statins, simvastatin, and atorvastatin); losoxantrone; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; martynidine A; martilmasat; masoprocol; maspin; matrix metalloproteinase inhibitor; menogaril; merbarone; melatonin; methioninase; metoclopramide; MIF inhibitor; mifepristone; milifuosine; miltefosine; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody to human chorionic gonadotropin; monophosphoryl lipid A + mycobacterial cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multikinase inhibitor; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neoadjuvanten; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitrooxy antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotide; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducator; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentastarch; pentoxifylline; perflurohalones; perfosfamide; pereirin; phenazinomycin; phenylacetate; phospholipase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim;Placetin A; Placetin B; Plasminogen activator inhibitor; Platinum complex; Platinum compound; Platinum-triamine complex; Porfimer sodium; Porfiromycin; Prednisone; Propyl bis-acridone; Prostaglandin J2; Proteasome inhibitor; Protein A based immunomodulator; Protein kinase C inhibitor; Protein kinase C inhibitor; Protozoan; Protein tyrosine phosphatase inhibitor; Purine nucleoside phosphorylase inhibitor; Purpurin; Pyrazoloacridine; Pyridoxalated hemoglobin polyoxyethylene conjugate; Raf antagonist; Raltitrexed; Ramucirumab; Ras farnesyl protein transferase inhibitor; Ras inhibitor; Ras-GAP inhibitor; Redihalseride; Rhenium Re 186 etidronate; Rhizoxin; Ribozyme; RII alitretinins; Roxadine; Roxilosine; Romurtide; Ruboxyl; Safingol; Saintopin; SarCNU; Sarcophytin A; Sargramostim; Sdi 1 mimetic; Semustine; Senescence derived inhibitor 1; Sense oligonucleotide; Signal transduction inhibitor; Signal transduction modulator; Single-chain antigen binding protein; Sizofiran; Sobuzoxane; Sodium borocaptate; Sodium phenylacetate; Solverol; Somatomedin binding protein; Sonermin; Spharose acid; Spicamycin D; Spiromustine; Splenopentin; Spongistatin 1; Squaramine; Stem cell inhibitor; Stem cell division inhibitor; Stipiamide; Stromelysin inhibitor; Sulfinosine; Super active vasoactive intestinal peptide antagonist; Suradista; Suramin; Swainsonine; Synthetic glycosaminoglycan; Talisomycin; Tamoxifen methiodide; Tauromustine; Tazarotene; Tecogalan sodium; Tecogalan sodium; Tellurapyrylium; Telomerase inhibitor; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxid; Tetrazomine; Thaliblastine; Thiocoraline; Thrombopoietin; Thrombopoietin mimetic; Thymalfasin; Thymidylate synthase inhibitor; Thymomegalmine; Thyrotropin; Tin ethyl etiopurpurin; Tirapazamine; Titanocene bichloride; Topsentin; Toremifene; Totipotent stem cell factor; Translation inhibitor; Tretinoin; Trichostatins; Tricyclodecanecarboxamide; Triptorelin; Tropisetron; Turosteride; Tyrosine kinase inhibitor; Tyrphostin; UBC inhibitor; Ubenimex; Urogenital sinus-derived growth inhibitory factor; Urokinase receptor antagonist; Valtorcitabine;variolin B; carrier systems; red blood cell gene therapy; verazol; veratramine; verdins; verteporfin; vinorelbine; vinxaltine; vorozole; zanotenum; zorbene; zilascorb; and zinecard. Other anticancer agents are 5-fluorouracil and folinic acid. These two agents are particularly useful when used in the methods using 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 prior to, during, or after surgery.

[0260] Methods of detecting TROP2 expression and diagnosis of TROP2-related cancers

[0261] According to another embodiment of the present disclosure, a kit for detecting TROP2 expression in vitro or in vivo is provided. The kit includes a TROP2 binding protein as previously described (e.g., a TROP2 binding protein comprising a labeled anti-TROP2 single-domain antibody or antigen-binding fragment thereof), and one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzymatic label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0262] 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, and pathology specimen. Biological samples also include tissue sections, e.g., frozen sections 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 a mammal, such as a human or non-human primate.

[0263] In one embodiment, a method of 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 is provided. An increase in binding of the antibody to the sample compared to binding of the antibody to a control sample determines that the subject has cancer.

[0264] In another embodiment, a method of confirming a diagnosis of cancer in a subject by contacting a sample from the subject diagnosed with cancer with an anti-TROP2 single-domain antibody disclosed herein and detecting binding of the antibody to the sample is provided. An increase in 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.

[0265] In some examples of the disclosed methods, the TROP2 single-domain antibody is directly labeled. In some examples, the method further comprises contacting a second antibody that specifically binds to the anti-TROP2 single-domain antibody with the sample; and detecting binding of the second antibody. An increase in binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects or confirms a diagnosis of cancer in the subject. In some cases, the cancer is a neuroendocrine cancer, a prostate cancer, a lung cancer, a gastric cancer, a squamous cell carcinoma, a pancreatic cancer, a cholangiocarcinoma, a triple negative breast cancer, or an ovarian cancer (such as an epithelial ovarian cancer), or any other type of cancer that expresses TROP2. In some examples, the control sample is a sample from a subject that does not have cancer. In particular examples, the sample is a blood or tissue sample.

[0266] In some cases, the antibody that binds (e.g., specifically binds) to TROP2 is directly labeled with a detectable label. In another embodiment, the antibody that binds (e.g., specifically binds) to TROP2 (the first antibody) is not labeled, while a second antibody or other molecule that can bind to the antibody that specifically binds to TROP2 is labeled. The second antibody is chosen so that it is able to specifically bind to the particular species and class of the first antibody. For example, if the first antibody is a llama IgG, the second antibody can be an anti-llama IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable labels for the antibody or the second antibody are described above and include various enzymes, prosthetic groups, fluorescent, luminescent, magnetic, and radioactive agents. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent agents include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent agent is luminol; a non-limiting exemplary magnetic agent is gadolinium; and non-limiting exemplary radioactive agents include 125 I, 131 I, 35 S or 3 H.

[0267] In an alternative embodiment, TROP2 can be measured in a biological sample by a competitive immunoassay employing 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 mixed together, 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.

[0268] 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 the production of TROP2 in cells in a 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 soluble TROP2 in a bodily fluid sample, such as a blood or serum sample).

[0269] In one embodiment, a kit for detecting TROP2 in a biological sample, such as a blood sample or a tissue sample, is provided. For example, to confirm a diagnosis of cancer in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be taken to detect the presence of soluble TROP2 protein or fragments. In accordance with the present disclosure, a kit for detecting a polypeptide will typically include a single domain antibody that specifically binds to TROP2. In some embodiments, an antibody fragment, such as a scFv fragment, a VH domain, or a Fab, is included in the kit. In further embodiments, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic label).

[0270] In one embodiment, the kit includes instructional materials that disclose the means for using the antibody that binds to TROP2. The instructional materials can be written, in electronic form (e.g., computer diskette or CD-ROM), can be visual (e.g., video file), or provided over an electronic network, such as via the Internet, World Wide Web, intranet, or other network. The kit can also include other components to facilitate the particular application for which the kit is designed. Thus, for example, the kit can additionally contain means for detecting the label (e.g., enzyme substrates for an enzymatic label, a filter set for detecting a fluorescent label, a suitable second label such as a second antibody, etc.). The kit can additionally include buffers and other reagents that are routinely used in the practice of a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0271] In one embodiment, the diagnostic kit comprises an immunoassay. While the details of the immunoassay can vary depending on the particular format employed, the method of detecting TROP2 in a biological sample generally comprises the step of contacting the biological sample with an antibody that specifically reacts with the TROP2 polypeptide under immuno- reactive conditions. The antibody is allowed to specifically bind under immuno- reactive conditions to form an immunocomplex, and the presence of the immunocomplex (bound antibody) is detected directly or indirectly.

[0272] Methods of determining the presence or absence of a cell surface marker 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, metallic compounds, radioactive compounds, or drugs. Antibodies can also be used in immunoassays, such as, but not limited to, radioimmunoassays (RIA), ELISA, or immunohistochemical assays. Antibodies can also be used in fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low angle and obtuse light scatter detection channels, and impedance channels, among other more complex detection levels, to separate or sort cells. See U.S. Patent No. 5,061,620. Any single domain antibody that binds TROP2, as disclosed herein, can be used in these assays. Thus, these antibodies can be used in routine immunoassays, including, but not limited to, ELISA, RIA, FACS, tissue immunohistochemistry, Western blotting, or immunoprecipitation.

[0273] Certain Definitions

[0274] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0275] The term "about" or "approximately" means 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 used or understood in the art - for example, measured versus. "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Where particular values are described in the application and claims, "about" should be understood to be an acceptable error range for that particular value, unless otherwise stated.

[0276] The terms“individual,”“patient,” or“subject” are used interchangeably. None of these terms requires or implies that a situation is characterized by the presence or absence of a health care worker (e.g., a physician, a registered nurse, a nurse practitioner, a physician’s assistant, a nursing attendant, or a hospice worker) or that a situation is characterized by the presence or absence of ongoing or intermittent monitoring by a health care worker.

[0277] An“antibody” generally refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and noncovalent interactions. Human light chains comprise a variable domain (VL) and a constant domain (CL), which can be readily classified as kappa or lambda based on amino acid sequence and locus. Each heavy chain comprises a variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD comprises three domains, designated CH1, CH2, and CH3, respectively (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 proline and cysteine-rich segment of variable length (typically about 10 to about 60 amino acids in IgG). The variable domains in both light and heavy chains are connected to the constant domains by a“J” region of about 12 or more amino acids, and the heavy chains also have a“D” region of about 10 additional amino acids. Each class of antibody further comprises inter- and intra-chain disulfide bonds formed by pairs of cysteine residues. There are two types of native disulfide bridges or disulfide bonds in immunoglobulin molecules: inter- and intra-chain disulfide bonds. The location and number of inter-chain disulfide bonds varies according to the class and species of immunoglobulin. Inter-chain disulfide bonds are located on the surface of the immunoglobulin, are solvent accessible, and are generally relatively easy to reduce. In human IgGl isotype, there are four inter-chain disulfide bonds, one from each heavy chain to a light chain, and two between the heavy chains. Inter-chain disulfide bonds are not required for chain association. It is well known that the cysteine-rich IgGl hinge region of the heavy chain is generally held to consist of three parts: the upper hinge, the core hinge, and the lower hinge. Those skilled in the art will appreciate that the IgGl hinge region contains cysteines in the heavy chain that make up inter-chain disulfide bonds (two heavy / heavy, two heavy / light) that provide structural flexibility that facilitates Fab movement. The inter-chain disulfide bonds between the light and heavy chains of IgGl are formed between C214 of the kappa or lambda light chain and C220 in the upper hinge region of the heavy chain. The inter-chain disulfide bonds between the heavy chains are located at positions C226 and C229 (all numbered according to the EU index, according to Kabat et al., infra).

[0278] As used herein, the term "antibody" includes polyclonal antibodies, multiple cloned antibodies, monoclonal antibodies, chimeric antibodies, de-immunized, 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: hclgG, V-NAR, Fv, Fd, Fab, F(ab')2, F(ab'), Fab2, Fab3 fragments, single chain fragments (e.g., di-scFv, ScFv, ScFvFc, scFv-zipper, scFab), disulfide bonded Fv (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single domain antibodies such as nanobodies or single variable domain antibodies containing only one variable domain such as sdAb (VH, VL or VHH domains), "rIgG" ("half-antibodies"), diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "minibodies", in some cases 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 so long as it comprises a domain with a binding site that preferentially associates or binds with a TROP2 protein. Furthermore, unless the context dictates otherwise, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG and IgM) and all subclasses (i.e., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The heavy chain constant domains that correspond to the different classes of antibodies are generally designated by the corresponding lower case Greek letters a, d, e, g and m, respectively. The light chains of an antibody from any vertebrate species can be assigned to one of two distinct types, called kappa (k) and lambda (l), based on the amino acid sequences 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 VH domain. In some examples, the engineered human VH domains are generated by panning phage display libraries. In some embodiments, the TROP2 binding protein comprises a VHH. The term "VHH" as used herein refers to a single chain antibody binding domain that does not contain a light chain.In some cases, the VHH is derived from a naturally light chain deficient type of antibody that can be found in Camelidae or cartilaginous fish, or from a synthetic and non-immune VHH that can be constructed accordingly. Each heavy chain comprises a variable region encoded by V-, D- and J- exons. In some cases, the VHH is a native VHH, such as a Camelidae-derived VHH, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camels, llamas, vicunas, guanacos and cartilaginous fish (such as, but not limited to, sharks). In another embodiment, the VHH is derived from an alpaca (such as, but not limited to, a Huacaya Alpaca or a Suri alpaca).

[0279] As used herein, "variable region" or "variable domain" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies, and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not even throughout the variable domains of the antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of the heavy and light chains each comprise four FR regions, mostly adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, or in some cases forming part of, the beta sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains, while not directly involved in the binding of antibody to antigen, exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. In some embodiments, unless otherwise specified, the amino acids are designated to each domain, framework region, and CDR according to one of the numbering schemes provided below: 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 comprises a heavy chain-only antibody, such as a VH or VHH domain, and comprises three CDRs. In some embodiments, such heavy chain-only antibodies bind TROP2 in monomeric form, with optimal binding affinity independent of dimerization with a VL (light chain variable) region.

[0280] “Variable domain residue numbering according to Kabat” or “amino acid position numbering according to Kabat” and variations thereof refer to the numbering system for the heavy chain variable domain or light chain variable domain used in the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and insertion of residues (e.g., residues 82a, 82b, and 82c, according to Kabat) after residue 82 of heavy chain FR. Kabat numbering of residues in a given antibody can be determined by alignment of the antibody sequence with the “standard” Kabat numbered sequence in regions of homology. This does not mean that the CDRs of the disclosure necessarily correspond to the Kabat numbering convention.

[0281] The term “framework” or “FR” residues (or regions) refers to the variable domain residues other than the CDR or hypervariable region residues defined herein. A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences.

[0282] The term “epitope” as used herein refers to a determinant that is the specific antigenic site on an antigen to which a particular antibody molecule's variable region binds. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are formed by spatial juxtaposition of amino acids from different segments of the linear polypeptide chain. Linear epitopes are formed by amino acid residues that are contiguous along the linear polypeptide chain. In certain instances, an epitope can include a sugar, phosphoryl, or sulfonyl moiety of an antigen.

[0283] As used herein, the term "percent (%) amino acid sequence identity" in reference to a sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0284] As used herein, "elimination half-life" is used in its ordinary sense as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is intended to include a quantitative measure of the time course of drug elimination. Elimination of most drugs is exponential (i.e., follows first order kinetics) because drug concentrations typically do not approach the concentrations required for saturation of the elimination process. The rate of an exponential process can be described by its rate constant, k, or by its half-life, t 1 / 2 , where the rate constant, k, represents the fractional change per unit time, and the half-life, t 1 / 2 , represents the time required for 50% completion of the process. The units of these two constants are time"1and time, respectively. The first order rate constant and half-life of a reaction are simply related (k x t 1 / 2 = 0.693) and can be interchanged accordingly. Since first order elimination kinetics indicates that a constant fraction of drug is lost per unit time, a plot of the logarithm of drug concentration versus time is linear after the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-life of drug elimination can be accurately determined from such a plot.

[0285] As used herein, the term "binding affinity" refers to the affinity of a protein described in the present disclosure for its binding target, and is expressed numerically using a "K D " value. If it is indicated that two or more proteins have comparable binding affinities for their binding targets, the KD If two or more proteins are shown to have comparable binding affinities for a single binding target, the K values ​​for each protein binding to the single binding target are calculated as follows: D If a protein is shown to bind two or more targets with comparable binding affinities, then the K values ​​for the protein binding to the two or more targets are within ±2-fold of each other. D values ​​are within ±2 times of each other. Generally, higher K D In some embodiments, "Kd" is measured by radiolabeled antigen binding assay (RIA) or surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In certain embodiments, BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) is also used to measure "Kd". TM -2000 or BIAcore TM The "on rate" or "rate of association" or "on rate" or "kon" and the "off rate" or "rate of dissociation" or "off rate" or "koff" are measured using a surface plasmon resonance technique using a BIAcore-3000 (BIAcore, Inc., Piscataway, NJ).

[0286] Systems (Pall Life Sciences) to determine "K D ”, “kon” and “koff”. In an exemplary method for measuring binding affinity using a ligand (e.g., biotinylated human or cynomolgus monkey TROP2) On the surface of the streptavidin capillary sensor tip, the streptavidin tip is then activated using about 20-50 μg / ml of human or cynomolgus monkey TROP2 protein according to the manufacturer's instructions. A PBS / casein solution is also introduced as a blocking agent. For association kinetics measurements, TROP2 binding protein variants are introduced at a concentration of about 10 ng / mL to about 100 μg / mL, about 50 ng / mL to about 5 μg / mL, or about 2 ng / mL to about 20 μg / mL. In some embodiments, a single domain protein that binds to TROP2 is used at a concentration of about 2 ng / mL to about 20 μg / mL. Complete dissociation was observed in the negative control, i.e., in the absence of a binding protein assay buffer. Appropriate tools such as ForteBio software are then used to determine the kinetic parameters of the binding reaction.

[0287] In some embodiments, "treatment" or "treatment" as used herein refers to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to enhance or improve a desirable physiological condition. For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state; and remission, whether partial or total, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without an unacceptably high level of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, "treatment" or "treatment" refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, e.g., in a person predisposed to the disease (e.g., an individual carrying a genetic marker for a disease such as breast cancer).

[0288] As used herein, "TriTAC," "TROP2-targeting TriTAC," or "Tri-specific protein targeting TROP2" refers to a tri-specific 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 and second target antigen binding domains comprises a TROP2 binding protein as described herein, and at least one of the first and second target antigen binding domains binds to CD3, such as human CD3.

[0289] As used herein, “ProTriTAC” or “pro-tri-specific protein targeting TROP2” refers to a conditionally activated tri-specific binding protein and comprises (i) a cleavable linker (e.g., comprising a sequence as set forth in SEQ ID NOs: 497-543), (ii) a binding moiety specific for a large volume serum protein and further comprising a masking moiety that prevents binding of the first and second target antigen binding domains to their targets (e.g., comprising a sequence as set forth in SEQ ID NO: 549), wherein at least one of the first and second target antigen binding domains comprises a TROP2 binding protein as described herein. In some cases, e.g., in a protease-rich environment, e.g., in a tumor microenvironment, the ProTriTAC protein of the disclosure is activated from a masked state to an active state by cleavage of the cleavable linker to form an active drug. In some cases, the active drug provided herein comprises a TROP2 binding domain of the disclosure and a CD3 binding domain of the disclosure. Examples of active drugs are provided in SEQ ID NOs: 229-264, or a sequence that is at least about 75% to 100% identical to a sequence selected from SEQ ID NOs: 229-264, such as a 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 a sequence selected from SEQ ID NOs: 229-264.

[0290] As used herein, “non-cleavable prodrug” refers to a ProTriTAC as described above, wherein the cleavable linker is replaced with a non-cleavable linker (e.g., a linker as in SEQ ID NO: 696). Examples of active drugs are provided in SEQ ID NOs: 229-264, or a sequence that is at least about 75% to 100% identical to a sequence selected from SEQ ID NOs: 229-264, such as a 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 a sequence selected from SEQ ID NOs: 229-264.

[0291] In non-beta-sandwich scaffolds (e.g., In the context of the affibody, "non-CDR loop" refers to a region that (1) is amenable to sequence randomization to allow for engineered specificity to a second antigen, and (2) is generally distal to the main specificity-determining regions used on the scaffold to allow for the scaffold to simultaneously engage both antigens without steric interference. To this end, the main specificity-determining regions can be defined using the framework definitions established in the Skrlec 2015 publication (Trends in Biotechnol, 33:408-418). An excerpt of the framework is listed below.

[0292]

[0293] As used herein, "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that provides antigen specificity to a cell, e.g., a T cell. A CAR comprises multiple domains, e.g., at least one target antigen binding domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. Each domain can be connected by a linker. As used herein, "ProCAR" refers to a conditionally activatable CAR comprising a TROP2 binding domain of the disclosure.

[0294] EMBODIMENT

[0295] The present application can be better understood by reference to the following non-limiting examples provided as exemplary embodiments of the present application. The following examples are presented in order to more fully illustrate embodiments of the application, however, they should not be construed as limiting the broadest scope of the application.

[0296] Example 1: Screening of phage display library for identification of TROP2 binding domains

[0297] Llamas were immunized with purified TROP2 dimer protein expressed in 293 cells. A phage display library for expression of heavy chain variable antibody domains was constructed from circulating B cells isolated in a humanized llama (van der Linden et al. 2000. J Immunol Methods 240: 185-195). Periplasmic extracts were prepared by expression of anti-llama TROP2 protein in E. coli, and phage clones were screened for binding to TROP2 dimer using a colorimetric ELISA. Fifty-two unique heavy chain-only sequences (SEQ ID NOs: 1-52) were identified that produced a signal in the ELISA screen relative to controls employing 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.

[0298] Table 3: Binding of llama anti-TROP2 antibodies to human or cynomolgus TROP2 in ELISA assay

[0299]

[0300]

[0301] The values in Table 1 represent absorbance readings for colorimetric ELISA.

[0302] Example 2: Introduction of single domain antibody with only TROP2 binding heavy chain into fusion protein and T cell dependent cytotoxicity assay Figures 1 to 12

[0303] Anti-TROP2 antibody sequences were cloned into DNA constructs for expression of recombinant fusion proteins (SEQ ID NOs: 229-258). The coding sequence for the fusion proteins included a signal peptide for secreted cell expression, a stub for a cleaved form of the 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 repeat of six histidine sequences (SEQ ID NO: 496). A linker sequence (SEQ ID NO: 497) was inserted at the junction between the antibody domains. These anti-CD3 / anti-TROP2 fusion protein constructs were transfected into Expi293 cells (Life Technologies). The amount of fusion protein in the transfected Expi293 cell conditioned media was quantified using an Octet instrument with a protein A or anti-6xHis tip, using a fusion protein with similar molecular weight to the anti-CD3 / anti-TROP2 proteins as a standard.

[0304] Conditioned media was tested in a T cell-dependent cellular cytotoxicity assay (TDCC) (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. 2015. J Biomol Screen. 20:519-27). In this assay, target cells (luciferase-labeled H292 cells or luciferase-labeled HT1376 cells, both of which express TROP2) are combined with purified human T cells, and the anti-CD3 / anti-TROP2 fusion protein is titrated. If the fusion protein directs the T cells to kill the target cells, the signal in a luciferase assay performed 48 hours after the start of the experiment should be reduced. Figure 5is a graph of TDCC viability results. EC50 values from TDCC assays are listed in Table 4. The most potent molecule had an EC50 of 37.4 pM in H292 cells and 152 pM in H1376 cells. The negative control for the TDCC assay was an anti-GFP / anti-albumin / anti-CD3 protein and this protein did not direct T cells to kill H292 cells, but it had slight activity at the highest concentration tested (e.g., Figure 11 and Example 3: Humanization of TROP2 binding antibodies and T cell dependent cytotoxicity assay ).

[0305] Table 4: EC50 values for llama anti-TROP2 sequence containing anti-CD3 / anti-TROP2 fusion proteins redirecting T cells to kill H292 cells

[0306]

[0307]

[0308] n / a: activity was insufficient to calculate an EC50 using the protein concentrations tested.

[0309] Figures 13 to 18

[0310] The llama anti-TROP2 antibodies were humanized by grafting the CDR sequences of the four llama anti-TROP2 antibody sequences (SEQ ID NOs: 16, 41, 43, and 52) onto human germline sequences, while retaining some llama framework sequences to ensure that the antibodies did not lose activity (SEQ ID NOs: 53-57). These humanized sequences were cloned into expression constructs for expression of anti-CD3 / anti-TROP2 fusion proteins in Expi293 cells (SEQ ID NOs: 259-263) and their parental constructs were prepared as previously described. The amount of anti-CD3 / anti-TROP2 fusion protein in the conditioned media was quantified as previously described. The conditioned media containing the anti-CD3 / anti-TROP2 fusion proteins was used in TDCC assays as previously described, using only the TROP2-expressing luciferase-tagged H292, HT1376, or HCC70 cell lines. The results of the TDCC assays are plotted in Figures 19-21 Figure 6, and the EC50 values for redirected T cell killing are listed in Table 3. Potent redirected T cell killing was observed using both the llama and humanized TROP2 antibodies.

[0311] The humanized binder 2TRH79B was used to make a fusion protein containing an anti-ALB domain containing a non-CDR loop mask that prevents CD3 binding linked to anti-CD3 and anti-TROP2 2TRH79B binders by a cleavable linker (SEQ ID NO: 498). The 2TRH79B binder and its parental constructs were purified and quantified. The anti-CD3 / anti-TROP2 fusion proteins were tested in TDCC assays using three different luciferase-labeled TROP2-expressing cell lines, HCC70, HPAF-II, or CAL27. The results of the TDCC assays are plotted in Figures 19-21 In the case of directed T cell killing, the EC50values are listed in Table 5.

[0312] Table 5: EC50values for llama or humanized anti-TROP2 sequence containing anti-CD3 / anti-TROP2 fusion proteins to indirectly direct T cell killing of H292, HT1376, or HCC70 cells

[0313]

[0314]

[0315] The humanized binder 2TRH79B is a sequence variant of the 2TRH79 binder. A DNA construct containing an anti-ALB domain containing a non-CDR loop mask that prevents CD3 binding linked to anti-CD3 and anti-TROP2 2TRH79B binder sequences by a cleavable linker (L040) (SEQ ID NO: 498) was made. This fusion construct was transfected into Expi293 cells and the conditioned media was collected a few 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 anti-CD3 domains linked to one of three anti-TROP2 sequences by a GGGGSGGGS linker were also transfected into Expi293 cells for protein expression: the llama anti-TROP2 binder sequence 2TRL79 and the humanized anti-TROP2 binder sequences 2TRH79 and 2TRH79B. The conditioned media was harvested five days after transfection 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 the 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 Example 4: Demonstration of tolerogenicity enhancement of exemplary anti-CD3 / anti-TROP2 fusion proteins in miceIn the anti-CD3:anti-TROP2 proteins with humanized anti-TROP2 domains, 2TRH79B had higher potency, which was only 2- to 3-fold less potent than the proteins with llama anti-TROP2 domains. Comparing the EC50values of anti-CD3:anti-TROP2 2TRH79B and anti-ALB:anti-CD3:anti-TROP2 proteins (Table 6), it can be seen that the killing potency of the anti-ALB:anti-CD3:anti-TROP proteins (2TRH79B L040 ProTriTAC) was 36- to 173-fold less potent than the anti-CD3:anti-TROP2 proteins (2TRH79B), which indicates that the presence of the anti-ALB domain, which has a non-CDR loop masking the binding to the anti-CD3 domain, reduces TDCC activity. This difference can be greater if the cell line used in this assay has partial activation of protease activity of the anti-ALB:anti-CD3:anti-TROP proteins.

[0316] Table 6: EC50values of anti-CD3 / anti-TROP2 fusion proteins containing llama or humanized anti-TROP2 sequences to indirectly direct T cell killing of HCC70, HPAF-II, or CAL27 cells

[0317]

[0318] nd = no data.

[0319] Figures 22-26

[0320] All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Harpoon Therapeutics (Protocol Number HAR-001-2019). Animals were purchased from Jackson Laboratory and then housed in the Harpoon Therapeutics’ pathogen-free animal facility according to IACUC guidelines. All studies were performed in 6, 7, or 11-week-old female NSG TM (NOD-SCID IL2Rgammanull) mice, n = 5-10 mice per group.

[0321] Human tumor cells expressing TROP2, HCC70 (10E6), CAL27 (5E6), or HPAF II (10E6), and activated and expanded human T cells (5E6, 2.5E6, or 5E6, respectively) were implanted subcutaneously in NSG TMRight flank (day 0). For HCC70 xenograft experiments 1 and 2, treatment was initiated 7 days post-implantation (day 7) once tumors were established (mean 184 mm3(HCC70 exp 1) or 155 mm3(HCC70 exp 2)). For CAL27 xenografts, treatment was initiated on day 4 once tumors were established (mean 120 mm3). For HPAF II xenografts, treatment was initiated on day 4 once tumors were established (mean 128 mm3). Mice were dosed intraperitoneally (qdx14) with negative control, non-Trop2-targeting anti-GFP TriTAC, anti-Trop2 2TRL79 (llama binder) ProTriTAC linker 040, anti-Trop2 2TRH79 (humanized binder) ProTriTAC linker 040, or anti-Trop2 2TRH79B (humanized binder) ProTriTAC linker 040. Mice were monitored at least twice per week for tumor growth as indicated. Mean tumor volumes shown were calculated from measurements on the last day of each xenograft model study. Statistical analysis was performed as RM one-way ANOVA with Dunnett’s post-test comparing all groups to the negative control anti-GFP TriTAC. Results from each combination therapy xenograft study are plotted in Example 5: Prodrugs and molar equivalents of active drugs can achieve comparable potent antitumor activity In summary, statistical analysis results for each xenograft model study are reported in Table 7.

[0322] Table 7: P-values for multiple rodent xenograft studies using anti-CD3 / anti-TROP2 fusion proteins containing human anti-TROP2 sequences 2TRL79, 2TRH79, and 2TRH79B L040 ProTriTAC

[0323]

[0324] HCC70 Experiment 1 2 HCC70 Experiment 2 a: anti; E: T; effector to target cell ratio; n / a: not applicable; ns: not statistically significant (P > 0.05); and P: probability value.

[0325] Figure 27

[0326] All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at Harpoon Therapeutics (Protocol Number HAR-001-2019). Animals were purchased from Jackson Laboratory and then housed in the Harpoon Therapeutics’ pathogen-free animal facility in accordance with IACUC guidelines. All studies were performed on 6, 7, or 11 -week-old female NSG mice (The Jackson Laboratory, Bar Harbor, ME) housed in groups of 3-5 in micro-isolator cages with a 12-hour light / dark cycle and ad libitum access to food and water. TMwere performed in NOD-SCID IL2Rgammanull mice, n=5-10 mice per group. For each experiment, mice were age-matched.

[0327] HCC70 (10E6) cells expressing TROP2 and activated and expanded human T cells (5E6) were implanted subcutaneously in NSG mice in a mixture at an E:T ratio (2.5 million: 5 million). The mice were monitored twice a week for tumor growth. The average tumor volume is calculated from the measurements on the last day of each xenograft model study. Statistical analysis was performed with RM one-way ANOVA with Dunnett’s post-test comparing all groups to the negative control anti-GFPTriTAC. Results from the mixed treatment xenograft rodent study are plotted in TM The mice were monitored twice a week for tumor growth. The average tumor volume is calculated from the measurements on the last day of each xenograft model study. Statistical analysis was performed with RM one-way ANOVA with Dunnett’s post-test comparing all groups to the negative control anti-GFPTriTAC. Results from the mixed treatment xenograft rodent study are plotted in Figure 27 The results show that the molar equivalents of prodrug and active drug show comparable potent antitumor activity Example 6: Assessment of binding activity and species cross-reactivity of TroP2 ProTriTAC linker 40 molecules by biolayer interferometry

[0328] Figures 28A-28B Figure 28A

[0329] ​Bio-Layer Interferometry (BLI) is a well-established analytical method for determining binding kinetics of specific bioanalytical interactions. One molecule (target ligand) is labeled or modified to enable specific capture and presentation to a second molecule (analyte) in solution. In some uses, 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 loading, the ligand is captured on the tip of a glass fiber biosensor that is chemically derivatized with a reagent capable of binding the labeled ligand with high affinity, i.e. metal-chelated nitrilotriacetic acid, anti-Fc monoclonal antibody, or streptavidin in previous examples. Using an instrument such as the Octet RED96 (Sartorius), white light is projected down the biosensor tip and reflected from two surfaces, one reference layer and one biocompatible surface with immobilized ligand. The distance reflected to the reference layer is constant, while the distance reflected from the surface immobilized ligand changes with binding or dissociation of the analyte, resulting in an interference of the light wave that causes a change in the amplitude measured over time. The resulting binding sensorgrams from serial dilutions of known analyte concentrations are then globally fit into a one-to-one binding model. This global fit determines the on-rate constant (kon) and off-rate constant (koff) used to calculate the binding dissociation constant (KD). Since the streptavidin-biotin interaction is one of the highest known affinities of biological molecules, to minimize the confounding effects of other ligand / surface chemistry, all ligands in this study were biotinylated.

[0330] To better characterize and aid in the identification of relevant toxicology species, the binding kinetics and affinity of the TroP2 ProTriTAC linker 40 molecule of tumor-associated calcium signal transducer 2 (TroP2) (2TRH79B) were evaluated. Biotinylated derivatives of the target ligand from human and non-human primate (NHP) species (cyno) were prepared. Thus, the binding of 2TRH79B and 2TRH79 to biotinylated target ligand was evaluated by bio-layer interferometry (BLI) in the absence or presence of calcium. Figure 28B It was shown that 2TRH79B has comparable affinity to human (Table 8) and cynomolgus monkey (cyno) Trop2 (Table 8) in the absence or presence of calcium (47-51 nM or 50 nM). Table 8 summarizes the results from the binding kinetics studies. Human TroP2 ) and cyno Trop2 ( Cyno TroP2 ) in the absence or presence of calcium. Table 8 summarizes the results from the binding kinetics studies.

[0331] Table 8: Binding kinetics values of anti-CD3 / anti-TROP2 fusion protein containing human anti-TROP2 sequence 2TRH79B, L040 ProTriTAC to human and Cyno Trop2

[0332] kon(1 / Ms)e5 Koff(1 / s) KD(nM) 0.9 0.8 Example 7: One month dose escalation / marginally tolerated dose study 4.1 x 10 -3 ]]> 5.7 x 10 -3 ]] Figure 29 48 73

[0333] In addition, the second TroP2 ProTriTAC linker 40 molecule (2TRH79), a related construct, was evaluated by BLI for binding kinetics and affinity to human and cyno Trop2 in the absence or presence of calcium, and the results were similar. 2TRH79 bound to human and cyno Trop2 with comparable affinity in the absence or presence of calcium (168-186 nM or 122-179 nM, respectively) (data not shown).

[0334] Figure 30

[0335] In this dose escalation / margin of safety study, the TroP2 ProTriTAC linker 40 molecule (2TRH79B) was used for intravenous injection (slow bolus) in cynomolgus monkeys.

[0336] The primary objective of this GLP-compliant escalating repeat-dose study was to assess the potential toxicity and systemic exposure of TroP2 ProTritTAC in cynomolgus monkeys when administered as a slow bolus intravenous (iv) injection at doses of 20, 60, 180, and 540 pg / kg on days 1, 8, 15, and 22, respectively. SEQUENCE LISTING The experimental design is shown. In addition, the toxicokinetic (TK) profile of TroP2 ProTriTAC was determined. Study endpoints included: mortality, clinical observations (daily cage-side, post-dose, and weekly detailed observations), body weight, qualitative food consumption, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), bioanalysis and TK parameters, cytokine analysis, organ weights, and macroscopic and microscopic examinations.

[0337] TroP2 ProTriTAC was well tolerated at the highest tested dose of 540 pg / kg. PK was assessed by capture with a biotinylated anti-idiotype antibody recognizing the aCD3 domain and detection with a sulfo-tagged anti-idiotype antibody raised against the aALB domain. ​ TroP2-ProTritTAC showed good pharmacokinetics, half-life, and systemic accumulation. Hematological changes included mild to moderate reductions in neutrophils at all dose levels, and transient reductions in lymphocytes and basophils at week 3 of the study. No significant findings were observed for plasma cytokines (IFNy, IL-1b, IL-2, IL-6, IL-10, IL-8, TNF-a) throughout the study, for coagulation, clinical chemistry, or urinalysis parameters, and for histopathology based on organ weights and gross examination.

[0338] ​

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

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[0358]

[0359]

[0360]

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[0362]

[0363]

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[0366]

[0367] While preferred embodiments of the application have been shown and described herein, it is to be understood that the application is not limited to these embodiments. Since modifications, changes, and substitutions are obvious foreign to those skilled in the art, it is intended that the application embrace all such changes, modifications, and substitutions that fall within the scope of the appended claims. Various alternatives to the embodiments described herein are now apparent to those skilled in the art. It is intended that the application embrace all such alternatives. The claims are intended to cover all methods and structures falling within their scope.

Claims

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

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

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

4. The TROP2 binding domain of any one of claims 1-3, 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 pharmaceutical form.

7. The TROP2 binding domain of any one of claims 4-6, 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 serum albumin.

9. The TROP2 binding domain of claim 8, wherein the serum albumin comprises human serum albumin.

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

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

494.

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

13. The TROP2 binding domain of any one of claims 7-9, wherein the bulk serum protein binding domain is a binding moiety comprising a linker and a masking moiety, wherein the masking moiety masks 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 7-9 and 13, wherein the multispecific protein comprises an unclippable prodrug form.

15. The TROP2 binding domain of claim 13 or 14, wherein the masking moiety comprises a sequence selected from SEQ ID NOs: 550 and 558-560, or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 550 and 558-560.

16. The TROP2 binding domain of any one of claims 13-15, wherein the linker comprises a sequence selected from SEQ ID NOs: 497-545, or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 497-545.

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

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

494.

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

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

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

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

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

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

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

26. The TROP2 binding domain of claim 25, wherein the non-CDR loop provides a binding site specific for a bulky serum protein.

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

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

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

30. The TROP2 binding domain of any one of claims 23-29, wherein the ProCAR further comprises a costimulatory domain, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137), and an amino acid sequence having at least one but not more than 20 modifications thereto.

31. The TROP2 binding domain of claim 30, wherein the at least one but not more than 20 modifications comprise modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to binding of an encoded T cell receptor fusion protein.

32. The TROP2 binding domain of any one of claims 23-31, wherein the CAR or the ProCAR further comprises the 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 an amino acid sequence having at least one but not more than 20 modifications thereto.

33. The TROP2 binding domain of any one of claims 23-32, wherein the CAR or the ProCAR further comprises the intracellular signaling domain, and the intracellular signaling domain is derived from CD3s, CD3y, CD35, CD3a, CD3b, or a combination thereof.

34. The TROP2 binding domain of claim 33, wherein the intracellular signaling domain is derived from CD3s.

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

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, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228, wherein the non-CDR loop is capable of binding the TROP2 binding domain or the second target antigen binding domain, and wherein the binding moiety is capable of masking the binding of the TROP2 binding domain or the second target antigen binding domain to its target.

38. The conditionally active TROP2 binding protein of claim 37, wherein the binding moiety comprises a masking moiety, and wherein the masking moiety comprises a sequence selected from the group consisting of SEQ ID NO: 550 or 558-560, or a sequence comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 550 and 558-560.

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 comprising one or more substitutions relative to a sequence selected from the group consisting of SEQ ID NOs: 497-543.

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

493.

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

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

494.

43. A method of 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 of any one of claims 37-42, or a pharmaceutical composition comprising the same.

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

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

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

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

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

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

50. The TROP2 binding domain of any one of claims 1-3 and 47-49, wherein the binding domain binds TROP2 with a binding affinity (K D ) of about 0.001 nM to about 500 nM.

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

52. A multi-specific protein comprising a TROP2 binding domain, wherein the TROP2 binding domain is according to any one of claims 1-3 and 47-51.

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

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

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

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

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

494.

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

493.

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

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

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

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

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

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

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

66. An active pharmaceutical comprising a sequence as set forth in any one of SEQ ID NOS: 229-264.

67. An active pharmaceutical comprising a sequence as set forth in any one of SEQ ID NOS: 1-57.

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

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

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

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 the TROP2 binding domain of any one of claims 1-34 and 47-51 or the pharmaceutical composition of claim 68.

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

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

74. The method of claim 73, wherein the method further comprises administering an agent, wherein the agent is a biologic 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 71-74, wherein the TROP2 binding domain selectively binds to a tumor cell expressing TROP2.

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

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

78. The method of any one of claims 71-77, wherein the neoplastic disease is 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. The method of any one of claims 43-46, wherein the method further comprises administering an agent, wherein the agent is a biologic 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. The method of claim 79, wherein the TROP2 binding domain selectively binds to a tumor cell expressing TROP2.

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

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

83. The method of any one of claims 79-82, 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 of producing a conditionally active TROP2 binding protein, the method 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 of any one of claims 37-42 under conditions permitting expression of the conditionally active TROP2 binding protein, and recovering and purifying the produced protein from the culture.

85. A method of producing a multivalent protein, the method 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 permitting expression of the multivalent protein, and recovering and purifying the produced protein from the culture.

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

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

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

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

90. A method of making a CAR or ProCAR comprising transfecting a cell of any one of claims 87-89 with a vector or RNA comprising a nucleotide sequence encoding the CAR or the ProCAR.

91. The conditionally active TROP2 binding protein of any one of claims 37-42, 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: 229-264.

92. The conditionally active TROP2 binding protein of any one of claims 37-42, 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: 229-264.

93. The conditionally active TROP2 binding protein of any one of claims 37-42, 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: 229-264.

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

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

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

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

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

100. The method of any one of claims 97-99, wherein the neoplastic disease is 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. A method of 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 loop comprises a binding site specific for the TROP2 binding domain, - wherein the TROP2 binding domain is masked from binding its target by the binding moiety, and - wherein the TROP2 binding domain binds its target upon cleavage of the cleavable linker.

102. The method of claim 101, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 115-171; and the CDR3 comprises a sequence selected from SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 172-228.

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. The method of any one of claims 101-103, 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. The method of any one of claims 101-105, wherein the cleavable linker comprises a sequence selected from SEQ ID NOs: 497-543 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 497-543.

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

108. The method of any one of claims 101-107, 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: 229-264.

109. The method of any one of claims 101-107, 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: 229-264.

110. The method of any one of claims 101-107, 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: 229-264.

111. The method of any one of claims 101-110, 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. The method of claim 111, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228.

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 111-113, 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: 1-57.

115. The method of any one of claims 111-113, 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: 1-57.

116. A method of improving 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 target antigen binding domain and the second target antigen binding domain comprises a TROP2 binding domain, the method comprising conjugating the first target antigen binding domain or the second target antigen binding domain to a binding moiety comprising a cleavable linker and a non-CDR loop, - 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 its target by the binding moiety, and - wherein the masked first or second target antigen binding domain binds its target upon cleavage of the cleavable linker.

117. The method of claim 116, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 115-171; and the CDR3 comprises a sequence selected from SEQ ID NOs: 172-228 or a sequence comprising one or more substitutions in a sequence selected from SEQ ID NOs: 172-228.

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

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

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. The method of any one of claims 116-121, 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 116-122, wherein the TROP2 binding domain comprises a sequence that is at least 75% identical to a sequence selected from SEQ ID NOs: 1-57.

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

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

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

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

128. A method of improving 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. The method of claim 128, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in 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. 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 128-131, 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: 1-57.

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 in a sequence selected from the group consisting of SEQ ID NOs: 497-543.

134. The method of any one of claims 128-133, wherein the TROP2 binding domain is part of a conditionally active multi-specific protein, and the conditionally active multi-specific 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. The method of any one of claims 128-133, wherein the conditionally active multi- specific protein comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 229-264.

136. The method of any one of claims 128-133, wherein the conditionally active multi- specific 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. A TROP2-targeted conditionally active multi-specific protein, comprising: a TROP2 binding domain, a CD3 binding domain, 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, wherein the TROP2 binding domain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 58-114 or a sequence comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 58-114; the CDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 115-171 or a sequence comprising one or more substitutions in 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 comprising one or more substitutions in a sequence selected from the group consisting of SEQ ID NOs: 172-228.

138. The TROP2-targeted conditionally active multi-specific 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. The TROP2-targeted conditionally active multi-specific protein of claim 137 or claim 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. The TROP2-targeted conditionally active multi-specific protein of any one of claims 137-139, 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 in a sequence selected from the group consisting of SEQ ID NOs: 497-543.

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

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

143. The TROP2-targeted conditionally active multispecific protein of any one of claims 137-140, 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: 229-264.

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

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

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

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

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

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

150. The method of any one of claims 147-149, wherein the neoplastic disease is 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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