TROP2-binding agents and conjugates thereof

Abstract

TROP2-binding agents and variants thereof are described. In specific embodiments, TROP2-binding agents that are antibodies that preferentially bind to high-expressing TROP2 cells over low-expressing TROP2 cells, and conjugates thereof, including the TROP2-binding agent conjugated to a payload, are described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 497,994, filed April 24, 2023, the entire contents of which are incorporated herein by reference.

[0002] Reference to an electronically submitted sequence listing The contents of the electronic sequence listing (25665-WO-PCT_SL.xml; size: 253 Kb; created on February 27, 2024) are incorporated herein by reference in their entirety.

[0003] The present invention relates to TROP2-binding agents and variants thereof. In specific embodiments, the present invention relates to TROP2-binding agents and conjugates thereof that are antibodies that preferentially bind to high-TROP2-expressing cells over low-TROP2-expressing cells, including TROP2-binding agents conjugated to a payload. [Background technology]

[0004] Proteins that play a role in breast cancer growth, differentiation, invasion, and / or metastasis may influence tumor biology and therefore provide important prognostic information. One such candidate is TROP2 (GA733-1, EGP-1). TROP2 is a 45 kDa monomeric transmembrane glycoprotein belonging to the TACSTD gene family, specifically TACSTD2, and is expressed in human epithelial cells at various stages of differentiation. Overexpression of TROP2 has been demonstrated to be both necessary and sufficient to stimulate tumor growth and is associated with poor overall prognosis. TROP2 expression is associated with poor prognosis in several human cancers, including oral, pancreatic, gastric, ovarian, colorectal, breast, and lung tumors. For example, TROP2 overexpression was observed in 55% of pancreatic cancer patients tested and was positively correlated with metastasis, tumor grade, and poor progression-free survival in patients undergoing curative surgery. Similarly, in gastric cancer, 56% of patients could demonstrate TROP2 overexpression on their tumors, which also correlated with shorter disease-free survival and worse prognosis in patients with lymph node involvement of TROP2-positive tumor cells.

[0005] Given these characteristics and the fact that TROP2 is associated with so many intractable cancers, it is an attractive target for therapeutic intervention. Nevertheless, TROP2 is also expressed in some normal tissues, although usually at much lower levels compared with those in neoplastic tissues, and is often expressed in areas of tissue with limited vascular access.

[0006] Several monoclonal antibodies against TROP2 have been established. Some anti-TROP2 monoclonal antibodies, such as 77220, are commercially available as reagents. Some of these established anti-TROP2 monoclonal antibodies are being investigated for the treatment of cancer.

[0007] Patent application WO 9714796 describes a monoclonal antibody, BR110, known to bind to TROP2 on the cell surface and internalize it within the cell. Patent applications WO 2003074566, U.S. Patent Application Publication No. 2004001825, U.S. Patent Application Publication No. 2007212350, and U.S. Patent Application Publication No. 2008131363 teach RS7 antibodies and their use for treating or diagnosing tumors. These patent applications further relate to humanized, human, and chimeric RS7 antigen-binding proteins (hRS7) and the use of such binding proteins in diagnosis and therapy. The anti-TROP2 monoclonal antibody AR47A 6.4.2 is disclosed in WO 2007095748, and AR52A301.5 is disclosed in WO 2007095749; both are antibodies that specifically bind to TROP2-expressing cancer cells.

[0008] Patent application WO 2008 / 144891 teaches a humanized version of AR47A 6.4.2 as an anti-TROP2 monoclonal antibody for treating tumors. Patent application WO 2011155579 teaches a monoclonal antibody or antibody fragment thereof that binds with high affinity to the extracellular domain of human TROP2 and exhibits high ADCC activity and high anti-tumor activity. Patent application WO 2013077458 teaches anti-human TROP2 antibodies with anti-tumor activity, particularly humanized antibodies including Huk5-70-2, and particularly anti-human TROP2 antibodies with in vivo anti-tumor activity. Patent application WO 2013068946 teaches antibodies that specifically bind to TROP2.

[0009] A promising application of antibodies for targeted tumor therapy involves the conjugation of multiple (2-8) highly toxic payloads to the antibody, thereby generating antibody-drug conjugates (ADCs). ADCs are well known in the art, as described, for example, by Chari et al. (Angew. Chem. Int. Ed. 53:3796 (2014)) and Beck et al. (Nat. Rev. Drug Discov. 16:315-37 (2017)). Mechanistically, antibodies are designed to bind with high specificity to tumor-associated receptors that are overexpressed relative to healthy tissue. ADCs are thought to be internalized into tumor cells after binding to the receptor and then release the toxic payload upon degradation of the antibody and / or linker in the lysosome.

[0010] ADCs targeting TROP2 are known in the art and are in various stages of clinical development. DS-1062a, an ADC derived from the humanized antibody hTINA conjugated to the camptothecin analog exatecan via a protease-sensitive cleavable linker, is currently undergoing clinical evaluation for the treatment of solid tumors. PF-06664178 is an ADC derived from the tail monoclonal antibody RN926 site-specifically conjugated to the auristatin analog PF-06380101 under the action of microbial transglutaminase. PF-06664178 was evaluated in a Phase I clinical trial in patients with advanced or metastatic solid tumors, but development was discontinued due to toxicity at high dose levels with only moderate antitumor activity.

[0011] Sacituzumab govitecan-hziy (TRODELVY, Immunomedics, Inc.) (SG) was approved in April 2020 for patients with metastatic triple-negative breast cancer (TNBC) who have received at least two prior therapies for metastatic disease (Bardia et al., N. Engl. J. Med. 380:741-51 (2019)). SG is an antibody-drug conjugate (ADC) consisting of the humanized anti-TROP2 monoclonal antibody (mAb), hRS7, linked to approximately eight molecules of SN-38, the active metabolite of irinotecan and a potent inhibitor of topoisomerase 1 (Thomas et al., Clin. Cancer Res. 25:6581-9 (2019)). Notably, until SG, no topoisomerase I inhibitors had been used in metastatic triple-negative breast cancer (TNBC), and SG effectively constitutes a new cytotoxic drug for treating a disease that is still heavily dependent on chemotherapy. However, the effectiveness of SG has been hampered by its toxicity.

[0012] The SG-targeting epitope in TROP2 may further limit its efficacy. hRS7 mAb has been shown to bind to the same epitope as T16, 162-46.2 (Alberti et al., Hybridoma; 11:539-45 (1992); Ikeda et al., Biochem Biophys. Res. Commun. 458:877-82 (2015)) and E1 mAb (Trerotola et al., Neoplasia 23:415-28 (2021)). Thus, RS7 joins a list that includes most anti-TROP2 antibodies, including MOv16 (Alberti et al., Hybridoma; 11:539-45 (1992)), cAR47A 6.4.2 (Truong et al., Mol. Cancer Ther. 6:3334 (2007)), 77220, MM0588, and YY-01 (Ikeda et al., Biochem Biophys. Res. Commun. 458:877-82 (2015)), which have been shown to bind to an immunodominant epitope (D146-T274) located in the N-terminal region of the stem domain of TROP2 (Ikeda et al., Biochem Biophys. Res. Commun. 458:877-82 (2015)) (Alberti et al. al., Hybridoma; 11:539-45 (1992); Ikeda et al., Biochem Biophys. Res. Commun. 458:877-82 (2015)). This epitope has been shown to be equally available in cancer cells and normal tissues (Trerotola et al., Oncogene 32:222-33 (2013); Alberti et al., Hybridoma 11:539-45 (1992); Stepan et al., J. Histochem. Cytochem. 59:701-10 (2011); Kaufmann et al., Arch. Dermatol. Res. 286:6-11 (1994)), thus raising the question of its lack of cancer specificity (Trerotola et al., Biochim. Biophys. Acta 1805:119-20(2010)).The Rinat-Pfizer RN926 anti-Trop-2 mAb, developed with the PF-06664178 / Aur0101 ADC, was also shown to bind to this immunodominant region of TROP2 (domain 3, residues 152–206, and domain 4, residues 209–274; WO 2013 / 068946). PF-06664178 showed early promise (Strop et al., Mol. Cancer Ther.; 15:2698–708 (2016)). A phase I, open-label, dose-escalation study of PF-06664178 was conducted in patients with advanced solid tumors. Doses of 3.60, 4.2, and 4.8 mg / kg were found to be intolerable due to skin rash, mucosal lesions, and neutropenia. PF-06664178 showed modest antitumor activity and was ultimately discontinued (King et al., Invest. New Drugs 36:836-47 (2018)). Thus, exposure of normal tissues to anti-TROP2 ADCs with high-potency payloads can result in unmanageable toxicity.

[0013] Furthermore, SN38 has a short half-life in patients (Ocean et al., Cancer 123:3843-54 (2017)), necessitating frequent dosing, which can lead to side effects such as neutropenia and diarrhea, which have been suggested to be due to the release of SN38 as free drug in the circulation (Santi et al., Ann. Transl. Med. 9:1113 (2021)). In light of the above, improvements in cell targeting and ADC half-life are necessary for anti-TROP2 ADCs to reach their full potential for treating cancers associated with high TROP2 expression. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 9714796 [Patent Document 2] International Publication No. 2003074566 [Patent Document 3] U.S. Patent Application Publication No. 2004001825 [Patent Document 4] U.S. Patent Application Publication No. 2007212350 [Patent Document 5] U.S. Patent Application Publication No. 2008131363 [Patent Document 6] International Publication No. 2007095748 [Patent Document 7] International Publication No. 2007095749 [Patent Document 8] International Publication No. 2008144891 [Patent Document 9] International Publication No. 2011155579 [Patent Document 10] International Publication No. 2013077458 [Patent Document 11] International Publication No. 2013068946 [Patent Document 12] International Publication No. 2015098099 [Non-patent literature]

[0015] [Non-Patent Document 1] Chari et al.,Angew.Chem.Int.Ed.53:3796(2014) [Non-patent document 2] Beck et al.,Nat.Rev.Drug Discov.16:315-37(2017) [Non-patent document 3] Bardia et al.,N.Engl.J.Med.380:741-51(2019) [Non-patent document 4] Thomas et al.,Clin.Cancer Res.25:6581-9(2019) [Non-Patent Document 5] Alberti et al., Hybridoma 11:539-45 (1992) [Non-patent document 6] Ikeda et al., Biochem. Biophys. Res. Commun. 458:877-82(2015)

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

[0017] The present invention further provides ADCs comprising the TROP2-binding agents of the present invention conjugated to a payload. The TROP2-binding agents and ADCs of the present invention are capable of targeting TROP2-expressing cells, particularly TROP2 高 They are useful for treating, imaging, diagnosing, preventing, containing, and reducing the growth of cells and TROP2-expressing tumors.

[0018] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences NYGMN (SEQ ID NO: 4), WINTYTGEPTYTDDFKG (SEQ ID NO: 5), GGFGSSYWYFDV (SEQ ID NO: 6), KASQDVSIAVA (SEQ ID NO: 7), SASDRYT (SEQ ID NO: 10), and QQHYITPLT (SEQ ID NO: 9), respectively. In a further embodiment, the antibody is a humanized antibody, and the antigen-binding fragment of the antibody is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0019] In a further embodiment, an antibody or antigen-binding fragment thereof is provided that exhibits reduced binding to low TROP2-expressing cells compared to high TROP2-expressing cells and has reduced hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

[0020] In further embodiments of the antibody or antigen-binding fragment thereof, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 or 14 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or 16.

[0021] In further embodiments of the antibody or antigen-binding fragment thereof, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3, or the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 14 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 16.

[0022] In further embodiments of the antibody or antigen-binding fragment thereof, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, 17, or 18.

[0023] In further embodiments of the antibody or antigen-binding fragment thereof, the antibody comprises (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 18.

[0024] In further embodiments of the antibody or antigen-binding fragment thereof, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58, 59, or 60.

[0025] In further embodiments of the antibody or antigen-binding fragment thereof, the antibody comprises (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 59; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 60.

[0026] In further embodiments of the antibody or antigen-binding fragment thereof, the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain and positions 165 and 168 of the light chain constant domain, wherein the numbering of the heavy chain constant domain positions is according to Eu numbering and the numbering of the light chain constant domain positions is according to consecutive numbering of the entire light chain sequence.

[0027] In a further embodiment of the antibody or antigen-binding fragment thereof, the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

[0028] In embodiments of the antibody or antigen-binding fragment thereof, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, 20, 61, or 62.

[0029] In further embodiments of the antibody or antigen-binding fragment thereof, the antibody is conjugated to a payload. In further embodiments of the antibody or antigen-binding fragment thereof, a cysteine ​​or non-standard amino acid is conjugated to the payload. In further embodiments of the antibody or antigen-binding fragment thereof, the payload is a therapeutic moiety, a detectable label, a radionuclide, or a protecting group. In further embodiments of the antibody or antigen-binding fragment thereof, the therapeutic moiety is a cytotoxic moiety, an anti-inflammatory moiety, a peptide, a nucleic acid molecule, or a nucleic acid analog. In further embodiments of the antibody or antigen-binding fragment thereof, the cytotoxic moiety is selected from the group consisting of taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (such as etoposide or etoposide phosphate), vinblastine, vincristine, vindesine, taxanes (such as taxol), taxotere arethinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, maytansinoids (DM1, DM2, DM3, DM4, etc.), auristatins (including monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethylauristatin D (MMAD)), 7-ethyl-10-hydroxy-camptothecin (SN-38), anthracyclines, alkylcyclines, and their derivatives.

[0030] In a further embodiment of the antibody or antigen-binding fragment thereof, the cytotoxic moiety is an inhibitor of topoisomerase I, topoisomerase II, or microtubule polymerization.

[0031] In a further aspect, the present invention provides the use of the aforementioned antibody.

[0032] In one aspect, the present invention provides a method of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain comprising CDRL1, CDRL2, and CDRL3, wherein each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:9, respectively, for treating cancer, and wherein the cancer is a cancer that overexpresses TROP2.

[0033] In further embodiments of the method, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0034] The present invention further provides an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:9, respectively, for use in the manufacture of a medicament for treating cancer that overexpresses TROP2.

[0035] In further embodiments of the use, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0036] The present invention further provides an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:9, respectively, for treating cancer that overexpresses TROP2.

[0037] In further embodiments, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0038] The present invention further provides a combination therapy for treating cancer comprising an antibody or antigen-binding fragment thereof that specifically binds to human TROP2 and a therapeutic agent, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain comprising CDRL1, CDRL2, and CDRL3, and wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:9, respectively, and wherein the cancer is a cancer that overexpresses TROP2.

[0039] In further embodiments of the combination therapy, the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody. In further embodiments, the therapeutic antibody is a checkpoint inhibitor. In further embodiments, the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

[0040] In further embodiments of the combination therapy, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0041] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 15.

[0042] In a further embodiment, the antibody or antigen-binding fragment thereof exhibits reduced hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

[0043] In further embodiments, the antigen-binding fragment of an antibody is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0044] In a further embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:21 and the heavy chain comprises the amino acid sequence of SEQ ID NO:17 or 18.

[0045] In a further embodiment, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:21 and a heavy chain comprising the amino acid sequence of SEQ ID NO:59 or 60.

[0046] In a further embodiment, the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain and positions 165 and 168 of the light chain constant domain, wherein the numbering of the heavy chain constant domain positions is according to Eu numbering and the numbering of the light chain constant domain positions is according to consecutive numbering of the entire light chain sequence.

[0047] In a further embodiment, the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

[0048] In further embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, 20, 61, or 62.

[0049] In further embodiments, the antibody is conjugated to a payload. In further embodiments, a cysteine ​​or non-standard amino acid is conjugated to the payload. In further embodiments, the payload is a therapeutic moiety, a detectable label, a radionuclide, or a protecting group. In further embodiments, the therapeutic moiety is a cytotoxic moiety, an anti-inflammatory moiety, a peptide, a nucleic acid molecule, or a nucleic acid analog. In further embodiments, the cytotoxic moiety is selected from the group consisting of taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (such as etoposide or etoposide phosphate), vinblastine, vincristine, vindesine, taxanes (such as taxol), taxoteretinoid acid , butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, maytansinoids (DM1, DM2, DM3, DM4, etc.), auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD)), 7-ethyl-10-hydroxy-camptothecin (SN-38), anthracyclines, alkylcyclines, and their derivatives.

[0050] In a further embodiment, the cytotoxic moiety is an inhibitor of topoisomerase I, topoisomerase II, or microtubule polymerization.

[0051] The present invention further provides a method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, the antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, to treat the cancer, wherein the cancer is a TROP2-overexpressing cancer.

[0052] In further embodiments of the method, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0053] The present invention further provides use of an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, for the manufacture of a medicament for treating cancer that overexpresses TROP2.

[0054] In further embodiments of the use, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0055] The present invention further provides an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, for treating cancer that overexpresses TROP2.

[0056] In further embodiments, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0057] The present invention further provides a combination therapy for treating cancer comprising an antibody or antigen-binding fragment thereof that specifically binds to human TROP2, the antibody or antigen-binding fragment comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2.

[0058] In a further embodiment of the combination therapy, the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

[0059] In a further embodiment of the combination therapy, the therapeutic antibody is a checkpoint inhibitor.

[0060] In a further embodiment of the combination therapy, the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

[0061] In further embodiments of the combination therapy, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0062] In a further aspect, the invention provides an ADC (MMAE-conjugated ADC) comprising an antibody that specifically binds human TROP2 conjugated to a linker-monomethyllauristatin E (linker-MMAE) payload, wherein the antibody comprises two heavy chains, each heavy chain comprising a variable domain and a constant domain, the variable domain comprising complementarity-determining regions (CDRs) H1, CDRH2, and CDRH3, and two light chains, each light chain comprising a variable domain and a constant domain, the variable domain comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of NYGMN (SEQ ID NO: 4), WINTYTGEPTYTDDFKG (SEQ ID NO: 5), GGFGSSYWYFDV (SEQ ID NO: 6), KASQDVSIAVA (SEQ ID NO: 7), SASDRYT (SEQ ID NO: 10), and QQHYITPLT (SEQ ID NO: 9), respectively.

[0063] In further embodiments of the MMAE-conjugated ADC, the antibody or antigen-binding fragment thereof exhibits decreased binding to low TROP2-expressing cells compared to high TROP2-expressing cells and has decreased hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

[0064] In a further embodiment of the MMAE-conjugated ADC, the antibody or antigen-binding fragment thereof is humanized.

[0065] In further embodiments of the MMAE-conjugated ADC, the antigen-binding fragment of the antibody is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0066] In further embodiments of the MMAE-conjugated ADC, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 or 14 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or 16.

[0067] In further embodiments of the MMAE-conjugated ADC, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3, or the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 14 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 16.

[0068] In further embodiments of the MMAE-conjugated ADC, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, 17, or 18.

[0069] In further embodiments of the MMAE-conjugated ADC, the antibody comprises (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 18.

[0070] In further embodiments of the MMAE-conjugated ADC, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58, 59, or 60.

[0071] In further embodiments of the MMAE-conjugated ADC, the antibody comprises (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 59; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 60.

[0072] In further embodiments of the MMAE-conjugated ADC, the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain and positions 165 and 168 of the light chain constant domain, where the numbering of the heavy chain constant domain positions is according to Eu numbering and the numbering of the light chain constant domain positions is according to consecutive numbering of the entire light chain sequence.

[0073] In a further embodiment of the MMAE-conjugated ADC, the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

[0074] In further embodiments of the MMAE-conjugated ADC, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, 20, 61, or 62.

[0075] In further embodiments of the MMAE-conjugated ADC, the linker-MMAE payload is conjugated to a cysteine ​​or a non-standard amino acid.

[0076] In a further embodiment of the MMAE-conjugated ADC, the antibody is one in which its SH groups are represented by the formula: MP-AA-PABC-MMAE: [ka] CM2P-AA-PABC-MMAE: [ka] CM3P-AA-PABC-MMAE: [ka] a linker comprising a cysteine ​​residue conjugated to an MMAE payload;

[0077] In a further embodiment of the MMAE-conjugated ADC, the ADC has the formula: [ka] (wherein Ab is an anti-TROP2 antibody and p is an integer from 1 to 8, where S is derived from the side chain of a cysteine ​​residue of the antibody).

[0078] In a further embodiment of the MMAE-conjugated ADC, the ADC has the formula: [ka] wherein Ab is an anti-TROP2 antibody comprising a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue, wherein the anti-TROP2 antibody comprising an engineered cysteine ​​residue is (A): (a) αTROP2 (HC:Sac-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 64 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 65 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (c) αTROP2 (HC:Sac-E171C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 66 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (d) αTROP2 (HC:Sac-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 67 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (e) αTROP2 (HC:Sac-E173C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 68 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (f) αTROP2 (HC:Sac-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 69 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (g) αTROP2 (HC:Sac-YTE-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 71 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (h) αTROP2 (HC:Sac-YTE-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 72 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (i) αTROP2 (HC:Sac-YTE-E171C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 73 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (j) αTROP2 (HC:Sac-YTE-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 74 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (k) αTROP2 (HC:Sac-YTE-E173C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 75 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (l) αTROP2 (HC:Sac-YTE-S375C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 76 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:58 and two light chains having the amino acid sequences set forth in SEQ ID NO:50; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 58 and two light chains having the amino acid sequences set forth in SEQ ID NO: 51; (o) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 63 and two light chains having the amino acid sequences set forth in SEQ ID NO: 50; (p) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 63 and two light chains having the amino acid sequences set forth in SEQ ID NO: 51; (q) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 83 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (r) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 78 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (s) αTROP2 (HC:BSM-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 79 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (t) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 80 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (u) αTROP2 (HC:BSM-E172C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 81 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (v) αTROP2 (HC:BSM-E173C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 82 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (w) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 83 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (x) αTROP2(HC:BSM)(LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:54; (y) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 59 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (z) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 60 and two light chains having the amino acid sequences set forth in SEQ ID NO: 54; (aa) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 58 and two light chains having the amino acid sequences set forth in SEQ ID NO: 57; (bb) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 85 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (cc) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 86 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (dd) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 87 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (ee) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 88 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (ff) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 89 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; and (gg) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 90 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1 or 2; or (B): (a) αTROP2 (HC:Sac-E152C-S375C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 70 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-YTE-E152C-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 77 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 84 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; and (d) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:91 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4; Includes:

[0079] In a further embodiment of the MMAE-conjugated ADC, the ADC has the formula: [ka] wherein Ab is an anti-TROP2 antibody comprising a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue, wherein the anti-TROP2 antibody comprising an engineered cysteine ​​residue is (A): (a) αTROP2 (HC:Sac-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 24 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 25 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (c) αTROP2 (HC:Sac-E171C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 26 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (d) αTROP2 (HC:Sac-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 27 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (e) αTROP2 (HC:Sac-E173C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 28 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (f) αTROP2 (HC:Sac-S375C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 29 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (g) αTROP2 (HC:Sac-YTE-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 31 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (h) αTROP2 (HC:Sac-YTE-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 32 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (i) αTROP2 (HC:Sac-YTE-E171C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 33 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (j) αTROP2 (HC:Sac-YTE-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 34 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (k) αTROP2 (HC:Sac-YTE-E173C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 35 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (l) αTROP2 (HC:Sac-YTE-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 36 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:11 and two light chains having the amino acid sequence set forth in SEQ ID NO:52; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 11 and two light chains having the amino acid sequence set forth in SEQ ID NO: 53; (o) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 23 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (p) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 23 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (q) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 19 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (r) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 38 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (s) αTROP2 (HC:BSM-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 39 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (t) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 40 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (u) αTROP2 (HC:BSM-E172C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 41 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (v) αTROP2 (HC:BSM-E173C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 42 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (w) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 19 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (x) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (y) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 54; (z) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (aa) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 44 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (bb) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:45 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (cc) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 46 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (dd) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:47 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (ee) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:48 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; and (ff) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 20 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1 or 2; or (B) (a) αTROP2 (HC:Sac-E152C-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 30 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-YTE-E152C-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 37 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:43 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; and (d) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:49 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4; Includes:

[0080] In a further embodiment of the MMAE-conjugated ADC, the ADC has the formula: [ka] wherein Ab is an anti-Trop2 antibody comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:20 and two light chains having the amino acid sequences set forth in SEQ ID NO:22, p is 1 or 2, and S is derived from the side chain of the cysteine ​​residue at position 375 of the constant domain of the heavy chain as defined according to Eu numbering.

[0081] The present invention further provides a compound of formula: [ka] wherein Ab is an anti-Trop2 antibody comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:20 and two light chains having the amino acid sequence set forth in SEQ ID NO:22, p is 1 or 2, and S is derived from the side chain of the cysteine ​​residue at position 375 of the constant domain of the heavy chain as defined according to Eu numbering.

[0082] In a further embodiment of the MMAE-conjugated ADC, the ADC is [ka] where Ab is an anti-TROP2 antibody, p is 1 or 2, and S is derived from the side chain of the cysteine ​​residue at position 375 of the constant domain of the heavy chain as defined according to Eu numbering. Includes:

[0083] The present invention further provides a first composition comprising one or more of the above-described MMAE-conjugated ADCs and a pharmaceutically acceptable carrier. In further embodiments, the predominant ADC species in the composition comprises (i) antibodies whose heavy chain C-terminus lacks a lysine residue, (ii) antibodies whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate, or (iii) antibodies whose heavy chain C-terminus lacks a lysine residue and whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate.

[0084] The present invention further provides a method for treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of any one of the aforementioned MMAE-conjugated ADCs or the aforementioned first composition to treat the cancer, wherein the cancer is a cancer that overexpresses TROP2. In further method embodiments, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0085] The present invention further provides a use of any one of the above-described MMAE-conjugated ADCs or the above-described first composition for the manufacture of a medicament for treating a cancer that overexpresses TROP2. In a further embodiment of the use, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0086] The present invention further provides any one of the above-mentioned MMAE-conjugated ADCs or the above-mentioned first composition for treating a cancer that overexpresses TROP2. In further embodiments, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0087] The present invention further provides a combination therapy for treating cancer comprising any one of the aforementioned MMAE-conjugated ADCs or the aforementioned first composition and a therapeutic agent, wherein the cancer is a TROP2-overexpressing cancer. In a further embodiment of the combination therapy, the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody. In a further embodiment of the combination therapy, the therapeutic antibody is a checkpoint inhibitor. In a further embodiment of the combination therapy, the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody. In further embodiments of the combination therapy, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0088] In a further aspect, the invention provides an ADC (second MMAE-conjugated ADC) comprising an antibody that specifically binds to human TROP2 conjugated to a linker-MMAE payload, wherein the antibody comprises two heavy chains, each heavy chain comprising a variable domain and a constant domain, wherein the variable domain comprises the amino acid sequence of SEQ ID NO: 14, and two light chains, each light chain comprising a variable domain comprising the amino acid sequence of SEQ ID NO: 15.

[0089] In a further embodiment of the second MMAE-conjugated ADC, the antibody exhibits reduced hydrophobicity relative to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

[0090] In further embodiments of the second MMAE-conjugated ADC, the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain and positions 165 and 168 of the light chain constant domain, wherein the numbering of the heavy chain constant domain positions is according to Eu numbering and the numbering of the light chain constant domain positions is according to consecutive numbering of the entire light chain sequence.

[0091] In a further embodiment of the second MMAE-conjugated ADC, the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

[0092] In further embodiments of the second MMAE-conjugated ADC, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 59, 60, or 61, and an LC comprising the amino acid sequence of SEQ ID NO: 21.

[0093] In further embodiments of the second MMAE-conjugated ADC, the linker-MMAE payload is conjugated to a cysteine ​​or a non-standard amino acid.

[0094] In a further embodiment of the second MMAE-conjugated ADC, the antibody is one in which its SH groups are represented by the formula: MP-AA-PABC-MMAE: [ka] ; CM2P-AA-PABC-MMAE: [ka] CM3P-AA-PABC-MMAE: [ka] a linker comprising a cysteine ​​residue conjugated to an MMAE payload;

[0095] In a further embodiment of the second MMAE-conjugated ADC, the ADC has the formula: [ka] where Ab is an antibody of the second MMAE-conjugated ADC that specifically binds to human TROP2, p is an integer from 1 to 8, and S is derived from the side chain of a cysteine ​​residue of the antibody.

[0096] In a further embodiment of the second MMAE-conjugated ADC, the ADC has the formula: [ka] wherein Ab is an antibody of the second MMAE-conjugated ADC that specifically binds to human TROP2, and the antibody comprises a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue, wherein the antibody comprising the engineered cysteine ​​residue is (A): (a) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:78 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (b) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:79 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (c) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 80 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (d) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 81 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (e) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 82 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (f) αTROP2(HC:BSM)(LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:52; (g) αTROP2(HC:BSM)(LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:53; (h) αTROP2 (HC:BSM-YTE) (LC:BSM-E165C) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 60 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (i) αTROP2 (HC:BSM-YTE) (LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 60 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (j) αTROP2(HC:BSM-YTE-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 85 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (k) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 86 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (l) αTROP2(HC:BSM-YTE-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 87 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (m) αTROP2(HC:BSM-YTE-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 88 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (n) αTROP2(HC:BSM-YTE-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 89 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; and (o) αTROP2(HC:BSM-YTE-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:90 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4; or (B) (a) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 84 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; and (b) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 91 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4; Includes:

[0097] In a further embodiment of the second MMAE-conjugated ADC, the ADC has the formula: [ka] wherein Ab is an antibody of the second MMAE-conjugated ADC that specifically binds to human TROP2, and the antibody comprises a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue, wherein the antibody comprising the engineered cysteine ​​residue is (A): (a) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:38 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (b) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:39 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (c) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:40 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (d) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:41 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (e) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:42 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (f) αTROP2(HC:BSM)(LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (g) αTROP2(HC:BSM)(LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (h) αTROP2 (HC:BSM-YTE) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (i) αTROP2 (HC:BSM-YTE) (LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (j) αTROP2(HC:BSM-YTE-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:44 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (k) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:45 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (l) αTROP2(HC:BSM-YTE-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:46 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (m) αTROP2(HC:BSM-YTE-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:47 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (n) αTROP2(HC:BSM-YTE-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:48 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; and (o) αTROP2(HC:BSM-YTE-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:20 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1 or 2; or (B) (a) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 43 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; and (b) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:49 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; wherein S is derived from the side chain of an engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4; Includes:

[0098] In a further embodiment of the second MMAE-conjugated ADC, the ADC has the formula: [ka] where Ab is an antibody of the second MMAE-conjugated ADC that specifically binds to human TROP2, and p is 1 or 2, where S is derived from the side chain of a cysteine ​​residue of the antibody.

[0099] The present invention further provides a second composition comprising one or more of the second MMAE-conjugated ADCs described above and a pharmaceutically acceptable carrier. In further composition embodiments, the predominant ADC species in the composition comprises (i) antibodies whose heavy chain C-terminus lacks a lysine residue, (ii) antibodies whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate, or (iii) antibodies whose heavy chain C-terminus lacks a lysine residue and whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate.

[0100] The present invention further provides a method for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of any one of the second MMAE-conjugated ADCs or the second composition to treat the cancer, wherein the cancer is a cancer that overexpresses TROP2. In further method embodiments, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0101] The invention further provides the use of any one of the second MMAE-conjugated ADCs described above or the second compositions described above for the manufacture of a medicament for treating a cancer that overexpresses TROP2. In further embodiments of the use, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0102] The present invention further provides any one of the second MMAE-conjugated ADCs or second compositions described above for the treatment of a cancer that overexpresses TROP2. In further embodiments, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0103] The present invention further provides a combination therapy for treating cancer comprising any one of the second MMAE-conjugated ADCs or the second compositions described above and a therapeutic agent, wherein the cancer is a TROP2-overexpressing cancer. In a further embodiment of the combination therapy, the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody. In a further embodiment of the combination therapy, the therapeutic antibody is a checkpoint inhibitor. In a further embodiment of the combination therapy, the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

[0104] In further embodiments of the combination therapy, the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0105] The invention further provides a pharmaceutically acceptable salt or solvate of any one of the aforementioned ADCs. [Brief explanation of the drawings]

[0106] [Figure 1]Figure 1 shows an ELISA demonstrating that the Y53D substitution in CDR2 of the sacituzumab light chain produces an anti-TROP2 antibody that preferentially binds to cells expressing high levels of TROP2 on the cell surface compared to cells expressing low levels of TROP2 on the cell surface. BxPC3 cells express high levels of TROP2 on the cell surface, while MDA-MB-231 cells express low levels of TROP2 on the cell surface. [Figure 2] FIG. 2 shows a comparison of the performance of sacituzumab (hRS7 clinical sequence) and sacituzumab BSM (hRS7 BSM sequence) on reversed-phase high-performance liquid chromatography (RP-HPLC) at 70° C. [Figure 2-1] Figure 2-1 shows a comparison of the performance of sacituzumab (hRS7 clinical sequence) and sacituzumab BSM (hRS7 BSM sequence) in reversed-phase high-performance liquid chromatography (RP-HPLC) at 70° C. AU on the Y-axis refers to absorbance units. [Figure 3] Figure 3 shows a comparison of the hydrophobicity of the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody and the αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) antibody, and the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y105S) antibody and the αTROP2(HC:BSM-S375C)(LC:BSM-Y105S) antibody. [Figure 4] Figure 4 shows a comparison of the hydrophobicity of the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody and the αTROP2(HC:BSM-S375C)(LC:BSM) antibody. [Figure 5] Figure 5 shows the rhesus monkey pharmacokinetics (PK) of several framework antibody variants after a 3 milligram per kilogram (mpk) intravenous (IV) bolus administration. The antibodies were sacituzumab (αTROP2), αTROP2(HC:Sac-S375C) (LC:Sac), and αTROP2(HC:BSM-S375C) (LC:BSM). [Figure 6]Figure 6 shows rhesus monkey PK of light chain Y53D and heavy chain Y105S antibody affinity mutants after a 1 mpk IV bolus administration. The antibodies were sacituzumab (αTROP2), αTROP2(HC:BSM-S375C)(LC:BSM-Y53D), and αTROP2(HC:BSM-S375C-Y105S)(LC:BSM). [Figure 7] Figure 7 shows rhesus monkey PK of antibody affinity mutants after a single 20 mpk IV dose. The antibodies were αTROP2(HC:BSM-YTE-S375C)(LC:BSM) and αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D). [Figure 8] Figure 8 shows rhesus monkey PK of antibody affinity mutants after a single 20 mpk IV dose. The antibodies were αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) and αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D). [Figure 9] Figure 9 shows the immunogenicity risk profile of the heavy and light chain amino acid sequences of αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) compared to the immunogenicity risk profile of the heavy and light chains of sacituzumab. [Figure 10] Figure 10 shows a comparison of the RP-HPLC performance of αTROP2(HC:BSM-YTE-S375C-MMAE) (LC:BSM) ADC and αTROP2(HC:Sac-S375C-MMAE) (LC:Sac) ADC. [Figure 11] Figure 11 shows the mass spectrometry (MS) profile of a composition comprising αTROP2-MP-AA-PABC-MMAEADC, with positions for DAR0, DAR1, DAR2, and DAR3 indicated. The αTROP2 antibody comprises HC:BSM-YTE-S375C-MMAE and LC:BSM-Y53D. [Figure 12]Figure 12 shows the hydrophobicity chromatography (HIC) profile of a composition containing αTROP2-MP-AA-PABC-MMAEADC, with positions for DAR0, DAR1, DAR2, and DAR3 indicated. The αTROP2 antibody contains HC:BSM-YTE-S375C-MMAE and LC:BSM-Y53D. The control antibody profile is unconjugated antibody. [Figure 13] Figure 13 shows a comparison of the antitumor efficacy of several binding-tuned sacituzumab variants conjugated to maleimide-C2-Ala-Ala-PABC-MMAE in the BxPC3 mouse model. The graph shows the change in tumor volume over time after a single dose on day 0. [Figure 14] Figure 14 shows a comparison of the antitumor efficacy of αTROP2 (HC:BSM-YTE-S375C-MMAE) (LC:BSM-Y53D) ADC at four doses in a BxPC3 mouse model. The graph shows the change in tumor volume over 35 days after a single dose on day 0. [Figure 15] Figure 15 shows rat PK for αTROP2 (HC:BSM-YTE-S375C-MMAE) (LC:BSM-Y53D). [Figure 16] Figure 16 shows the VH and VL of αTROP2(HC:BSM)(LC:BSM-Y53D). CDRs as defined by Kabat are underlined, and BSM amino acid substitutions are shown in bold. The numbering and consecutive numbering of VH according to Kabat are shown. The consecutive numbering of VL is also shown. DETAILED DESCRIPTION OF THE INVENTION

[0107] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0108] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0109] As used herein, the term "TROP2" refers to trophoblast surface antigen 2, also known as tumor-associated calcium signal transducer 2 (TACSTD2) or epithelial glycoprotein-1 antigen (EGP-1). TROP2 is a protein encoded by the TACSTD2 gene in humans. This intronless gene encodes a cancer-associated antigen defined by the monoclonal antibody GA733. This antigen is a member of a family containing at least two type I membrane proteins. It transduces intracellular calcium signals and acts as a cell surface receptor. TROP2 expression was first reported in trophoblast cells (placenta) and fetal tissues (e.g., lung). Later, its expression was also described in normal stratified squamous epithelium of the skin, cervix, esophagus, and tonsillar crypts. TROP2 plays a role in tumor progression by actively interacting with several key molecular signaling pathways traditionally associated with cancer development and progression. Aberrant overexpression of TROP2 has been described in several solid tumors, including colorectal, renal, lung, bladder, and breast cancers. TROP2 expression has also been described in several rare and aggressive malignancies, such as salivary duct, anaplastic thyroid, uterine / ovarian, and neuroendocrine prostate cancers.

[0110] As used herein, an antigen and an antigen-binding polypeptide (K D The term "affinity" is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antibody (or fragment thereof), expressed in terms of the equilibrium constant for dissociation of the antigen with the antigen, K D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can be expressed as 1 / K D The affinity constant (K AAffinity can be determined by known methods depending on the specific antigen of interest. For example, K D can be determined by surface plasmon resonance (SPR; Biacore™). -6 Less than K D The value will be considered to indicate binding. Specific binding of an antibody or fragment thereof to an antigen or antigenic determinant can be determined by any suitable known method, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, equilibrium dialysis, equilibrium binding, gel filtration, enzyme-linked immunosorbent assays (ELISA), SPR or spectroscopy (e.g., using fluorescence assays), and various modifications thereof known in the art.

[0111] As used herein, the term "avidity" is a measure of the strength of binding between an antibody or fragment thereof and its associated antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antibody and the number of associated binding sites present on the antibody. Avidity affects both the association and dissociation steps of the binding reaction. The association rate increases as the antibody can bind to several sites, which simply increases the association rate constant due to the multiplicity of the reaction. For example, a typical IgG antibody is bivalent for a specific target; each arm of the antibody contains a Fab portion that can bind independently to the target. Following initial association, the other Fab portion can bind to an adjacent copy of the target in an intramolecular reaction called ring closure. Because ring closure occurs intramolecularly, it is concentration-independent. Instead, it depends on the structure of the antibody and antigen, which together define the effective concentration (Mack et al., J. Am. Chem. Soc. 133:11701-11715 (2011); Mack et al., J. Am. Chem. Soc. 134:333-345 (2012)). Dissociation from the two targets requires the simultaneous release of both Fab moieties and is therefore dependent on the ring closure equilibrium and effective concentration. In principle, the avidity of a bivalent interaction could be predicted from the effective concentration of ring closure (Bobrovnik, J. Mol. Recognit. 20:253-262 (2007)). Effective concentrations and binding activities have been previously studied using either model systems (Mack et al., supra) or theoretical models (Diestler & Knapp, Phys. Rev. Lett. 100:178101 (2008); Diestler & Knapp, J. Phys. Chem. C 114(12), 5287-5304 (2010); Numata et al., J. Phys. Chem. B 116:2595-2604 (2012)).

[0112] As used herein, the terms "administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition, including a human TROP2-binding agent or ADC disclosed herein, with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid in which the fluid is in contact with the cell. "Administration" and "treatment" also refer to in vitro and ex vivo treatment of a cell, for example, with a reagent, diagnostic, binding compound, or another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., a human, rat, mouse, dog, cat, rabbit). In a preferred embodiment, the term "subject" refers to a human.

[0113] As used herein, the term "amino acid" refers to a simple organic compound containing both a carboxyl (-COOH) group and an amino (-NH2) group. Amino acids are the building blocks of proteins, polypeptides, and peptides. Amino acids exist in L- and D-forms, with the L-form being the naturally occurring proteins, polypeptides, and peptides. The amino acids and their code names are listed in Table 1 below.

[0114] [Table 1]

[0115] As used herein, the term "antibody" or "immunoglobulin" refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC contains a heavy chain variable region or domain (V H ) and a heavy chain constant region or domain. Each light chain is composed of a LC variable region or domain (V L) and LC constant domains. In certain naturally occurring IgG, IgD, IgE, IgM, and IgA antibodies, the heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Generally, the basic antibody structural unit of an antibody is a Y-shaped tetramer containing two HC / LC pairs (2H). Each tetramer contains two identical pairs of polypeptide chains, each pair having one LC (approximately 25 kDa) and one HC chain (approximately 50-70 kDa) (H+L). Each HC:LC pair is composed of one V H :One V L Includes a pair of V H :One V L The pair is sometimes referred to by the term "Fab." Thus, each antibody tetramer contains two Fabs, one in each arm of the Y-shaped antibody.

[0116] The LC constant domain is composed of one domain, CL. H The seven family members are: H 1. V H 2. V H 3. V H 4. V H 5. V H 6, and V H 7, including human V L The following 16 family members: κ 1. V κ 2. V κ 3. V κ 4. V κ 5. V κ 6. V λ 1. V λ 2. V λ 3. V λ 4. V λ 5. V λ 6. V λ 7. V λ 8. V λ 9, and V λ 10. Each of these family members can be further divided into specific subtypes. H and V Lcan be further subdivided into regions of hypervariability called complementarity-determining regions (CDR) regions, interspersed with more conserved regions called framework regions (FR). H and V L is composed of three CDR regions and four FR regions arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The numbering of the amino acids in VH can be determined using the Kabat numbering scheme. See Beranger, et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG, created 17 / 05 / 2001, version: 08 / 06 / 2016, which is accessible at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0117] The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Typically, the numbering of amino acids in the heavy chain constant domain begins at number 118 according to the Eu numbering scheme. The Eu numbering scheme is based on the human IgG1 (Eu) amino acid sequence, with the constant domain starting at position 118 (Eu) of the IgG1 amino acid sequence described in Edelman et al., Proc. Natl. Acad. Sci. USA 63:78-85 (1969), and for IgG1, IgG2, IgG3, and IgG4 constant domains, as shown in Beranger et al. (supra).

[0118] The variable regions of the heavy and light chains contain binding domains that contain CDRs that interact with antigens. Several methods for defining CDR sequences of antibody variable domains are available in the art (see Dondelinger et al., Frontiers in Immunol. 9:Article 2278 (2018)). Common numbering schemes include: The Kabat numbering scheme is based on sequence variability and is the most commonly used (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (defining the CDR regions of an antibody by sequence); the Chothia numbering scheme is based on the location of structural loop regions (see Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997)); the AbM numbering scheme is a comparison between the two used by Oxford Molecular's AbM antibody modeling software (Karu et al., ILAR Journal 37:132-141 (1995); the contact numbering scheme is based on the analysis of available complex crystal structures (see www.bioinf.org.uk; Prof. Andrew C. Martin's Group; Abhinandan & Martin, Mol. Immunol. 45:3832-3839 (2008)); the IMGT (ImMunoGeneTics) numbering scheme is a standardized numbering system for all protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains and T-cell receptor chains from different species, counting residues consecutively from 1 to 128 based on germline V sequence alignments (Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509 (1997); Lefranc et al., Dev Comp Immunol. 27:55-77 (2003).

[0119] The following general rules, as disclosed in www.bioinf.org.uk: Prof. Andrew C. Martin's Group and reproduced below in Table 2, may be used to define CDRs in an antibody sequence that contain amino acids that specifically interact with amino acids that comprise the epitope in the antigen to which the antibody binds. There are rare instances where these generally consistent features do not occur. However, Cys residues are the most conserved features.

[0120] [Table 2]

[0121] V H The entire amino acid sequence of V is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any one of the aforementioned numbering schemes. H The numbering of amino acid positions in the V region may start at amino acid position 1 and continue consecutively to the end of the sequence, or may be consecutive according to Kabat. Figure 16 shows the V region of αTROP2 BSM numbered consecutively according to Kabat. H Unless otherwise specified, V H and V L The amino acid positions in are defined according to consecutive numbering.

[0122] The numbering of amino acid positions in the heavy chain constant domain may start at amino acid position 1 and continue consecutively to the end of the sequence, or may continue according to Eu numbering. The IgG1 heavy chain constant domain amino acid sequence has 330 amino acids numbered consecutively from 1 to 330. The corresponding sequence numbered according to Eu starts at position 118 and ends at position 447. Unless otherwise specified, the amino acid positions of the heavy and light chains herein are defined according to consecutive numbering.

[0123] As used herein, the term "Fc domain" or "Fc" refers to a crystallizable fragment domain or region obtained from an antibody, comprising the antibody CH2 and CH3 domains. In an antibody, the two Fc domains are held together by two or more disulfide bonds and the hydrophobic interactions of the CH3 domain. The Fc domain can be obtained by digesting an antibody with the protease papain. Typically, the amino acids in the Fc domain are numbered according to the EU numbering convention (see Edelmann et al., Biochem. 63:78-85 (1969)).

[0124] As used herein, the term "antigen" as used herein refers to any foreign substance that induces an immune response in the body.

[0125] As used herein, the term "antigen-binding fragment" refers to one or more polypeptides comprising a fragment of a full-length antibody that retains the ability to specifically bind to the antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv regions, and scFvs.

[0126] As used herein, "specifically binds," with respect to a target antigen, refers to the fact that all or part of the binding agent preferentially associates with the target antigen and does not preferentially associate with other molecules, particularly molecules found in human blood or serum. The binding agents described herein typically bind to 10 -7 ~10 -11 The dissociation constant (K D ) specifically binds to the target antigen with high affinity, reflected by approximately 10 -6 Any K greater than M D As used herein, a binding agent that "specifically binds" or "specifically binds" to a target antigen refers to a binding agent that binds to the target antigen with high affinity, which is generally considered to indicate non-specific binding. -7 K below M D, particularly in the embodiment 10 -8 M or less, or 5 x 10 -9 M or less, or 10 -8 M~10 -11 K below M D "An antibody specifically binding to human TROP2 may also bind to homologs of human TROP2, such as rhesus monkey TROP2 and rat TROP2, as long as it is specific for the TROP2 homolog. This term means having a specific binding affinity to a target antigen, but does not bind to non-target antigens with measurable binding as determined by cell ELISA or surface plasmon resonance (SPR) assays using 10 μg / mL of antibody. This term does not exclude antibodies that bind to homologs of the target. For example, an antibody that specifically binds to human TROP2 may also bind to homologs of human TROP2, such as rhesus monkey TROP2 and rat TROP2, as long as it is specific for the TROP2 homolog.

[0127] As used herein, the term "Fab fragment" refers to a fragment that contains one antibody light chain and the CH1 and V of one antibody heavy chain. H The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fab fragment" can be the product of papain cleavage of an antibody.

[0128] As used herein, the term "Fab' fragment" refers to a fragment comprising one antibody light chain and a V(ab')2 heavy chain, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. H and a portion or fragment of one antibody heavy chain comprising the CH1 domain up to the region between the CH1 and CH2 domains.

[0129] As used herein, the term "F(ab')2 fragment" refers to a fragment of an antibody that has been isolated from two antibody light chains and up to the region between the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. H and two heavy chains containing a CH1 domain. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains. An "F(ab')2 fragment" may be the product of pepsin cleavage of an antibody.

[0130] As used herein, the term "Fv region" refers to an antigen-binding agent that comprises the variable regions from both the heavy and light chains of an antibody, but lacks the constant regions.

[0131] As used herein, the term "ScFv" or "single-chain variable fragment" refers to VFvs fused or linked to each other by a short linker peptide of 10 to about 25 amino acids. H and V L The linker is typically rich in glycine for flexibility, rich in serine or threonine for solubility, and V H N-terminus of V L The C-terminus of the immunoglobulin can be linked to the C-terminus of the immunoglobulin, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker.

[0132] As used herein, the term "diabody" refers to a group of molecules that are composed of the same polypeptide chain (V H -V L or V L -V H ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H (Diabodies) refers to antigen-binding agents comprising small antibody fragments having two antigen-binding regions comprising a nucleotide sequence (nucleotide sequence) and a nucleotide sequence (nucleotide sequence). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding regions. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0133] These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0134] As used herein, the term "binding agent" refers to an antibody or antigen-binding fragment thereof.

[0135] The term "antibody-drug conjugate" or "ADC" refers to an antibody or binding agent conjugated to one or more (typically 1 to 8) payloads, each attached via a linker to a specific site on the antibody or binding agent. The antibodies are typically monoclonal antibodies specific for a cancer antigen and are capable of delivering the payload to cells expressing the cancer antigen on their extracellular surface.

[0136] As used herein, the term "DAR" or "drug-antibody ratio" refers to the average number of linker-to-payload moieties attached to an antibody present in a composition. For a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is the average of the DARs (of all linker-payload moieties of individual antibody-drug conjugate molecules present in the composition), this average expressed as a decimal. Thus, in some embodiments of a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal between 0 and 24, 0 and 8, 0 and 7, 0 and 6, 0 and 5, 0 and 4, 0 and 3, 0 and 2, and 0 and 1. In further embodiments, for a composition comprising an antibody-drug conjugate of the present disclosure, the DAR of the composition is a decimal between 1 and 4, 2 and 5, 3 and 6, 4 and 7, 5 and 8, and 6 and 8. In other embodiments, for compositions comprising an antibody-drug conjugate of the disclosure, the DAR of the composition is a decimal number between 1 and 3, 2 and 4, 3 and 5, 4 and 6, 5 and 7, and 6 and 8. In further embodiments, for compositions comprising an antibody-drug conjugate of the disclosure, the DAR of the composition is a decimal number between 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, and 7 and 8. In certain embodiments, the DAR of the composition is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, The average DAR is 0.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. As used above, the term "composition" is understood to encompass pharmaceutical compositions. The average DAR can be determined by a variety of conventional means, such as UV spectroscopy, mass spectrometry, ELISA assay, radiometric methods, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0137] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular T cell receptor activation signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activating stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells, triggering proliferation, cytokine production, phagocytosis, and / or production of molecules that can mediate cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0138] As used herein, the terms "extracellular antigen-binding domain," "extracellular domain," or "extracellular ligand-binding domain" when used in reference to a CAR refer to the portion of a CAR that is located on the outside of the cell membrane and is capable of binding to an antigen, target, or ligand.

[0139] As used herein, the term "hinge region" when used in reference to a CAR refers to the portion of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain.

[0140] As used herein, the term "transmembrane domain" refers to the portion of a CAR that extends across and anchors the CAR to the cell membrane.

[0141] As used herein, the terms "intracellular T cell receptor activation signaling domain," "cytoplasmic signaling domain," or "intracellular signaling domain" refer to the portion of a CAR that is located inside the cell membrane and is capable of transducing an effector signal.

[0142] As used herein, the term "isolated" antibody or antigen-binding fragment thereof is at least partially free from other biological molecules from the cell or cell culture in which it is produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth medium. An isolated antibody or antigen-binding fragment may also be at least partially free from expression system components, such as biological molecules from a host cell or its growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of a pharmaceutical formulation that includes the antibody or fragment.

[0143] As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible minor naturally occurring mutations. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). See also Presta, J. Allergy Clin. Immunol. 116:731 (2005).

[0144] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, a gene includes coding and / or regulatory sequences necessary for its expression. For example, "gene" refers to an mRNA, functional RNA, or nucleic acid fragment that expresses a specific protein, including regulatory sequences. "Gene" also includes non-expressed DNA segments that form, for example, recognition sequences for other proteins. "Gene" can be obtained from a variety of sources, including cloning from a desired source or synthesis from known or predicted sequence information, and can include sequences designed to have desired parameters. Genes include both naturally occurring nucleotide sequences encoding a molecule of interest and synthetically derived nucleotide sequences encoding a molecule of interest, such as complementary DNA (cDNA) derived from a messenger RNA (mRNA) nucleotide sequence.

[0145] As used herein, the term "polynucleotide" discussed herein forms part of the present invention. A "polynucleotide," "nucleic acid," or "nucleic acid molecule" includes single- or double-stranded DNA and RNA. For example, a polynucleotide encoding an immunoglobulin chain or component of the antibody display system of the present invention may, in one embodiment of the present invention, be adjacent to its natural regulatory (expression control) sequences or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5' and 3' non-coding regions, and the like.

[0146] For example, a polynucleotide encoding an immunoglobulin chain or component of an antibody or ADC of the invention can be operably associated with a promoter. In one embodiment of the invention, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding a cellular RNA polymerase (e.g., directly or via a protein or substance bound to another promoter) and initiating transcription of a coding sequence. A promoter sequence is generally attached at its 3' end to a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence, a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1) and protein binding domains (consensus sequences) involved in the binding of RNA polymerase can be found. A promoter can be operably associated with other expression control sequences, including enhancer and repressor sequences, or with a nucleic acid of the invention.Promoters that can be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequence of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); the β-lactamase promoter (Villa-Komaroff et al., Nature 296:39-42 (1982)); Prokaryotic expression vectors such as the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also "Useful proteins from recombinant bacteria" in Scientific American 242:74-94 (1980)); and promoter elements derived from yeast or other fungi, such as the Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, or alkaline phosphatase promoter.

[0147] As used herein, the terms "vector," "cloning vector," and "expression vector" include vehicles (e.g., plasmids) capable of introducing DNA or RNA sequences into host cells, which may transform the host and promote the expression and / or replication of the introduced sequences. Polynucleotides encoding immunoglobulin chains or components of antibodies or ADCs of the invention may, in one embodiment of the invention, be present in a vector.

[0148] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that not all progeny of a parent cell have precisely identical DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a clear designation is intended, it will be clear from the context.

[0149] As used herein, the term "control sequence" or "regulatory sequence" refers to the DNA sequence required for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for expression in eukaryotes include, for example, promoters, operator or enhancer sequences for the expression of messenger RNA encoding protein, transcription termination sequences, and polyadenylation sequences, and ribosome binding sites for promoting the translation of messenger RNA.

[0150] As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, e.g., a regulatory sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0151] As used herein, the term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" are degenerate versions of each other and include all nucleotide sequences that encode the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can contain introns.

[0152] As used herein, the term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0153] As used herein, the term "TROP2-binding agent" refers to the anti-TROP2 antibodies and antigen-binding fragments thereof of the present invention. This term specifically excludes other anti-TROP2 antibodies, such as sacituzumab.

[0154] As used herein, the terms "treat" or "treating" refer to the topical, subcutaneous, intramuscular, intradermal, intravenous, or systemic administration of a therapeutic moiety, such as a composition containing any of the anti-TROP2 binding agents or ADCs of the present invention, to an individual in need thereof. The amount of a therapeutic moiety effective to treat cancer or a proliferative disease in an individual may vary according to factors such as the individual's injury or disease state, age and / or weight, and the ability of the therapeutic agent to elicit a desired response in the individual. Achievement of a therapeutic goal can be assessed by personal and / or any clinical measurements routinely used by physicians or other skilled health care providers to assess the severity or progress of treatment. Thus, these terms indicate that a beneficial result has been or will be imparted to a human or animal individual in need thereof. Treatment may be therapeutic or prophylactic.

[0155] As used herein, the term "treatment" as applied to a human or veterinary individual refers to therapeutic or prophylactic treatment, as well as diagnostic applications. "Treatment," when applied to a human or veterinary individual, encompasses contacting a human or animal subject with a TOP2-binding agent or ADC of the invention.

[0156] As used herein, the term "therapeutically effective amount" refers to an amount of a particular substance sufficient to achieve a desired effect in the treated individual. For example, this may be the amount necessary to inhibit or reduce the severity of a disease or disorder in the individual.

[0157] As used herein, the term "combination therapy" refers to the treatment of a human or animal individual that involves administering a first therapeutic agent and a second therapeutic agent to the individual, either sequentially or simultaneously. Generally, the first and second therapeutic agents are administered to the individual separately, rather than as a mixture. However, there may be embodiments in which the first and second therapeutic agents are mixed prior to administration.

[0158] As used herein, the term "solvate" refers to a physical association of an ADC disclosed herein with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, a solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of solvates include ethanolates, methanolates, and the like. A "hydrate" is a solvate in which the solvent molecule is water.

[0159] One or more ADCs disclosed herein may be converted into solvates. The preparation of solvates is generally known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci., 93(3), 601-611 (2004) describes the preparation of solvates of the antifungal fluconazole in ethyl acetate and water. Similar preparations of solvates, hemisolvates, hydrates, etc. are described in Tonder et al., AAPS PharmSciTechours., 5(1), article 12 (2004); and A.L. Bingham et al., Chem. Commun., 603-604 (2001). A typical, non-limiting method involves dissolving a compound of the present invention in a desired amount of a desired solvent (organic or aqueous, or a mixture thereof) at a temperature above room temperature, cooling the solution at a rate sufficient to form crystals, and then isolating them by standard methods. Analytical techniques such as, for example IR spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

[0160] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and base salts.

[0161] Exemplary acid addition salts include acetate, ammonium, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphor, camphorsulfonate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate (also known as mesylate), naphthalenesulfonate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (also known as tosylate), etc. Additionally, acids generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds can be found, for example, in P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2 nd Revised Ed. (2011) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986)-33 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, DC on their website), the disclosures of which are incorporated herein by reference. In one embodiment, the acid salt is an ammonium salt or a diammonium salt.

[0162] Exemplary base salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as dicyclohexylamine, t-butylamine, choline, and the like, and salts with amino acids such as arginine, lysine, etc. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, and dibutyl sulfate), long-chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides, and iodides), aralkyl halides (e.g., benzyl bromide and phenethyl bromide), and the like.

[0163] All such acid and base salts are intended to be pharmaceutically acceptable salts within the scope of this disclosure, and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of this disclosure.

[0164] Introduction: The present invention relates to low TROP2 expressing cells (TROP2 低 cells) than high TROP2 expressing cells (TROP2 高 The present invention provides a TROP2-binding agent that preferentially binds to TROP2-expressing cells (e.g., TROP2-positive cells), and a conjugate comprising the TROP2-binding agent conjugated to a payload. In certain embodiments, the TROP2-binding agent is an anti-TROP2 antibody of the present invention conjugated to a payload to provide an anti-TROP2 antibody-drug conjugate (ADC) of the present invention. As shown in the Examples, the anti-TROP2 ADC is stable and effective in mouse and non-human primate (NHP) models. The ADC of the present invention is useful for treating, imaging, diagnosing, preventing the growth of, containing, and reducing TROP2-expressing cells, particularly TROP2-expressing tumors.

[0165] The ADCs of the present invention can be used to treat disorders involving cells that overexpress TROP2 on the cell surface. Examples of such disorders include, but are not limited to, breast cancer, triple-negative breast cancer (TNBC), ovarian cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, neuroendocrine cancer, prostate cancer, sarcoma, gastric cancer, esophageal cancer, and cervical cancer.

[0166] TROP2-binding agents The present invention provides a method for the detection of TROP2, as determined by cell-based enzyme-linked immunosorbent assay (ELISA). 低 TROP2 over cells 高 Provided are TROP2 binding agents (anti-TROP2 antibodies and antigen-binding fragments thereof) that preferentially bind to cells and exhibit reduced hydrophobicity compared to sacituzumab, as determined by hydrophobic interaction chromatography (HIC). Sacituzumab is a humanized anti-TROP2 antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 12. Sacituzumab is disclosed in U.S. Patent No. 9,745,380 (SEQ ID NO: 14 (V H ) and 13(V L ) have been disclosed in the literature. The antibody-drug conjugate (ADC) sacituzumab govitecan-hziy is currently available commercially under the trade name TRODELVY.

[0167] In general, TROP2 is overexpressed in various cancers, such as colorectal cancer, pancreatic cancer, gastric cancer, oral squamous cell carcinoma, ovarian cancer, bladder cancer, and breast cancer, compared with the expression in corresponding normal tissues and various other tissues (Ohmachi et al., Clin Cancer Res. 12:3057-63 (2006); Fong et al., Br J Cancer. 99:1290-5 (2008); Lin et al., Exp Mol Pathol. 94:73-8 (2013); Bignotti et al., Eur J Cancer. 46:944-53 (2010); Muhlmann et al., J Clin Pathol. 62:152-8 (2009); Fong et al., Mod Pathol. 21:186-91 (2008)). In these studies, carcinomas with high TROP2 expression showed poor prognosis. TROP2 高 The preferential binding of the TROP2-binding agents of the present invention to cells reduces the risk of off-target binding, thus limiting undesirable adverse events (AEs) during treatment regimens. Selectivity is particularly advantageous for use in cancer treatment regimens targeting cancers that overexpress TROP2. Thus, ADCs comprising the anti-TROP2 antibodies of the present invention conjugated to a therapeutic moiety, e.g., a cytotoxin such as an inhibitor of topoisomerase I or II or an inhibitor of microtubule assembly, are particularly useful in treatment regimens targeting cancers that overexpress TROP2.

[0168] The TROP2-binding agent of the present invention has a reduced binding activity to TROP2 when a tyrosine is introduced into the aspartic acid amino acid substitution at position 53 of the light chain of sacituzumab (Y53D amino acid substitution). 低 TROP2 over cells 高Incorporating the discovery that a modified sacituzumab (αTROP2(HC:Sac)(LC:Sac-Y53D) antibody) is provided that has preferential or selective binding to cells. Furthermore, unexpectedly, the Y53D amino acid substitution was found to reduce the hydrophobicity of the αTROP2(HC:Sac)(LC:Sac-Y53D) antibody compared to sacituzumab, as determined by hydrophobic interaction chromatography (HIC). The reduced hydrophobicity may provide a TROP2-binding agent that has a reduced tendency to aggregate, allowing for the preparation of concentrated aqueous solutions of the antibody with reduced viscosity.

[0169] In an exemplary embodiment, the invention provides a heavy chain variable domain (V) having the amino acid sequence set forth in SEQ ID NO:1. H ) and a light chain variable domain (V) comprising the amino acid sequence set forth in SEQ ID NO: 3 L ) and a TROP2-binding agent with modulated binding. This exemplary anti-TROP2 antibody has the V H and the V of sacituzumab with the Y53D amino acid substitution L In a further exemplary embodiment, the invention provides a TROP2-binding agent that is an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13. This exemplary anti-TROP2 antibody comprises the amino acid sequence of the heavy chain of sacituzumab and the amino acid sequence of the light chain of sacituzumab with a Y53D amino acid substitution.

[0170] Thus, the binding-modulating TROP2-binding agents of the present invention comprise: (i) a heavy chain variable domain (V) comprising heavy chain complementarity-determining regions (HC-CDRs) 1, 2, and 3 as shown in the amino acid sequence of SEQ ID NO: 1; H ) (CDRs are defined according to Kabat, ABM, IMGT, Chothia or Contact); and (ii) a light chain variable domain (V) comprising light chain complementarity determining regions (LC-CDRs) 1, 2, and 3 as set forth in the amino acid sequence of SEQ ID NO: 3. L ) (CDRs are defined according to Kabat, ABM, IMGT, Chothia or Contact).

[0171] In a further embodiment, the binding-modulated TROP2-binding agent of the invention comprises (i) a V-CDR1 comprising the amino acid sequence NYGMN set forth in SEQ ID NO: 4, a HC-CDR2 comprising the amino acid sequence WINTYTGEPTYTDDFKG set forth in SEQ ID NO: 5, and a HC-CDR3 comprising the amino acid sequence GGFGSSYWYFDV set forth in SEQ ID NO: 6. H , (CDRs are defined according to Kabat); and (ii) a V comprising an LC-CDR1 having the amino acid sequence KASQDVSIAVA set forth in SEQ ID NO:7, an LC-CDR2 having the amino acid sequence SASDRYT set forth in SEQ ID NO:10, and an LC-CDR3 having the amino acid sequence QQHYITPLT set forth in SEQ ID NO:9. L (CDRs are defined according to Kabat).

[0172] In a further embodiment, the binding-modulating TROP2-binding agent of the invention comprises (i) a V H and (ii) V comprising the amino acid sequence set forth in SEQ ID NO:3. L In certain embodiments, V H is linked to the heavy chain constant domain of an IgG1, IgG2, IgG3, or IgG4 isotype, and V L is linked to a light chain constant domain of the human kappa or human lambda isotype. H is linked to the heavy chain constant domain of the IgG1 or IgG4 isotype, and V L is linked to a light chain constant domain of the human kappa or human lambda isotype. In further embodiments, the IgG1 or IgG4 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and / or deletions compared to the native human IgG1 or IgG4 isotype. In certain embodiments, the heavy chain constant domain is of the IgG1 isotype and may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or a combination thereof compared to the amino acid sequence of the native IgG1 isotype.

[0173] In certain embodiments, V H is linked to the constant domain of human IgG1 comprising the amino acid sequence set forth in SEQ ID NO: 92, or a variant thereof comprising the S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 103.

[0174] In a further embodiment, the constant domain of human IgG1 comprises substitutions of amino acids at positions 252, 254 and 256 of the constant domain of the heavy chain with amino acids Tyr (Y), Thr (T) and Glu (E), respectively (M252Y, S254T, T256E substitutions) (numbering according to Eu) (sequentially numbered positions are 256, 258, 260, respectively) to provide a heavy chain constant domain comprising a "YTE" substitution and having the amino acid sequence set forth in SEQ ID NO: 93, or a variant thereof comprising a S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 104.

[0175] In a further embodiment, the human IgG1 heavy chain constant domain comprises E233A and L235A amino acid substitutions (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 94, or a variant thereof comprising the S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 105.

[0176] In a further embodiment, the human IgG1 heavy chain constant domain comprises L234A L235A D265S substitutions (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 95, or a variant thereof comprising an S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 106.

[0177] In a further embodiment, the human IgG1 heavy chain constant domain comprises L234A L235A P329G substitutions (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 96, or a variant thereof comprising an S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 107.

[0178] In a further embodiment, the human IgG1 heavy chain constant domain comprises a L235E substitution (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 97, or a variant thereof comprising a S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 108.

[0179] In a further embodiment, the human IgG1 heavy chain constant domain comprises a D265A substitution (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 98, or a variant thereof comprising an S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 109.

[0180] In a further embodiment, the human IgG1 heavy chain constant domain comprises a D265A N297G substitution (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 99, or a variant thereof comprising an S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 110.

[0181] In a further embodiment, the human IgG1 heavy chain constant domain comprises N297X, where X is any amino acid other than the N substitution, to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 100, or a variant thereof comprising a S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 111 (numbering according to Eu).

[0182] In a further embodiment, the human IgG1 heavy chain constant domain comprises N297A / D356E / L358M substitutions (numbering according to Eu) to provide a heavy chain constant domain having the amino acid sequence set forth in SEQ ID NO: 101, or a variant thereof comprising an S375C substitution and having the amino acid sequence set forth in SEQ ID NO: 112.

[0183] In certain embodiments of the present invention, the IgG1 or IgG4 heavy chain constant domains disclosed herein may contain a C-terminal lysine or may lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments, the N-terminal amino acid of an antibody variable domain may undergo cyclization to pyroglutamate. Thus, in compositions comprising specific antibodies disclosed herein, the composition may contain a population of antibody species, each of which may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain an N-terminal glutamine or glutamic acid, or may contain cyclization of the N-terminal amino acid to pyroglutamate.

[0184] The invention further provides TROP2-binding agents that are rehumanized variants of sacituzumab, where the rehumanization process unexpectedly produces antibodies with reduced hydrophobicity relative to that of sacituzumab, as can be determined by hydrophobic interaction chromatography (HIC), and have a more human-like sequence than sacituzumab, with reduced predicted epitope content compared to sacituzumab, as can be determined in silico, for example, using immunogenicity prediction programs.

[0185] These rehumanized TROP2-binding agents have amino acid sequences that, compared to the amino acid sequence of the heavy chain of sacituzumab having the amino acid sequence set forth in SEQ ID NO: 11, include: (i) a light chain that has amino acid substitutions S20T, D60S, V85T, and A100P (positions defined by consecutive numbering) compared to the amino acid sequence of the light chain of sacituzumab set forth in SEQ ID NO: 12; and (ii) amino acid substitutions Q5L, K38R, A69S, T78Q, D89S, T90Q, D91Q, D92Q, D93Q, D94Q, D95Q, D96Q, D97Q, D98Q, D9 ... The rehumanized sacituzumab comprises the amino acid sequence of sacituzumab modified to include a heavy chain having the following mutations (positions defined by consecutive numbering): E, F95Y, S115T, R218K, E360D, and M362L (the equivalent positions in the VH as defined by Kabat numbering are Q5L, K38R, A68S, T77Q, D85E, F91Y, and S107T; and R214K, E256D, and M359L in the heavy chain constant domain as defined by Eu numbering). These mutations, when present together on the heavy or light chain, are referred to herein as "Best Single Mutations" (BSM). In an exemplary embodiment, the rehumanized sacituzumab is αTROP2(HC:BSM) (LCBSM), which comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 or 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 21. Rehumanized sacituzumab αTROP2(HC:BSM)(LC:BSM) exhibits a higher degree of humanization compared to sacituzumab and is less hydrophobic than sacituzumab, as determined by hydrophobic interaction chromatography (HIC).

[0186] In further embodiments, the heavy chain comprising the TROP2-binding agent disclosed herein contains a YTE substitution in the constant domain. The YTE substitution provides the TROP2-binding agent with improved PK in humans and non-human primates, but appears to have reduced PK compared to that of sacituzumab in rodents. The YTE substitution promotes FcRn-mediated recycling to minimize ADC catabolism in non-tumor normal tissues. An exemplary TROP2-binding agent is αTROP2(HC:BSM-YTE)(LC:BSM), which comprises a light chain having the amino acid sequence set forth in SEQ ID NO:21 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:18 or 62, and exhibits less hydrophobicity than sacituzumab as determined by HIC and has a longer serum half-life than sacituzumab.

[0187] In a further embodiment, the αTROP2(HC:BSM-YTE) (LC:BSM) binder comprises a light chain further comprising a Y53D amino acid substitution, providing the TROP2 binder αTROP2(HC:BSM-YTE) (LC:BSM-Y53D). These TROP2 binders exhibit (i) preferential binding to cells expressing high amounts of TROP2, such as found in TROP2-expressing cancer cells, over cells expressing low amounts of TROP2, such as found in non-cancer cells, (ii) reduced hydrophobicity compared to sacituzumab, and (iii) reduced immunogenicity potential compared to sacituzumab.

[0188] Antibody-drug conjugates The present invention further provides antibody-drug conjugates (ADCs) comprising an anti-TROP2 antibody of the invention conjugated to one or more payload molecules via a linker.

[0189] The payload used in the present invention is not particularly limited.The payload used in the present invention includes cytotoxic moieties, particularly those used in cancer treatment.Such cytotoxic moieties include, but are not limited to, DNA damaging agents, DNA binding agents, metabolic antagonists, enzyme inhibitors such as thymidylate synthase inhibitors and topoisomerase inhibitors, tubulin inhibitors, and toxins (for example, toxins of bacterial, fungal, plant or animal origin).

[0190] Illustrative examples of cytotoxic moieties include, but are not limited to, taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (e.g., etoposide or etoposide phosphate), vinblastine, vincristine, vindesine, taxanes (e.g., taxol), and taxoteretinoid acid. , butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamicin, ene-diyne, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, maytansinoids (including DM1, DM2, DM3, DM4), auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD)), 7-ethyl-10-hydroxy-camptothecin (SN-38), anthracyclines, alkylcyclines, or derivatives thereof. The therapeutic moiety can be linked to the linker via any suitable method known in the art.

[0191] The payload used in the present invention can be linked to the anti-TROP2 antibody via a linker. Various linkers for ADCs are known in the art. The linker useful in the present invention is not particularly limited, as long as it contains a moiety that can react with a thiol group on the antibody and thereby bind to the antibody. In certain embodiments, the linker is a maleimide or haloacetyl-functionalized linker. Examples of linkers include, but are not limited to, linkers having the following structure: i.-MC-vc-PABC- ("MC": maleimidocaproyl (maleimide-C6); "-vc-": dipeptide of -Val-Cit-; "PABC": para-aminobenzylcarbamate), ii.-MC-GGFG-("-GGFG-": tetrapeptide-Gly-Gly-Phe-Gly-)GGFG is disclosed as SEQ ID NO: 185; iii.-MC-vc-, iv.-MP- ("MP": maleimidopropanyl (maleimide-C3)), v.-MP-GGGG-("-GGGG-": tetrapeptide-Gly-Gly-Gly-Gly)GGGG is disclosed as SEQ ID NO: 186. vi.-MP-GGG- ("-GGG-": tripeptide -GGG-), vii.-MP-GGG-PABC-, viii.-MC-, ix.-SMCC-(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), x.-MP-AA-PABC-("Ala-Ala" dipeptide), xi.-CM2P-(5-cyano-6-methylsulfonyl 2-pyridinecarboxamide), xii.-CM3P-(5-cyano-6-methylsulfonyl 3-pyridinecarboxamide), xiii.CM2P-AA-PABC-, and xiv.CM3P-AA-PABC-, Here, the proximal end of the linker containing the reactive group MP, MC, CM2P, CM3P or SMCC can be conjugated to a reactive group (thiol group) on the antibody, and the distal end of the linker can be linked to a payload.

[0192] In further embodiments, the payload provides for conjugation in the form of a linker-payload compound intermediate having one of the following structures: I.MC-vc-PABC-Payload, II.MC-GGFG-Payload, III.MC-vc-payload, IV.MP-Payload, V.MP-GGG-Payload, VI.MP-GGGG-Payload, VII.MP-GGG-PABC-Payload, VIII.MC - Payload, IX.SMCC-Payload, X.MP-AA-PABC-Payload, XI.-CM2P-Payload, XII.-CM3P-Payload, XIII.CM2P-AA-PABC-Payload, and XIV.CM3P-AA-PABC-Payload, Here, the proximal end of the linker, which contains the reactive group MP, MC, CM2P, CM3P, or SMCC, can be conjugated to a reactive group (thiol group) on the antibody.

[0193] In certain embodiments of the foregoing linker payloads, the payload is selected from the group consisting of taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (etoposide or etoposide phosphate), and the like. The payload may be a benzophenone-3-one (e.g., benzocaine), vinblastine, vincristine, vindesine, taxanes (e.g., taxol), taxoteretic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, DM1, DM2, DM3, DM4, MMAE, MMAF, MMAD, SN-38, anthracyclines, alkylcyclines, or derivatives thereof. The aforementioned payloads further include pharmaceutically acceptable salts and solvates thereof. Exemplary linker-payloads include, but are not limited to, MC-vc-PABC-MMAE, MP-AA-PABC-MMAE, CM2P-AA-PABC-MMAE, and CM3P-AA-PABC-MMAE.

[0194] The linker-payload can form salts or solvates, which are also within the scope of this disclosure. Exemplary linker payloads further include compounds of Formula I, or pharmaceutically acceptable salts, solvates, or stereoisomers thereof: [ka] (In the formula, R 1 teeth, [ka] [ka] The wavy lines indicate the covalent binding sites, and R 2 is the cytotoxic moiety and R3 and R 4 independently, C 1~3 represents an alkyl or a naturally occurring or unnatural amino acid side chain, and n is an integer from 1 to 4).

[0195] In certain embodiments, R 2 is selected from anthracyclines, auristatins, camptothecins, duocarmycins, etoposide, maytansinoids, pyrrolobenzodiazepine dimers, DNA minor groove binders, taxanes, enediynes, antitubulins, and vinca alkaloids. 2 are auristatin T, auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-AE ester, monomethyl auristatin F, lipophilic MMAF, MMAE, lexitropsin, duocarmycin, paclitaxel and docetaxel, T67 (Tularik), vincristine, vinblastine, vindesine, vinorelbine, nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), tubulysin M, and selected from alkylcyclines, melphalan, methotrexate, mitomycin C, etoposide, CC-1065 analogs, calicheamicin, maytansine, dolastatin 10 analogs, rhizoxin, palytoxin, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colchimide, estramustine, cryptophycin, cemadotin, maytansinoids, combretastatin, discodermoid, tesirin, and eleusrobin.

[0196] In certain embodiments, R 3 and R 4 is C 1~3 alkyl, or both are CH. An exemplary linker payload is of Formula II: [ka] (In the formula, R 1 and R 2is as described herein). In certain embodiments, R 2 is an auristatin selected from auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-AE ester, MMAF, MMAE, or pyrrolobenzodiazepine dimer.

[0197] Exemplary linker payloads comprising MMAE derivatives include, but are not limited to, the following compounds: MP-AA-PABC-MMAE: [ka] ; CM2P-AA-PABC-MMAE: [ka] CM3P-AA-PABC-MMAE: [ka] .

[0198] Exemplary linker payloads further include pharmaceutically acceptable salts and solvates of the following compounds: MP-AA-PABC-MMAE: [ka] ; CM2P-AA-PABC-MMAE: [ka] CM3P-AA-PABC-MMAE: [ka] .

[0199] In some embodiments, the above exemplary linker-payloads are linked to anti-TROP2 antibodies of the invention via cysteine ​​residues provided by selected interchain disulfide bonds opened by reduction of the anti-TROP2 antibody to provide ADCs of the invention. In some embodiments, the ADCs may contain 1, 2, 3, 4, 5, 6, 7, or 8 payloads conjugated thereto. For compositions or mixtures of ADCs, the mixtures or compositions may have a drug-to-antibody (DAR) ratio ranging from about 2 to about 8. In certain embodiments, the DAR may be about 2 to about 6, and in certain embodiments, the DAR may be about 2 or about 1 to 2. This ratio may refer to an average ratio in a population, such as an average DAR of 2 for a population of ADCs. In certain embodiments, the conjugates contain payloads primarily attached to the Fab domains, and in some cases, all four payloads attached to the Fab domains.

[0200] In further embodiments, provided herein are compositions or mixtures comprising or consisting of ADCs of the invention, wherein at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the ADCs in the mixture or composition have a DAR of about 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the mixture of ADCs has a DAR of about 4, with the majority of the ADCs in the mixture having four payload molecules linked thereto. In other words, the predominant species of ADC in the mixture or composition comprises four payload molecules.

[0201] In some embodiments, the anti-TROP2 antibodies of the present invention are engineered to contain one or more cysteine ​​or non-standard amino acid substitutions at defined positions within the anti-TROP2 antibody. These cysteine ​​or non-standard amino acid residues can then be conjugated to a linker payload via the sulfhydryl group of the cysteine ​​residue or the reactive group of the non-standard amino acid.

[0202] Thus, the anti-TROP2 antibody of the present invention may further comprise one or more substitutions of amino acids in its heavy or light chain with cysteine ​​residues or non-canonical amino acid residues, which can be used to conjugate a payload thereto. In certain embodiments, the amino acid positions that can be substituted are selected from positions 152, 153, 171, 172, 173, and 375 (numbering according to the Eu numbering scheme) of the heavy chain constant domain and positions 165 and 168 (numbering starting with amino acid 1 at the N-terminus) of the light chain constant domain. In certain embodiments, cysteine ​​may substitute for an amino acid at one or more of positions 152, 153, 171, 172, 173, and 375 (numbering according to the Eu numbering scheme) of the heavy chain constant domain and positions 165 and 168 (numbering starting with amino acid 1 at the N-terminus) of the light chain constant domain. In certain embodiments, the anti-TROP2 antibody comprises an S375C amino acid substitution. In certain embodiments, the antibody comprises an S375C amino acid substitution and an E152C amino acid substitution. In certain embodiments, the antibody comprises an S375C amino acid substitution and an S168C amino acid substitution. Tables 3-8 show non-exclusive lists of exemplary embodiments of anti-TROP2 antibodies of the invention that comprise substitutions of one or more amino acids therein with cysteines that can be conjugated to the aforementioned payloads.

[0203] [Table 3]

[0204] [Table 4]

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8]

[0209] In an exemplary embodiment, the ADC comprises an anti-TROP2 antibody of the invention conjugated to an exemplary linker-payload disclosed herein and has the following structure: a) Ab-MC-vc-PABC-payload; b) Ab-MC-GGFG-payload; c) Ab-MC-vc-payload; d) Ab-MP-payload; e) Ab-MP-GGG-payload; f) Ab-MP-GGGG-payload; g) Ab-MP-GGG-BCP-payload; h) Ab-MP-GGG-PABC-payload; i) Ab-MP-GGG-EDA-payload; j) Ab-MC-payload; k) Ab-SMCC-payload; l) Ab-MP-AA-PABC-payload; m) Ab-CM2P-AA-PABC-payload, or n) Ab-CM3P-AA-PABC-payload (wherein Ab is an anti-TROP2 antibody of the present invention).

[0210] In certain embodiments of the aforementioned ADCs, the payload is selected from the group consisting of taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (etoposide or etoposide phosphate, etc.), and the like. ), vinblastine, vincristine, vindesine, taxanes (such as taxol), taxotereticinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, DM1, DM2, DM3, DM4, MMAE, MMAF, MMAD, 7SN-38, anthracyclines, alkylcyclines, or derivatives thereof. Exemplary linker payloads include, but are not limited to, Ab-MC-vc-PAB-MMAE, Ab-MP-AA-PABC-MMAE, Ab-CM2P-AA-PABC-MMAE, and Ab-CM3P-AA-PABC-MMAE.

[0211] The present invention further provides exemplary ADCs comprising an anti-TROP2 antibody of the invention conjugated to a linker payload having the formula shown in Formula III, or a stereoisomer thereof: [ka] (In the formula, R 1 teeth, [ka] is selected from [ka] One wavy line represents -(CH2) n indicates the covalent binding site to [ka] The double wavy line indicates the covalent binding site to the sulfur of the cysteine ​​residue of Ab, R 2 is the cytotoxic moiety and R 3 and R 4 independently, C 1~3 represents an alkyl or a naturally occurring or unnatural amino acid side chain, n is an integer from 1 to 4, Ab is an anti-TROP2 binding agent of the invention, and p is a positive rational number from 1 to 24, including fractions and decimals. 2 is an auristatin drug selected from auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvaleric acid-AE ester, MMAF, MMAE, or pyrrolobenzodiazepine dimer, and R 3 and R 4 are both CH3.

[0212] In a further embodiment, the invention further provides an ADC comprising an anti-TROP2 antibody of the invention conjugated to a linker-MMAE payload, the ADC having the formula: [ka] wherein Ab is an anti-Trop2 antibody of the invention, S is the sulfur atom of a cysteine ​​residue present in the heavy or light chain of the Ab, p is an integer from 1 to 8, and the linker-MMAE payload is conjugated to the SH group of the cysteine ​​residue of the Ab. In a further embodiment, S is provided by the side chain of the cysteine ​​residue at position 375 of the heavy chain constant domain (this position is defined by Eu numbering).

[0213] In certain embodiments, the invention provides an ADC comprising an anti-TROP2 antibody of the invention having an engineered cysteine ​​residue conjugated to a linker-MMAE payload, the ADC having the formula: [ka] wherein Ab is an anti-Trop2 antibody of the invention comprising two heavy chains and two light chains, wherein the heavy or light chain comprises an engineered cysteine, and wherein the heavy and light chains are (A) (a) αTROP2 (HC:Sac-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 64 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 65 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (c) αTROP2 (HC:Sac-E171C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 66 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (d) αTROP2 (HC:Sac-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 67 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (e) αTROP2 (HC:Sac-E173C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 68 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (f) αTROP2 (HC:Sac-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 69 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (g) αTROP2 (HC:Sac-YTE-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 71 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (h) αTROP2 (HC:Sac-YTE-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 72 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (i) αTROP2 (HC:Sac-YTE-E171C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 73 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (j) αTROP2 (HC:Sac-YTE-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 74 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (k) αTROP2 (HC:Sac-YTE-E173C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 75 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (l) αTROP2 (HC:Sac-YTE-S375C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 76 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:58 and two light chains having the amino acid sequences set forth in SEQ ID NO:50; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 58 and two light chains having the amino acid sequences set forth in SEQ ID NO: 51; (o) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 63 and two light chains having the amino acid sequences set forth in SEQ ID NO: 50; (p) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 63 and two light chains having the amino acid sequences set forth in SEQ ID NO: 51; (q) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 78 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (r) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:79 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (s) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 80 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (t) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 81 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (u) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 82 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (v) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 83 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (w) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 78 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (x) αTROP2 (HC:BSM-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 79 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (y) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 80 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (z) αTROP2 (HC:BSM-E172C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 81 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (aa) αTROP2 (HC:BSM-E173C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 82 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (bb) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 83 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (cc) αTROP2(HC:BSM)(LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:52; (dd) αTROP2(HC:BSM)(LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:53; (ee) αTROP2(HC:BSM)(LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:54; (ff) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 59 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (gg) αTROP2 (HC:BSM-YTE) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 60 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (hh) αTROP2 (HC:BSM-YTE) (LC:BSM-E168C) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 60 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (ii) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 60 and two light chains having the amino acid sequences set forth in SEQ ID NO: 54; (jj) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 58 and two light chains having the amino acid sequences set forth in SEQ ID NO: 57; (kk) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 85 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (ll) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:86 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (mm) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 87 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (nn) αTROP2(HC:BSM-YTE-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 88 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (oo) αTROP2(HC:BSM-YTE-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:89 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (pp) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 90 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (qq) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 85 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (rr) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 86 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (ss) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 87 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (tt) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 88 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (uu) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 89 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; and (vv) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 90 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; wherein S is the sulfur atom of an engineered cysteine ​​conjugated to a linker-MMAE payload and p is an integer selected from 1 or 2; or (B) (a) αTROP2 (HC:Sac-E152C-S375C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 70 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-YTE-E152C-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 77 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 84 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (d) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 84 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (e) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 91 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; and (f) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 91 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; wherein S is the sulfur atom of an engineered cysteine ​​conjugated to a linker-MMAE payload and p is an integer selected from 1, 2, 3, or 4. is.

[0214] In certain embodiments, the invention provides an ADC comprising an anti-TROP2 antibody of the invention having an engineered cysteine ​​residue conjugated to a linker-MMAE payload, the ADC having the formula: [ka] wherein Ab is an anti-Trop2 antibody of the invention comprising two heavy chains and two light chains, wherein the heavy or light chain comprises an engineered cysteine, and the heavy and light chains are (A) (a) αTROP2 (HC:Sac-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 24 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 25 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (c) αTROP2 (HC:Sac-E171C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 26 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (d) αTROP2 (HC:Sac-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 27 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (e) αTROP2 (HC:Sac-E173C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 28 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (f) αTROP2 (HC:Sac-S375C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 29 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (g) αTROP2 (HC:Sac-YTE-E152C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 31 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (h) αTROP2 (HC:Sac-YTE-E153C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 32 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (i) αTROP2 (HC:Sac-YTE-E171C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 33 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (j) αTROP2 (HC:Sac-YTE-E172C) (LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 34 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (k) αTROP2 (HC:Sac-YTE-E173C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 35 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (l) αTROP2 (HC:Sac-YTE-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 36 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:11 and two light chains having the amino acid sequence set forth in SEQ ID NO:52; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 11 and two light chains having the amino acid sequence set forth in SEQ ID NO: 53; (o) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 23 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (p) αTROP2 (HC:Sac-YTE) (LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 23 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (q) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 38 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (r) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:39 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (s) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:40 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (t) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:41 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (u) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:42 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (v) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 19 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (w) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 38 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (x) αTROP2 (HC:BSM-E153C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 39 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (y) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 40 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (z) αTROP2 (HC:BSM-E172C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 41 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (aa) αTROP2 (HC:BSM-E173C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 42 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (bb) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 19 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (cc) αTROP2(HC:BSM)(LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (dd) αTROP2(HC:BSM)(LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (ee) αTROP2(HC:BSM)(LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 54; (ff) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (gg) αTROP2 (HC:BSM-YTE) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (hh) αTROP2 (HC:BSM-YTE) (LC:BSM-E168C) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (ii) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 54; (jj) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (kk) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 44 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (ll) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:45 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (mm) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 46 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (nn) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 47 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (oo) αTROP2(HC:BSM-YTE-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:48 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (pp) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 20 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (qq) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 44 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (rr) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:45 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (ss) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 46 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; (tt) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:47 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (uu) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 48 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; and (vv) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 20 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; wherein S is the sulfur atom of the side chain of an engineered cysteine ​​conjugated to a linker-MMAE payload and p is an integer selected from 1 or 2; or (B) (a) αTROP2 (HC:Sac-E152C-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 30 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (b) αTROP2 (HC:Sac-YTE-E152C-S375C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 37 and two light chains having the amino acid sequences set forth in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 43 and two light chains having the amino acid sequences set forth in SEQ ID NO: 21; (d) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:43 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (e) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:49 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; and (f) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:49 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; wherein S is the sulfur atom of the side chain of an engineered cysteine ​​conjugated to a linker-MMAE payload and p is an integer selected from 1, 2, 3, or 4; is.

[0215] In a further embodiment, the invention further provides an ADC comprising an anti-TROP2 antibody of the invention having an engineered cysteine ​​residue at position 375 of the heavy chain constant domain conjugated to a linker payload, wherein the ADC has the formula: [ka] (wherein Ab is an anti-TROP2 antibody of the present invention comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 20 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22, where p is 1 or 2, and where S is the sulfur atom of the side chain of the cysteine ​​residue at amino acid position 375 as defined by Eu numbering).

[0216] In the ADCs of the invention, the maleimide residue of the MP-AA-PABC-MMAE linker payload undergoes a ring-opening reaction when conjugated to a cysteine ​​residue in an antibody, resulting in a more stable linkage between the antibody and the maleimide group. [ka]

[0217] Because ring-opening occurs more rapidly than that of vedotin (MC-vc-PABC-MMAE), less deconjugation occurs prior to ring-opening of the ADCs of the invention compared to ADCs conjugated to vedotin. Accordingly, disclosed herein are compositions of any one of the aforementioned ADCs of the invention having an anti-TROP2 antibody disclosed herein conjugated to MP-AA-PABC-MMAE, wherein a portion of the ADC in the composition has the following structure: [ka] where p is 1 or 2, and S is the sulfur atom of a cysteine ​​residue present in the heavy or light chain of the Ab. In certain embodiments, the ADC comprises a cysteine ​​residue at position 375, the sulfur atom of the cysteine ​​residue is conjugated to an MP-AA-PABC-MMAE linker payload, and the amino acid numbering of the heavy chain constant domain is according to the Eu numbering scheme.

[0218] In further embodiments, provided herein are pharmaceutical compositions comprising any one of the above-described ADCs or a mixture thereof and a pharmaceutically acceptable carrier. In certain embodiments of the pharmaceutical composition, the predominant species of ADCs comprises antibodies whose heavy chains comprise a C-terminal lysine. In certain embodiments of the pharmaceutical composition, the predominant species of ADCs comprises antibodies whose heavy chains lack a C-terminal lysine. In certain embodiments of the pharmaceutical composition, the predominant species of ADCs comprises antibodies whose heavy chains lack a C-terminal glycine-lysine dipeptide. In certain embodiments of the pharmaceutical composition, the predominant species of ADCs comprises antibodies whose heavy chain N-terminal amino acid is glutamine. In certain embodiments of the pharmaceutical composition, the predominant species of ADCs comprises antibodies whose heavy chain N-terminal amino acid is glutamic acid. In certain embodiments of the pharmaceutical composition, the predominant species of ADCs comprises antibodies whose heavy chain N-terminal amino acid is glutamine cyclized to pyroglutamate. In certain embodiments of the pharmaceutical composition, the predominant species of ADC comprises antibodies whose heavy chain N-terminal amino acid is glutamic acid cyclized to pyroglutamate. In certain embodiments of the pharmaceutical composition, the predominant species of ADC comprises antibodies whose heavy chain N-terminal amino acid is pyroglutamate. In certain embodiments of the pharmaceutical composition, the predominant species of ADC comprises antibodies whose heavy chain N-terminal amino acid is pyroglutamate and whose heavy chain C-terminus lacks lysine. In certain embodiments of the pharmaceutical composition, the predominant species of ADC comprises antibodies whose heavy chain N-terminal amino acid is pyroglutamate and whose heavy chain C-terminus lacks glycine-lysine dipeptide.

[0219] The invention further provides a pharmaceutically acceptable salt or solvate of any one of the aforementioned ADCs.

[0220] ScFv fusion protein that binds to TROP2 In certain embodiments, the V L and V H is V L and V H The domains are expressed as ScFv fusion proteins, with the domains linked together by a peptide linker. H -V L The carboxyl terminus of one variable domain is joined to the amino terminus of the other variable domain without compromising the fidelity of the antigen-binding site. L The C-terminus of V is connected to H or a fusion protein linked to the N-terminus of V H The C-terminus of V is connected to L The peptide linker for connecting the variable domains can vary from 10 to 25 amino acids in length and typically, but not always, contains glycine (G) and serine (S) with the structure G4S (SEQ ID NO: 187), e.g., (G4S) n (SEQ ID NO: 188) (where n is 1, 2, 3, 4, or 5). Peptide linkers of shorter lengths (0-4 amino acids) have also been used, but ScFvs with shorter linkers can form multimers. Commonly, (G4S)3 peptides containing three repeating G4S units ("(G4S)3" disclosed as SEQ ID NO: 189) are used as ScFv peptide linkers (see, e.g., Leath et al., Int. J. Oncol. 24:765-771 (2004); Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Iliades et al., FEBS Lett. 409:437-441 (1997)).

[0221] An exemplary ScFv fusion protein has the structure V L -(G4S) n -V H or V H -(G4S) n -V L Including V HThe domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, and V L The domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, wherein the CDR sequences are defined by the Kabat numbering scheme. In certain embodiments, n is 1, 2, 3, 4, or 5.

[0222] An exemplary ScFv fusion protein has the structure V L -(G4S) n -V H or V H -(G4S) n -V L Including V H comprises the amino acid sequence set forth in SEQ ID NO: 1, and V L comprises the amino acid sequence set forth in SEQ ID NO:3, where n is 1, 2, 3, 4, or 5.

[0223] An exemplary ScFv fusion protein has the structure V L -(G4S) n -V H or V H -(G4S) n -V L Including V H comprises the amino acid sequence set forth in SEQ ID NO: 14, and V L comprises the amino acid sequence set forth in SEQ ID NO: 15, wherein n is 1, 2, 3, 4, or 5.

[0224] An exemplary ScFv fusion protein has the structure V L -(G4S) n -V H or V H -(G4S) n -V L Including V H comprises the amino acid sequence set forth in SEQ ID NO: 14, and V L comprises the amino acid sequence set forth in SEQ ID NO: 16, where n is 1, 2, 3, 4, or 5.

[0225] The ScFvs disclosed herein can be provided in a bispecific format comprising a CD3 binder (ScFv) linked by a peptide linker to a TROP2-binding ScFv disclosed herein. These molecules, called bispecific T cell engagers (BiTEs), bind to CD3 on T cells and TROP2 expressed on the surface of the cells, bringing the T cells to the tumor site.

[0226] The ScFvs disclosed herein can also be fused to cytotoxins, radioisotopes, cytokines, and enzymes for cancer, autoimmune, and / or inflammatory therapeutic applications. In certain embodiments, the peptide linker can comprise 1 to 10 G4S peptide units of SEQ ID NO: 190.

[0227] In further embodiments, the ScFvs disclosed herein can be linked or inserted at different positions in an intact IgG molecule to confer dual epitope binding. For example, a bispecific antibody can be provided that comprises two heterodimeric heavy chain constant domains, where the N-terminus of one heavy chain constant domain is fused to the C-terminus of an ScFv disclosed herein and the N-terminus of the other heavy chain constant domain is fused to the C-terminus of an ScFv that targets an antigen other than TROP2 or a Fab' that targets an antigen other than TROP2.

[0228] Nucleic acid molecules encoding the TROP2-binding agents of the present invention The present invention further provides nucleic acid molecules encoding the TROP2-binding agents of the present invention. In certain embodiments, the TROP2-binding agent is a V encoded by a first nucleic acid molecule. H and V encoded by a second nucleic acid molecule L In certain embodiments, the TROP2-binding agent is an antibody whose heavy chain is encoded by a first nucleic acid molecule and whose light chain is encoded by a second nucleic acid molecule.

[0229] In certain embodiments, the heavy and light chains (or V H and VL ) are heavy and light chains (or V H and V L ) is expressed as a fusion protein, in which the N-terminus of the ScFv fusion protein is fused to a leader peptide at the N-terminus to facilitate transport of the TROP2-binding agent through the secretory pathway. In certain embodiments, the N-terminus of the ScFv fusion protein is fused to a leader or signal peptide at the N-terminus to facilitate transport of the ScFv through the secretory pathway. Examples of leader / signal peptides that can be used include those comprising the amino acid sequence set forth in SEQ ID NO: 56 or SEQ ID NO: 57. Thus, in certain embodiments, the aforementioned nucleic acid molecule can comprise a polynucleotide encoding a leader peptide linked to the 5' end of the nucleic acid molecule encoding the anti-TROP2-binding agent.

[0230] The nucleic acid molecules disclosed herein may contain one or more substitutions that optimize one or more codons to enhance expression of the nucleic acid molecule in a particular host cell, such as a yeast or fungal host cell, a non-human mammalian host cell, a human host cell, an insect host cell, or a prokaryotic host cell.

[0231] Methods for producing the TROP2-binding agents of the present invention The present invention provides a recombinant method for producing a TROP2-binding agent of the invention, comprising: (i) a V H and V L or (ii) an expression vector comprising one or more nucleic acid molecules encoding the heavy and light chains of the anti-TROP2 binding agent, or (iii) introducing into a host cell two expression vectors comprising nucleic acid molecules, one of which encodes the V of the TROP2 binding agent. H or a nucleic acid molecule encoding the heavy chain of an anti-TROP2 binding agent, and the other vector contains a nucleic acid molecule encoding the V L or a nucleic acid molecule encoding the light chain of the TROP2-binding agent. H , V LThe nucleic acid molecule or polynucleotide encoding the heavy or light chain is operably linked to a promoter and other transcriptional and translational regulatory sequences. The host cell is cultured under conditions and for a period of time suitable for expression of the nucleic acid molecule, followed by isolation of the TROP2-binding agent from the host cell and / or the medium in which the host cell is grown. See, for example, WO 2004041862, WO 2006122786, WO 2008020079, WO 2008142164, or WO 2009068627. The expression vector may be a plasmid or a viral vector. The present invention also relates to the expression of such nucleic acid molecules (V) encoding TROP2-binding agents. H and V L a host cell containing a nucleic acid molecule encoding a heavy chain or a nucleic acid molecule encoding a light chain) or a component thereof (V H or heavy chain only or V L or a host cell containing a nucleic acid molecule encoding only the light chain).

[0232] Eukaryotic and prokaryotic host cells, including mammalian cells, for expression of TROP2-binding agents are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Thus, mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Cell lines are selected by determining which cell lines have particularly high expression levels. Other cell lines that can be used include insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Saccharomyces cerevisiae, and Trichoderma reesei. The present invention further includes any host cell that contains a TROP2-binding agent of the invention, or that contains one or more nucleic acid molecules encoding such a TROP2-binding agent, or that contains an expression vector that contains one or more nucleic acid molecules encoding such a TROP2-binding agent.

[0233] Furthermore, expression of TROP2-binding agents from manufacturing cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is described in whole or in part in connection with European Patent Nos. 0216846B1, 0256055B1, 0323997B1, and 0338841B1. Thus, in one embodiment of the present invention, a mammalian host cell lacks a glutamine synthetase gene and is grown in the absence of glutamine in the culture medium, while a nucleic acid molecule encoding an immunoglobulin chain contains a glutamine synthetase gene that complements the lack of the gene in the host cell. Such host cells comprising the TROP2-binding agents or nucleic acid molecule(s) or expression vector(s) discussed herein, as well as the expression methods discussed herein for producing TROP2-binding agents using such host cells, are part of the present invention.

[0234] The invention further includes methods for purifying a TROP2-binding agent, comprising introducing a sample containing the TROP2-binding agent (e.g., culture medium, cell lysate, or a cell lysate fraction, e.g., the soluble fraction of the lysate) into a purification medium (e.g., a cation exchange medium, an anion exchange medium, and / or a hydrophobic exchange medium) and recovering the purified TROP2-binding agent from the flow-through fraction of the sample that does not bind to the medium, or discarding the flow-through fraction and eluting the bound TROP2-binding agent from the medium and recovering the eluate. In one embodiment of the invention, the medium is in a column to which the sample is applied. In one embodiment of the invention, the purification method is carried out after recombinant expression of the TROP2-binding agent in host cells; for example, the host cells may be first lysed and the lysate purified from insoluble material and then purified in the medium, or the TROP2-binding agent may be secreted by the host cells into the medium and the medium, or a fraction thereof, applied to the purification medium.

[0235] Generally, glycoproteins produced in a particular cell line or transgenic animal have a glycosylation pattern characteristic of the glycoprotein produced in that cell line or transgenic animal. Therefore, the specific glycosylation pattern of a TROP2-binding agent depends on the particular cell line or transgenic animal used to produce the TROP2-binding agent. TROP2-binding agents containing only nonfucosylated N-glycans are part of the present invention and can be advantageous because nonfucosylated antibodies have typically been shown to exhibit stronger efficacy than their fucosylated counterparts both in vitro and in vivo (e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patent Nos. 6,946,292 and 7,214,775). These TROP2-binding agents with nonfucosylated N-glycans are unlikely to be immunogenic because their carbohydrate structures are normal components of the population present in human serum IgG.

[0236] The present invention includes TROP2-binding agents that contain N-linked glycans typically added to immunoglobulins produced in Chinese hamster ovary cells (CHON-linked glycans) or engineered yeast cells (engineered yeast N-linked glycans), such as Pichia pastoris. For example, in one embodiment of the present invention, the TROP2-binding agent contains one or more "engineered yeast N-linked glycans" or "CHO ​​N-linked glycans" (e.g., G0 and / or G0-F and / or G1 and / or G1-F and / or G2-F and / or Man5). In one embodiment of the present invention, the TROP2-binding agent contains engineered yeast N-linked glycans, i.e., G0 and / or G1 and / or G2, and optionally further includes Man5. In one embodiment of the present invention, the TROP2-binding agent contains CHO N-linked glycans, i.e., G0-F, G1-F, and G2-F, and optionally further includes G0 and / or G1 and / or G2 and / or Man5. In one embodiment of the present invention, about 80% to about 95% (e.g., about 80-90%, about 85%, about 90%, or about 95%) of the total N-linked glycans on the TROP2-binding agent are engineered yeast N-linked glycans or CHO N-linked glycans. See Nett et al. Yeast. 28:237-252 (2011); Hamilton et al. Science. 313:1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5):387-392 (2007). For example, in one embodiment of the present invention, the engineered yeast cells are GFI5.0 or YGLY8316, or strains described in U.S. Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also International Patent Application Publication No. 2013066765.

[0237] Pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the present invention The TROP2-binding agents or ADCs of the present invention disclosed herein can be provided in suitable pharmaceutical compositions comprising one or more TROP2-binding agents or ADCs of the present invention and a pharmaceutically acceptable carrier. The carrier can be a diluent, adjuvant, excipient, or vehicle in which the TROP2-binding agents or ADCs of the present invention are administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions can contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and coloring agents. The concentration of the TROP2-binding agent or ADC of the invention in such pharmaceutical formulations can vary widely, i.e., less than about 0.5% by weight, usually at least about 1% by weight, up to as much as 15 or 20% by weight, and will be selected primarily based on the required dose, fluid volume, viscosity, etc., according to the particular mode of administration selected. For suitable vehicles and formulations containing other human proteins, such as human serum albumin, see, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DBed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, especially pp. 958-989.

[0238] The mode of administration of the TROP2-binding agents or ADCs of the invention, or pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the invention, can be any suitable route, for example, parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), or other means recognized by those skilled in the art and known in the art.

[0239] The TROP2-binding agents or ADCs of the invention can be administered to an individual (e.g., a patient) by any suitable route, for example, parenterally, intramuscularly or subcutaneously by intravenous (iv) infusion or bolus injection, or intraperitoneally. The iv infusion can be administered over, for example, 15, 30, 60, 90, 120, 180, or 240 minutes, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0240] Administration of a TROP2-binding agent or ADC of the invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the invention, may be repeated after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or more. Similar to chronic administration, repeated courses of treatment are also possible. Repeated administrations may be at the same dose or at different doses.

[0241] The TROP2-binding agents or ADCs of the invention, or pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the invention, may be administered as a maintenance therapy, for example, once weekly, for a period of six months or more.

[0242] The anti-TROP2 binding agents or ADCs of the invention, or pharmaceutical compositions comprising the TROP2 binding agents or ADCs of the invention, may also be administered prophylactically to reduce the risk of developing cancer, delay the occurrence of events in cancer progression, and / or reduce the risk of recurrence when the cancer is in remission. This may be particularly useful in patients in whom it is difficult to locate a tumor known to be present due to other biological factors.

[0243] The TROP2-binding agents or ADCs of the invention, or pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the invention, can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations, and well-known lyophilization and reconstitution techniques can be used.

[0244] Combination Therapy Treatment Combination therapies of the invention comprising a TROP2-binding agent or ADC of the invention, or pharmaceutical compositions comprising a TROP2-binding agent or ADC of the invention and another therapeutic agent (e.g., a small molecule or antibody), may be used in the treatment of any proliferative disease, particularly in the treatment of cancer. In certain embodiments, combination therapies of the invention include: It may be used to treat breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0245] Combination therapies and chemotherapy involving the TROP2-binding agents or ADCs of the invention The combination therapy of the present invention comprising a TROP2-binding agent or ADC of the present invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the present invention, can be administered to an individual with cancer in combination with chemotherapy. The individual may receive chemotherapy at the same time as the combination therapy of the present invention. The individual may receive the combination therapy of the present invention after the individual has completed chemotherapy. The individual may receive chemotherapy after completing the combination therapy. The combination therapy of the present invention can also be administered to an individual with recurrent or metastatic cancer with disease progression or recurrent cancer who is receiving or has completed chemotherapy.

[0246] The chemotherapy may include a chemotherapeutic agent selected from the group consisting of: (i) alkylating agents, including but not limited to bifunctional alkylating agents, cyclophosphamide, mechlorethamine, chlorambucil, and melphalan; (ii) monofunctional alkylating agents, including but not limited to dacarbazine, nitrosoureas, and temozolomide (oral dacarbazine); (iii) anthracyclines or alkylcyclines; (iv) cytoskeletal disrupting agents (taxanes), including but not limited to paclitaxel, docetaxel, abraxane, and taxotere; (v) epothilones, including but not limited to ixabepilone and utiderone; (vi) histone deacetylase inhibitors, including but not limited to vorinostat and romidepsin; (vii) inhibitors of topoisomerase I, including but not limited to irinotecan and topotecan; (viii) inhibitors of topoisomerase II, including but not limited to etoposide, teniposide, and tafluposide; (ix) kinase inhibitors, including but not limited to bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib; (x) Nucleotide analogs and precursor analogs, including but not limited to azacitidine, azathioprine, fluoropyrimidines (e.g., capecitabine, carmofur, doxifluridine, fluorouracil, and tegafur), cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly thioguanine); (xi) peptide antibiotics, including but not limited to bleomycin and actinomycin; platinum-based agents, including but not limited to carboplatin, cisplatin, and oxaliplatin; (xii) retinoids, including but not limited to tretinoin, alitretinoin, and bexarotene; and (xiii) Vinca alkaloids and derivatives, including but not limited to vinblastine, vincristine, vindesine, and vinorelbine.

[0247] The choice of chemotherapeutic agent dose for chemotherapy depends on several factors, including the serum or tissue turnover rate of the agent, the level of symptoms, the immunogenicity of the agent, and the availability of target cells, tissues, or organs in the individual being treated.

[0248] The dosage of additional therapeutic agents must be such that they provide acceptable levels of side effects.Therefore, the dosage and frequency of administration of each additional therapeutic agent depend in part on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient.Guidelines for selecting appropriate dosages of antibodies, cytokines, and small molecules are available. For example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker,New York,NY;Baert et al.(2003)New Engl.J.Med.348:601-608;Milgrom et al.(1999)New Engl.J.Med.341:1966-1973;Slamon et al.(2001)New Engl.J.Med.344:783-792;Beni Aminovitz et al. al.(2000)New Engl.J.Med.342:613-619;Ghosh et al.(2003)New Engl.J.Med.348:24-32;Lipsky et al.(2000)New Engl.J.Med.343:1594-1602;Physicians' Desk Reference 2003(Physicians' Desk Reference,57th Ed);Medical Economics Company;ISBN:1563634457;57th edition (November 2002).Determining an appropriate dosage regimen can be made by a clinician using, for example, parameters or factors known or suspected in the art that affect or are predicted to affect treatment, and depends, for example, on the individual's medical history (e.g., previous treatments), the type and stage of the cancer being treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0249] Thus, the present invention contemplates embodiments of the combination therapy of the present invention further comprising a chemotherapy step comprising either platinum-containing chemotherapy, e.g., pemetrexed and platinum chemotherapy, or carboplatin and paclitaxel, or nab-paclitaxel. In certain embodiments, the combination therapy with a chemotherapy step can be used to treat at least NSCLC and HNSCC.

[0250] The combination therapy further combined with chemotherapy can be used to treat any proliferative disease, particularly cancer.In certain embodiments, the combination therapy of the present invention can be used to treat breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, gastric cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0251] Combination therapy comprising a TROP2-binding agent or ADC and a therapeutic antibody The TROP2-binding agents or ADCs of the present invention, or pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the present invention, can be administered in combination with one or more therapeutic antibodies for treating cancer or proliferative diseases. An individual may receive a therapeutic antibody treatment at the same time as the individual receives the combination therapy of the present invention. An individual may receive a combination therapy of the present invention after completing treatment with a therapeutic antibody. An individual may receive a therapeutic antibody treatment after completing combination therapy. The combination therapy of the present invention can also be administered to individuals with recurrent or metastatic cancer with disease progression or recurrence who are undergoing or have completed chemotherapy. In certain embodiments, the therapeutic agent targets the programmed death 1 receptor or ligand, PD-1, and PD-L1, respectively.

[0252] Exemplary anti-PD-1 antibodies that may be used in combination therapy with the TROP2-binding agents or ADCs of the invention disclosed herein include any antibody that binds to PD-1 and inhibits PD-1 from binding to PD-L1 and / or PD-L2, or that binds to PD-L1 or PD-L2 and inhibits PD-1 from binding. In certain embodiments, an exemplary anti-PD-1 antibody is pembrolizumab (KEYTRUDA). In certain embodiments, an exemplary anti-PD-1 antibody is nivolumab (OPDIVO). In certain embodiments, an exemplary anti-PD-1 antibody is cemiplimab (LIBTAYO). In certain embodiments, an exemplary anti-PD-L1 antibody is durvalumab (IMFINZI). In certain embodiments, an exemplary anti-PD-L1 antibody is atezolizumab (TECENTRIQ). In certain embodiments, an exemplary anti-PD-L1 antibody is avelumab (BAVENCIO).

[0253] Injection device for administering a TROP2-binding agent or ADC The present invention also provides an injection device comprising any one of the TROP2 binding agents or ADCs of the present invention, or a pharmaceutical composition comprising any one of the anti-TROP2 binding agents or ADCs of the present invention. An injection device is a device for introducing a substance into a patient's body via a parenteral route, for example, intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector) that includes, for example, a cylinder or barrel for holding a fluid to be injected (e.g., a pharmaceutical composition comprising any one of the TROP2 binding agents or ADCs of the present invention), a needle for puncturing the skin and / or a blood vessel to inject the fluid, and a plunger for forcing the fluid from the cylinder through the needle hole. In one embodiment of the present invention, the injection device comprising any one of the TROP2 binding agents or ADCs of the present invention, or a pharmaceutical composition comprising any one of the TROP2 binding agents or ADCs of the present invention, is an intravenous (IV) injection device. Such devices include a composition comprising the TROP2-binding agent or ADC, or a pharmaceutical composition within a cannula or trocar / needle that may be attached to tubing that may be attached to a bag or reservoir for holding a fluid (e.g., saline; or lactated Ringer's solution, which contains NaCl, sodium lactate, KCl, CaCl, and may contain glucose) that is introduced into the subject's body through the cannula or trocar / needle.

[0254] In embodiments of the present invention, the TROP2-binding agents or ADCs of the present invention, or pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the present invention, can be introduced into a device by inserting a trocar and cannula into a subject's vein and removing the trocar from the inserted cannula. The IV device can be inserted, for example, into a peripheral vein (e.g., in the hand or arm); into the superior or inferior vena cava or the right atrium of the heart (e.g., a central IV); or into the subclavian, internal jugular, or femoral vein and advanced toward the heart until it reaches, for example, the superior vena cava or the right atrium (e.g., a central venous line). In one embodiment of the present invention, the injection device is an autoinjector, jet injector, or external infusion pump. Jet injectors use a high-pressure narrow jet of liquid to penetrate the epidermis and introduce the TROP2-binding agents or ADCs of the present invention, or pharmaceutical compositions comprising the TROP2-binding agents or ADCs of the present invention, into the patient's body. An external infusion pump is a medical device that delivers a TROP2 binding agent or ADC of the present invention, or a pharmaceutical composition containing a TROP2 binding agent or ADC of the present invention, into a patient's body in controlled amounts. External infusion pumps may be electrically or mechanically powered. Various pumps operate in different ways: for example, syringe pumps hold fluid in a syringe reservoir and a movable piston controls fluid delivery; elastomeric pumps hold fluid in a stretchable balloon reservoir and pressure from the balloon's elastic walls drives fluid delivery. In peristaltic pumps, a set of rollers squeezes a length of flexible tubing, forcing fluid forward. Multichannel pumps can deliver fluid from multiple reservoirs at multiple rates.

[0255] TROP2-binding agent or ADC or composition of the invention or kit containing said composition Further provided are kits comprising one or more components, including but not limited to, a TROP2-binding agent or ADC of the invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the invention in combination with one or more additional components, including but not limited to, an additional therapeutic agent, as discussed herein. Pharmaceutical compositions comprising a TROP2-binding agent or ADC of the invention, or a TROP2-binding agent or ADC of the invention and / or a therapeutic agent, can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

[0256] In one embodiment, the kit comprises a TROP2-binding agent or ADC of the invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the invention in one container (e.g., in a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0257] In another embodiment, the kit comprises a combination of the invention comprising a TROP2-binding agent or ADC of the invention, or a pharmaceutical composition combining a TROP2-binding agent or ADC of the invention with one or more therapeutic agents, optionally formulated together as a pharmaceutical composition in a single common container.

[0258] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for such administration. For example, the kit may include one or more hypodermic needles or other injection devices as described above. Thus, the present invention includes kits comprising an injection device and a TROP2-binding agent or ADC of the present invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the present invention, e.g., the injection device contains a TROP2-binding agent or ADC of the present invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the present invention, or the TROP2-binding agent or ADC of the present invention, or a pharmaceutical composition comprising a TROP2-binding agent or ADC of the present invention, are in separate containers.

[0259] The kit may include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. Generally, such information will assist patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information about the combination of the present invention may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, delivery method, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0260] The Examples describe the discovery of the novel class of TROP2-binding agents of the invention and the ADCs of the invention comprising the novel class of TROP2-binding agents.

[0261] General method Ultra-High Performance Size Exclusion Chromatography (UP-SEC) To measure aggregation by UP-SEC, 5 μg of purified antibody was injected at 0.5 mL / min onto an Acquity BEH200 SEC, 1.7 μm, 4.6 x 150 mm size-exclusion column (Waters Corporation, Milford, MA) equilibrated with 100 mM sodium phosphate, 200 mM sodium chloride, 0.02% sodium azide, pH 7, using a Waters H-Class ultra-performance chromatography (UPLC) column. Chromatograms were collected at both 215 nm and 280 nm wavelengths, and an integrated trace of the absorbance at 280 nm was performed using EMPOWER 2 (Waters).

[0262] Hydrophobic Interaction Chromatography (HIC) To determine the hydrophobicity of a given antibody or ADC using HIC, 50 µg of sample at approximately 0.5–1 mg / mL is mixed 1 / 1 (v / v) with a 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 / isopropanol (95:5 v / v) solution. The prepared sample is then filtered through a 0.22 µm polyvinylidene fluoride (PVDF) membrane and loaded onto a Thermo Fisher Scientific, Inc. (Waltham, MA, USA) MAbPac™ HIC-Butyl high-performance liquid chromatography (HPLC) column equilibrated with 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 / isopropanol (95:5 v / v) (mobile phase A) according to the manufacturer's manual. The sample is eluted using a reverse gradient from mobile phase A to 50 mM sodium phosphate, pH 7.0 / isopropanol (80:20 v / v) (mobile phase B). After elution, A280nm is recorded as a function of time, and the data is then exported and analyzed using Empower software. The retention time of each sample is compared to a standard, which is characteristic of the antibody's hydrophobicity; longer elution times correlate with higher hydrophobicity.

[0263] Reversed-phase high-performance liquid chromatography (RP-HPLC) To determine purity using RP-HPLC, 15 µg of purified antibody was loaded onto a POROS R2 / 10 2.1 x 30 mm column (Applied Biosystems, Thermo Fisher Corporation) and equilibrated with 30% acetonitrile, 0.2% trifluoroacetic acid at 70 °C and 2 mL / min. The sample was eluted with a linear gradient from 30 to 58% acetonitrile in 0.2% trifluoroacetic acid over 5 min. Chromatograms were collected at both 215 nm and 280 nm wavelengths, and the A280 nm trace was integrated using Chemstation Rev.B.04.01 (Agilent Technologies, Inc., Santa Clara, CA).

[0264] Tonset / Tm / Tagg measurement by nano-differential scanning fluorimetry (nano-DSF) Nano-DSF is a method for measuring ultra-high-resolution protein stability using intrinsic tryptophan or tyrosine fluorescence. All nano-DSF studies are performed using a NanoTemper Prometheus NT.48 instrument (NanoTemper Technologies, Inc., South San Francisco, CA). Samples (approximately 10 µL at 0.5–1 mg / mL) are loaded by capillary action into standard-grade nano-DSF capillaries, placed in a Prometheus capillary holder, and subjected to a 1 °C / min temperature ramp from 20 °C to 94.8 °C. Up to 48 samples can be analyzed in parallel and assessed for stability at each temperature for 3 seconds.

[0265] Melting temperature (Tm) onset (°C) and Tm (°C) values ​​indicate the structural stability of a sample and are obtained by monitoring intrinsic tryptophan and tyrosine fluorescence at emission wavelengths of 330 nm and 350 nm. To generate an unfolding curve, the ratio of fluorescence intensities (F350nm / F330nm) is plotted against temperature or time. The thermal stability of a sample is described by the thermal unfolding transition midpoint Tm (°C), at which half of the protein population is unfolded. Tm corresponds to the inflection point of the unfolding curve and is determined via the derivative of the curve.

[0266] The aggregation point, Tagg (°C), represents the colloidal stability of a sample and is obtained by monitoring the backreflection of near-ultraviolet (UV) light using backreflection optics. Backreflection optics utilizes near-UV light scattering by protein aggregates, so only unscattered light reaches the detector. Therefore, the reduction in backreflected light is a direct measure of aggregation in the sample.

[0267] Non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS NR) Five microliters of each sample at 1 mg / mL was mixed with 35 μL of loading buffer (HT Protein Express Sample Buffer) (PerkinElmer, Waltham, MA) containing either 50 mM iodoacetamide or 50 mM dithiothreitol in a 96-well plate. The plate was incubated at 70°C for 20 minutes, and 75 μL of water was added to each well. Each sample was analyzed on a LabChip GXII (PerkinElmer) using an HT Protein Express chip (PerkinElmer). Electropherograms were collected by measuring the fluorescence of the sample over time and integrated using LabChip GX software V 4.1.1619.0 SP1 (PerkinElmer).

[0268] Affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) The AC-SINS assay measures protein self-interactions by capturing antibodies on the surface of gold colloids, which exhibit surface resonant oscillations at visible light frequencies. When the immobilized antibodies self-interact, the colloids aggregate and shift their oscillation frequency to absorb at longer wavelengths. Gold nanoparticles are incubated overnight with an 80 / 20 (v / v) capture / non-capture antibody mixture. The coated gold nanoparticles are then spun down and resuspended in conjugation buffer (20 mM sodium acetate pH 5.5 and 1x PBS pH 7.4) to a final volume of 50 μL. Samples are diluted to 0.05 mg / mL in conjugation buffer, and 45 μL of each dilution is loaded into a 384-well plate. Five μL of pre-prepared gold nanoparticles are then added to each well of the plate, including the antibody and buffer controls. The plate is then covered with an aluminum lid, incubated at room temperature for 2 hours, and quickly spun down at 3000 rpm before reading the absorbance spectrum of each well from 450 nm to 650 nm using a plate reader. Each sample spectrum is recorded and analyzed for a red shift in the absorption peak maximum compared to buffer and antibody controls. The red shift and its intensity indicate the self-interaction tendency of the antibody sample tested.

[0269] Assessment of aggregation and self-interactions by dynamic light scattering (DLS) All DLS studies were performed on undiluted samples in glass-bottom 96-well plates at 25°C using a DynaPro Plate Reader II (Wyatt Technology Corporation, Santa Barbara, CA). Twenty acquisitions (5 s each) per well were averaged, and the hydrodynamic radius (Rh), % polydispersity, and % mass were modeled using Dynamics version 7.1.9.3 (Wyatt Technology Corporation) to assess aggregation.

[0270] To determine the self-interaction, the diffusion interaction parameter (k D ) is determined by DLS. High-concentration samples are diluted with the desired buffer (20 mM sodium acetate pH 5.5 and 10 mM histidine hydrochloride pH 6.5) to a concentration of 20 mg / mL, filtered through a 0.22 μm filter, and diluted with the filtration buffer (with the desired pH and ionic strength) to obtain lower-concentration samples (2, 5, 10, 15, 20 mg / mL), which are then added to the microplate. D is determined by a linear fit of the measured (mutual) diffusion coefficients as a function of concentration.

[0271] PEG 6000 solubility measurement To determine the PEG 6000 solubility of a given protein, the sample is dialyzed and diluted to 2 mg / mL in the desired filtration buffer (20 mM sodium acetate pH 5.5 and 10 mM histidine hydrochloride pH 6.5). A PEG 6000 concentration screen (0-40% w / v) is generated using an Andrew robot (Alliance, Geneva, Switzerland) by diluting a 40% w / v PEG 6000 stock solution in the desired buffer with the corresponding buffer stock solution. Ten (10) µL of the 2 mg / mL sample is loaded into a half-well UV microplate (Corning, Corning, NY) pre-filled with 90 µL of the previously prepared PEG 6000 concentration screen solution, mixed, and incubated at room temperature for 1 h. After incubation, the plate is read using an EPOCH / 2 Microplate Reader from BioTek Instruments, Inc. (Winooski, VT) to measure optical density at 320 nm wavelength. Optical density at 280 nm (OD280) can also be used to analyze filtered samples. The reported value is the PEG 6000 concentration at the midpoint where half of the protein population has precipitated, determined by derivative of the precipitation curve.

[0272] Viscosity Evaluation This method accurately measures the dynamic viscosity of antibody and protein formulations across a range of concentrations and viscosities (1-80 centipoise (cP)). Samples are evaluated for viscosity in three different formulations (10 mM sodium acetate pH 5.5, 10 mM histidine-HCl pH 6.5, and 1x PBS pH 7.4) and within a concentration range of 10 to 200 mg / ml. The prepared samples are then filtered using a 0.2 μm PVDF membrane before loading 60 μL into glass vials. The vials are quickly spun down and placed in a VROC starter (Rheosense Inc., San Ramon, CA) sample vial tray. 48 μL of sample is then injected into the instrument cell, and the viscosity of the solution is measured from 1 to 80 cP at 25 °C. Viscosity values ​​are then plotted as a function of protein concentration. Each reported viscosity value is the average of 10 measurements.

[0273] Evaluation of aggregate formation after low pH In this method, samples from small-scale purifications are rapidly buffer-exchanged using a 96-well ZEBASpin desalting plate (Thermo Fisher Scientific Corporation) before lowering the pH to 3.5 using 2 M acetic acid. The plate is then covered with a Roche LightCycler foil and incubated at room temperature for 30 min, after which the pH of the solution is adjusted to 5 using 1 M TRIS base. The sample is then rapidly spun down at 3000 rpm, after which 5–10 μg is injected onto a Waters BEH200 size-exclusion chromatography (SEC) column equilibrated with 100 mM sodium phosphate, 200 mM sodium chloride, and 0.02% sodium azide pH 7 using a Waters UPLC system to assess sample purity by UP-SEC.

[0274] Oxidation with azobis(2-amidinopropane) dihydrochloride (AAPH) Incubate 1-2 mg / mL of antibody in 1 mM AAPH at 40°C for 6 hours, protect from light, exchange the buffer into 20 mM sodium acetate (pH 5.5), and store at -80°C until analysis.

[0275] Light stress Place 1-2 mg / mL of antibody in a reusable quartz cuvette, expose to 1x light (200 W-h / m2 ultraviolet and 1200 k-lux visible light) at 25 °C, and store at -80 °C until analysis.

[0276] Evaluation of isoelectric points and charge variants by capillary isoelectric focusing (cIEF) To determine the isoelectric point (pI) by cIEF, samples were diluted to 0.2 mg / mL in a buffer containing 0.35% methylcellulose, 3 M urea, 1% Pharmalyte 3-10 (GE Healthcare), 0.5% Pharmalyte 5-8, 0.5% Pharmalyte 8-10, 0.5% pI marker 5.85 (ProteinSimple Inc., San Jose, CA), and 0.5% pI marker 9.77. Samples were run on an iCE3 (ProteinSimple) using FC-coated capillary focusing at 1500 V for 1 min, followed by 3000 V for 8 min. Data were exported and integrated using Empower 2 (Waters Corporation).

[0277] Antibody analysis by LC-MS / MS peptide mapping For peptide mapping by mass spectrometry, 100 μg of each sample was denatured with 30 μL of 8 M guanidine / 1 M Tris hydrochloride solution (15:1), reduced with 2 μL of 1 M dithiothreitol at 60 °C for 30 min, and alkylated with 5 μL of 1 M iodoacetamide for 45 min in the dark. Prior to digestion, samples were buffer-exchanged into 50 mM ammonium bicarbonate using a 7 kDa molecular weight cutoff ZEBA cartridge. Samples were digested with 2 μg of trypsin and chymotrypsin for 2 h at 37 °C. The digestion was quenched by adding 3 μL of 5 M hydrochloride to each sample. Data were acquired on a Dionex / QE Plus MS using a linear gradient from 2 to 36% acetonitrile in 0.1% formic acid over 50 min. Samples will be analyzed using PEAKS DB (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) for database searching and PepFinder (Thermo Fisher Scientific Corporation) and manual validation for rate of change assessment.

[0278] Analysis by surface plasmon resonance (SPR) The binding kinetics of antibodies to their targets are determined by SPR on a BIAcore T200 or BIAcore 4000 (GE Healthcare). The running buffer, 10 mM HEPES, 150 mM NaCl, 0.05% v / v surfactant P20, 3 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4 (HBS-EP+, GE Healthcare), is used for immobilization and reagent dilution. All binding kinetics are measured at 25 °C.

[0279] For each injection cycle, the antibody is first captured on a different flow cell (Series S CM5, GE Healthcare) using an anti-human Fc antibody (Human Antibody Capture Kit, GE Healthcare) immobilized on a sensor chip. A reference flow cell without captured antibody is also used. Serial dilutions of the target protein, ranging from 0.16 nM to 80 nM, and a buffer blank are injected over the captured antibody and reference surfaces in multiple cycles for 3 min of association, followed by 10 min of dissociation. After each cycle, the surface is regenerated by injecting 3 M MgCl2 for 30 s.

[0280] The double-reference titration data were globally fitted to a 1:1 Langmuir binding model to determine the association rate constant, k, using BIAcore T200 Evaluation Software Version 2.0 or BIAcore 4000 Evaluation Software Version 1.1 (GE Healthcare). a (M-1 s-1) and the dissociation rate constant k d Determine the equilibrium dissociation constant (s-1). D (M)=k d / k a It was calculated as:

[0281] Subvisible particle assessment after 10 days of incubation at 50°C by flow cytometry The Guava EasyCyte 5HT™ flow cytometer (GFC) used in this study was purchased from EMD Millipore Corp. (Billerica, MA). Briefly, the plate carrying the protein sample (160 μL of 1 mg / mL protein solution) was left overnight at 5°C to degas and minimize potential interference from microbubbles that may be trapped in the solution during sample preparation. Data was collected for 250 seconds, allowing for an analyzed sample volume of 60 μL, unless the particle count reached the instrument's 200,000 count limit before 250 seconds, in which case the instrument automatically terminated data collection and moved on to the next sample. The number of particles measured was limited by the analyzed sample volume (60 μL) or 200,000 counts. The reported number of particles in particles / mL is proportional to the volume multiplication factor and the measured volume. Instrument performance was confirmed with an Easy Check Kit (EMD Millipore Corporation, St. Louis, MO) prior to sample analysis. The sample plate is gently mixed manually before loading into the GFC instrument and assayed without the use of a GFC mixer to avoid generating bubbles, potentially altering the protein aggregate population.

[0282] [Example 1] Sacituzumab CDR and framework saturation mutagenesis. 57 V H and 43 V L Positions were selected in the CDR and framework regions of the sacituzumab (hRS7) sequence. For each position, each of the 17 amino acids was selected from 976 V H +731 V L A total of 1707 point mutants were tested for all sequence variations (excluding M, C, and W). Yeast cells expressing sacituzumab mutants (mAb) were sorted with increasing amounts of labeled TROP2 protein (10, 30, and 100 nM), and low affinity cells were selected by gating to the left of the wild-type sequence. The selected mutant sequences were sorted using 40 single-point (20 V) L +20 V H) were accurately measured. Mutants with reduced affinity were identified and evaluated for binding using surface plasmon resonance single cycle kinetics. The results are shown in Table 9. Unless otherwise indicated in the table, all light chain muteins (Lm) pair with a sacituzumab heavy chain containing an S375C substitution (Hm_S375C having the amino acid sequence set forth in SEQ ID NO: 162), and all Hm muteins contain a sacituzumab light chain (LC having the amino acid sequence set forth in SEQ ID NO: 12). The amino acid sequences comprising the antibodies disclosed in Table 9 can be found in Table 27.

[0283] [Table 9] TIFF2026505934000047.tif87167

[0284] [Example 2] On-cell ELISA binding affinity of sacituzumab mutants, comparing BxPC3 and MDA-MB-231 cell lines.

[0285] TROP2-expressing cell lines. Quantitative fluorescence-activated cell sorter (qFAC) analysis of BxPC3 and MDA-MB-231-KWL cell lines for TROP2 copy number expression (Table 10).

[0286] [Table 10]

[0287] Comparison of on-cell ELISA binding affinity of sacituzumab mutants in BxPC3 and MDA-MB-231 cell lines. Cells were seeded in duplicate in 96-well plates, and primary antibodies were diluted in cell culture medium at a 1:5 dilution to a starting concentration of 30 μg / mL (200 nM). After three washes, secondary antibodies were added at 1 μg / mL for 1 hour, washed three times, and absorbance was measured at 450 nm. Data were plotted and analyzed. EC50 (kD) was determined along with the area under the curve (AUC) for each cell line. The large difference between the AUC of BxPC3 binding and MDA-MB-231-KWL (ΔAUC / AUC(BxPC-3)) was used to select mutants for further investigation. Results are shown in Table 11. Unless otherwise indicated in the table, all light chain muteins (Lm) pair with a sacituzumab heavy chain (HC having the amino acid sequence set forth in SEQ ID NO: 11), and all Hm muteins contain a sacituzumab light chain (LC having the amino acid sequence set forth in SEQ ID NO: 12). The amino acid sequences comprising the antibodies disclosed in Table 11 can be found in Table 27.

[0288] [Table 11] TIFF2026505934000050.tif31155

[0289] Surface plasmon resonance (SPR) analysis of the Y53D substitution in CDR2 of the light chain showed an approximately 10-fold decrease in affinity when the antibody bound in bivalent mode, and an approximately 100-fold difference in affinity when the Fab of the antibody bound (monovalent mode). See Table 12.

[0290] [Table 12]

[0291] Figure 1 shows that the Y53D substitution in CDR2 of the sacituzumab light chain inhibits the expression of TROP2 in low-expressing MDA-MB-231-KWL cells (TROP2 低 Higher TROP2 expression in BxPC3 cells (TROP2 高The results show that the results provide binding-modulating anti-TROP2 antibodies that preferentially bind to IgG1-specific ...

[0292] [Example 3] Developmental characteristics of various mutants. The developmental characterization of various mutants was performed similarly to Bailly et al., Predicting Antibody Developability Profiles Through Early Stage Discovery Screening, MABS 12(1):e1743053 (2020) (doi:10.1080 / 19420862.2020.17430530), the entire contents of which are incorporated herein by reference. The various methods used can be found under General Methods herein.

[0293] The results of the various assays are shown in Tables 13, 14, and 15. Unless otherwise indicated in Tables 13, 14, and 15, all light chain muteins (Lm) pair with a sacituzumab heavy chain (HC having the amino acid sequence set forth in SEQ ID NO: 11) and all Hm muteins contain a sacituzumab light chain (LC having the amino acid sequence set forth in SEQ ID NO: 12). The amino acid sequences comprising the antibodies disclosed in Tables 13, 14, and 15 can be found in Table 27.

[0294] [Table 13] TIFF2026505934000053.tif92154

[0295] [Table 14] TIFF2026505934000055.tif114153

[0296] [Table 15]

[0297] [Table 16]

[0298] [Table 17]

[0299] [Table 18]

[0300] In the table, K DApp reflects the average of measurements at different tip loadings, so the "apparent" K D RU (response units) refers to the loading onto the chip (8RU and 28.8RU mean that measurements were taken at lower antigen density (8RU) and higher antigen density (28.8RU)). BC (binding capacity) is a measure of the number of molecules that remain on the chip relative to the reference (lower k D (usually equals a lower BC due to off-rate), this is a qualitative measure.

[0301] [Example 4] Sacituzumab rehumanization and identification of the best single mutation (BSM). The amino acid sequence of sacituzumab was evaluated using the software package BIOVIA Discovery Studio using the "Predict Humanizing Mutations" tool. Conformation was set to exclude substitutions in Vernier zones, Kabat, and IMGT CDR residues. Calculated mutation energy was set to True. Germline gene matches were IGKJ4 and IGKV1_39_01 for the light chain and IGHJ4 and IGHV7_4_1 for the heavy chain. BSM was determined by the sum of each individual point mutation in the framework region predicted to be stabilizing. Overall, BSM mutations were calculated to provide -9.03 kcal / mol of stabilization using Chemistry with Harvard Macromolecular Mechanics (CHARMM) energetics against the clinical sacituzumab sequence. The resulting sacituzumab BSM sequence has higher identity to the human germline with 83% identity to IGHV7-4-1*02 and 84% identity to IGKV1-13*02 compared to the clinical sequence (81% identity for both chains).

[0302] A comparison of the performance of sacituzumab and sacituzumab BSM on reversed-phase high-performance liquid chromatography (RP-HPLC) was comparable, as shown in Figure 2 and Figure 2-1. RP-HPLC was performed at 70°C using POROS™ resin as described in the General Methods.

[0303] Hydrophobicity determination was performed on various sacituzumab variants using HIC, which was performed using a butyl HIC column as described in General Methods.

[0304] Figure 3 shows a comparison of the hydrophobicity of the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) and αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) antibodies, and the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y105S) and αTROP2(HC:BSM-S375C)(LC:BSM-Y105S) antibodies. The YTE substitution in the heavy chain did not significantly increase hydrophobicity.

[0305] Figure 4 shows a comparison of the hydrophobicity of the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) and αTROP2(HC:BSM-S375C)(LC:BSM) antibodies. The αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody peak elution was at fraction 24.14, while the αTROP2(HC:BSM-S375C)(LC:BSM) antibody eluted at fraction 31.89. The Y53D substitution in the light chain resulted in a substantial decrease in hydrophobicity.

[0306] [Example 5] The αTROP2 mAb variants were administered intravenously to biologically naive male rhesus monkeys to evaluate their pharmacokinetics (PK). The study design is listed in Table 16. Blood samples were collected from peripheral blood vessels at the indicated time points, and serum was separated from blood cells for PK analysis by a ligand-binding assay using an anti-human IgG framework antibody. PK parameters were estimated by non-compartmental analysis using Phoenix WinNonlin (version 6.3, Certara).

[0307] After a single 3 mpk IV dose in rhesus monkeys, BSM, the S375C mutation did not significantly affect parental αTROP2 PK, as shown in Figure 5 (framework variant rhesus PK after a 3 mpk IV bolus dose), and clearance values ​​were 11–13 mL / day / kg, as shown by the data in Table 16. As shown in Figure 6 (affinity variant rhesus PK after a 1 mpk IV bolus dose) and the data in Table 17, the affinity variants Y53D (light chain) and Y105S (heavy chain) on the BSM backbone had significantly lower plasma clearance (approximately 5 mL / day / kg) compared to the parental αTROP2 mAb (approximately 18 mL / day / kg) after a single 1 mpk IV dose. As shown in Figure 7 (BSM-YTE and BSM-YTE-Y53D rhesus monkey PK after a single 20 mpk IV dose) and the data in Table 18, the Y53D mutation on the BSM-YTE backbone also improved monkey PK by decreasing clearance from 4.5 mL / day / kg for BSM-YTE to 3 mL / day / kg for BSM-YTE-Y53D, and increasing the terminal half-life from 1.3 to 4.3 days. The data indicate that the PK of αTROP2 mAbs can be improved by reducing avidity and increasing hydrophilicity. Introducing YTE mutations to improve FcRn-based antibody recycling actually improved the PK of BSM-Y53D by increasing the AUC from 3930 μg / mL* days to 6560 μg / mL* days and extending the half-life from 3.4 to 4.3 days, as shown in Figure 8 and the data in Table 19.

[0308] [Table 19]

[0309] [Table 20]

[0310] [Table 21]

[0311] [Table 22]

[0312] [Example 6] In silico immunogenicity analysis (protein immunogenicity score). Immunogenicity risk profiles of the amino acid sequences of various anti-TROP2 variants, including αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D), were performed using the Interactive Screening and Protein Reengineering Interface (ISPRI) from EpiVax (Providence, RI). Epitope prediction and self-homology assessment were performed using the EpiMatrix, ClusterMatrix, Antibody Analysis, and Janus Matrix Homology tools within the ISPRI software suite (Moise et al., Clin. Immunol. 142:320-331 (2012)).

[0313] First, variant amino acid sequences were screened for MHC class II epitope content using the EpiMatrix tool. Input amino acid sequences were evaluated for predicted binding to a panel of nine class II HLA-DRB1 alleles (*0101, *0301, *0401, *0701, *0801, *0901, *1101, *1301, and *1501). The ClustiMer algorithm was then used to identify regions of high epitope density within the protein sequence. ClustiMer searches for contiguous segments of 15–30 amino acids with high binding across common HLA-DR alleles. Subsequently, epitope clusters identified by ClustiMer were evaluated with the Janus Matrix algorithm to determine epitope autologousness and assess the potential for immune tolerance to a given epitope. We used the Janus Matrix Algorithm (Moise et al., Hum. Vaccin. Immunother. 9:1577-1586 (2013)) to identify epitopes that share T cell receptor (TCR) surface conservation (positions 2, 3, 5, 7, and 8) with epitopes restricted by the same allele found in the human proteome. Epitopes with identical TCR-facing residues that are also predicted to bind the same MHC allele are more likely to induce cross-reactive T cells. Furthermore, the Janus Matrix algorithm identifies epitopes known to correspond to immunosuppressive regulatory T cell responses (termed Tregitopes), which are thought to reduce the immunogenic potential of the construct. We applied a Janus Matrix homology score threshold of two (cross-conserved HLA allele-specific epitopes averaged over the length of the sequence) for cross-conservation with human (self) proteins to identify epitopes likely to be tolerated or actively regulatory.

[0314] Of the analyzed TROP2 variant sequences, αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) demonstrated the lowest predicted immunogenicity, demonstrating an overall Tregitope-regulated protein immunogenicity score of (-31.72), comparable to the immunogenicity profile of known non-immunogenic antibodies tested in clinical trials. The antibody analysis tool predicted a 1.85% ADA response in clinical populations due to its relatively low predicted effector epitope content and high predicted Tregitope content, predicting bucketed αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) as the "optimal antibody" construct. Overall, αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) was found to have less predicted epitope content than other candidate sequences, including wild-type sacituzumab (see Figure 9). The K38R mutation (HC:BSM-S375C) (LC:BSM-Y53D) in the heavy chain of αTROP2 disrupts two promiscuous non-self epitopes found in wild-type sacituzumab and introduces two Tregitopes with high HLA binding potential, reducing the total epitope content and predicted immunogenicity risk of this molecule.

[0315] [Example 7] Synthesis of 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (1) [ka]

[0316] Step A - Synthesis of Compound I1-b p-Aminobenzyl alcohol (154 g, 125 mmol) was added to a stirred mixture of ethyl 2-ethoxy-2H-quinoline-1-carboxylate (103 g, 418 mmol) and (2S)-2-[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanamido]propanoic acid I1-a (100 g, 261 mmol) in dichloromethane (DCM):MeOH (2:1) (3000 mL) at 0 °C. The reaction mixture was stirred at room temperature for 18 h and monitored by LCMS. The solvent was evaporated in vacuo, and the residue was diluted with methyl tertiary butyl ether (TBME) (2000 mL) and stirred for 30 min. The solid was collected by filtration, washed with TBME (1000 mL), and dried in vacuo to give 9H-fluoren-9-ylmethyl N-[(1S)-1-{[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]-carbamoyl}ethyl]carbamate (I1-b). LCMS: (ES, m / z): [M+H] + =488.

[0317] Step B—Synthesis of Compound I1-c Diethylamine (756 g, 10.3 mol) was added to a stirred mixture of 9H-fluoren-9-ylmethyl N-[(1S)-1-{[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]-carbamoyl}ethyl]carbamate (I1-b) (140 g, 287 mmol) in DMF (1.4 L) at room temperature. The mixture was stirred at room temperature for 18 hours. The reaction was monitored by LCMS. The solvent was evaporated, and the residue was diluted with ethyl acetate (EtOAc) (500 mL) and stirred for 30 minutes. The solid was collected by filtration, washed with EtOAc (300 mL), and dried in vacuo to give (2S)-2-[(2S)-2-aminopropanamido]-N-[4-(hydroxymethyl)phenyl]propanamide (I1-c). LCMS: (ES, m / z): [M+H] + =266.

[0318] Step C—Synthesis of Compound I1-d A mixture of 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxopyrrol-1-yl)propanoate (55.2 g, 207 mmol), I1-c (55.0 g, 207 mmol), and DIPEA (40.2 g, 311 mmol) in DMF (550 mL) was stirred at room temperature for 16 hours. The reaction mixture was added to HO (600 mL) with stirring. The solid was collected by filtration, washed with water (500 mL), and dried in vacuo to give (2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamide]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d), which was used directly in the next step.

[0319] Step D - Synthesis of Compound I1-e N,N-Diisopropylethylamine (DIPEA) (30.7 g, 237 mmol) was added to a stirred mixture of bis(4-nitrophenyl)carbonate (60.3 g, 198 mmol) and (2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamide]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d) (55 g, 32 mmol) in dimethylformamide (DMF) (550 mL) at room temperature. The mixture was stirred for 16 h and monitored by liquid chromatography-mass spectrometry (LCMS). The reaction solution was added to HO (400 mL) with stirring. The mixture was filtered, and the filter cake was washed with water (1 × 100 mL). The solid was collected by filtration and purified using reverse-phase chromatography (dynamic axial chromatography column C-18, eluting with 15%-65% ACN / water (containing 0.1% ammonium acetate (NH4OAc) as a modifier)). The resulting mixture was concentrated in vacuo, and the solid was dried in vacuo at 45 °C to give {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamido]propanamido]propanamido]phenyl}methyl-4-nitrophenyl carbonate (I1-e). LC-MS: (ES, m / z): [M+H] + =582.

[0320] Synthesis of Step E-1 To a 60 mL round-bottom flask under N2, {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrol-1-yl)propanamido]propanamido]propanamido]phenyl}methyl-4-nitrophenyl carbonate (I1-e) (3.00 g, 5.15 mmol) and DMF (45 mL) were added, followed by the addition of hydroxybenzotriazole (HOBt) (140 mg, 1.03 mmol). The reaction mixture was stirred at 25 °C for 10 min, after which MMAE (4.00 g, 5.57 mmol) was added at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was then purified using reverse-phase column chromatography (AQ C18 30%-60% acetonitrile (MeCN) / water with 0.1% ammonium acetate as a modifier). The MeCN was concentrated in vacuo, the remaining aqueous mixture was extracted with ethyl acetate (300 ml x 3), and the combined organics were concentrated in vacuo. Four batches were run in parallel, combined, dissolved in 200 mL of MeCN, 400 mL of water was added, the solution was frozen, and then lyophilized to give 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R ,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (1) was obtained. LC-MS: (ES, m / z): [M+H] + =1160. 1H NMR(400MHz,CD3OD)δ 7.53(br d,J=8.19Hz,2H),7.03-7.32(m,7H),6.67(s,2H),5.21-5.29(m,2H),4.90-5.14(m,2H ),4.28-4.66(m,4H),4.04-4.22(m,4H),3.52-3.81(m,4H),3.24-3.46(m,6H),3.17(s, 2H), 2.97-3.09 (m, 2H), 2.73-2.90 (m, 4H), 2.28-2.47 (m, 4H), 1.43-2.20 (m, 8H), 1.16-1.38 (m, 10H), 1.00-1.12 (m, 6H), 0.61-0.94 (m, 18H). Not all exchangeable protons are reported.

[0321] [Example 8] Synthesis of 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (2). [ka]

[0322] Step A—Synthesis of Compound I-2b 6-Chloro-5-cyanopicolinic acid (I-2a) (80 g, 0.44 mol) was dissolved in DMF (5000 mL), and sodium methanethiolate (77 g, 1.1 mol) was added to the mixture in batches. The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was then diluted with ethyl acetate and added to water. The mixture was extracted with ethyl acetate, and the aqueous phase was adjusted to pH 5 with 10% citric acid. The mixture was extracted three times with ethyl acetate, and the combined organics were concentrated in vacuo to give 5-cyano-6-(methylthio)picolinic acid (I-2b). LC-MS: (ES, m / z): [M+H] + =195.

[0323] Step B—Synthesis of Compound I-2c To 5-cyano-6-(methylthio)picolinic acid (I-2b) (66 g, 0.34 mol) dissolved in tetrahydrofuran (THF) (3000 mL) was added (1-[bis(dimethylamino)methylene]-1H-1,2,3 triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (155 g, 0.41 mol). The resulting mixture was stirred at 25 °C for 30 min. tert-Butyl 3-aminopropanoate HCl salt (67.7 g, 0.37 mol) was added to the mixture, which was then cooled to 10 °C. N,N-Diisopropylethylamine (DIEA) (175 g, 1.35 mol) was added dropwise over 1 h at 10 °C. The reaction mixture was stirred at 25 °C for 16 h, concentrated in vacuo, and diluted with ethyl acetate. The resulting solution was washed three times with water, and the combined organics were concentrated in vacuo. The residue was purified using silica gel column chromatography (ethyl acetate:petroleum ether 1:2) to give tert-butyl 3-(5-cyano-6-(methylthio)picolinamido)propanoate (I-2c). LC-MS: (ES, m / z): [M+Na] + =344

[0324] Step C-Synthesis of compound I-2d tert-Butyl 3-(5-cyano-6-(methylthio)picolinamido)propanoate (I-2c). I-2c (96 g, 0.30 mol) was dissolved in DCM (1500 mL), and a solution of meta-chloroperbenzoic acid (m-CPBA) (206 g, 1.19 mol) in DCM (1500 mL) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The resulting reaction mixture was diluted with DCM and poured into ice water. The organic phase was washed four times with 10% aqueous sodium bicarbonate solution, and the combined organic phases were dried and concentrated in vacuo to give tert-butyl 3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoate (I-2d). LC-MS: (ES, m / z): [M+Na] + =376

[0325] Step D - Synthesis of Compound I-2e To tert-butyl 3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoate (I-2d) (90 g, 0.25 ol) dissolved in 1,4-dioxane (1000 mL) was added 4 M HCl in 1,4-dioxane (2500 mL). The resulting mixture was stirred at 25 °C for 16 h and then filtered. The solid was washed with n-heptane and dried under nitrogen to give 3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoic acid (I-2e). LC-MS: (ES, m / z): [M+Na] + =320

[0326] Step E - Synthesis of Compound I-2f tert-Butyl L-alanyl-L-alaninate (iii) (50 g, 0.23 mol; Example 9) was dissolved in THF (2300 mL). To this mixture was added 3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoic acid (I-2e) (69 g, 0.23 mol) and HATU (106 g, 0.28 mol). The reaction mixture was stirred for 30 minutes and then cooled to 10°C. DIEA (90 g) was then added dropwise over 30 minutes. The reaction mixture was stirred at 20°C overnight. The reaction mixture was then concentrated in vacuo, and the resulting residue was diluted with ethyl acetate. The combined organics were washed three times with water, dried, and concentrated in vacuo. The resulting residue was purified using silica gel chromatography with DCM:MeOH (2:1) to give tert-butyl (3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alaninate (I-2f). LC-MS: (ES, m / z): [M+Na] + =518

[0327] Step F - Synthesis of Compound I-2g 4M HCl in 1,4-dioxane (1800 mL) was added to tert-butyl (3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alaninate (I-2f) (90 g, 0.18 mol) dissolved in MeCN (2000 mL). The reaction mixture was stirred at 25° C. for 16 hours and then concentrated in vacuo. The resulting solid was washed with MTBE and filtered. The solid was air-dried overnight to give (3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alanine (I-2g). LC-MS: (ES, m / z): [M+Na] + =462

[0328] Step G - Synthesis of compound I-2h A 500 mL four-neck round-bottom flask was purged with nitrogen and maintained under an inert atmosphere of nitrogen, and (3-(5-cyano-6-(methylsulfonyl)picolinamido)propanoyl)-L-alanyl-L-alanine (I-2g) (30 g, 0.65 mol), DMF (300 mL), and HATU (31.2 g, 0.780 mol) were added. The reaction mixture was stirred at room temperature for 30 minutes. Then, (4-aminophenyl)methanol (9.0 g, 0.068 mmol) was added at 20° C., and the reaction mixture was cooled to 10° C. DIEA (90 g, 0.19 mmol) was added dropwise to the reaction mixture over 30 minutes at 10° C., and the resulting mixture was stirred at 25° C. for 5 hours. Then, bis(4-nitrophenyl)carbonate (42 g, 0.13 mmol) was added to the reaction mixture at 25° C., and the mixture was stirred at 25° C. for 1 hour. The reaction mixture was purified using C-18 flash column chromatography (30%-60% ACN / water (containing 0.05% TFA as a modifier)) to give 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)benzyl(4-nitrophenyl)carbonate (I-2h). LC-MS: (ES, m / z): [M+Na] + =732

[0329] Step H - Synthesis of Compound 2 To 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)benzyl (4-nitrophenyl)carbonate (I-2h) (5.6 g, 7.9 mmol) and 1H-benzo[d][1,2,3]triazol-1-ol (0.213 g, 1.58 mmol) was added DMF (56.0 mL). MMAE (5.67 g, 7.89 mmol) was added to the reaction at 20 °C, and the reaction mixture was stirred at 40 °C for 16 h. The resulting mixture was analyzed by Prep-HPLC (10-95% MeCN / water, 0.05% HCl). TFA) to give 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamido)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (2). 1 H NMR(400MHz,DMSO-d6)δ 9.86(s,1H),9.14(t,J=6.0Hz,1H),8.79(d,J=8.1Hz,1H),8.38(d,J=8.1Hz,1H),8.33-7.97(m,3H),7.96-7.51 (m,3H),7.41-6.87(m,7H),6.09(s,1H),5.37(dd,J=26.2,5.0Hz,1H),5.04(tt,J=25.6,12.6Hz,2H),4.69(d,J= 44.2Hz,1H),4.59-4.16(m,5H),4.15-3.90(m,2H),3.80-3.43(m,8H),3.36-2.95(m,9H),2.94-2.58(m,2H),2.5 0-2.20(m,3H),2.19-1.79(m,3H),1.65-1.37(m,3H),1.32-1.18(m,9H),1.06-0.96(m,7H),0.89-0.59(m,20H).

[0330] [Example 9] Preparation of intermediate tert-butyl L-alanyl-L-alaninate (iii) [ka] Step A - Synthesis of Compound II To (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (i) (140 g, 450 mmol) dissolved in DCM (4500 mL) was added PyBOP (281 g, 540 mmol, 1.2 equiv.) at room temperature. The reaction mixture was stirred for 20 minutes. tert-Butyl L-alaninate HCl (85.6 g, 471 mmol) was added and the solution was cooled to 0°C. DIEA (175 g, 1.35 mol) was then added dropwise to the reaction over 1 hour, which was then stirred at 20°C overnight. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate, and washed with aqueous sodium carbonate (1 M), potassium bisulfate (1 M), water, and brine. The organic phase was then concentrated in vacuo and evaporated to 25% of the original solvent volume. MTBE (1400 mL) was added dropwise and stirred for 5 hours. The suspension was filtered and the solid was washed with MTBE to give tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (ii). LCMS: (ESI, m / z): [M+H] + =439.2.

[0331] Step B - Synthesis of Compound iii To tert-butyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanyl-L-alaninate (ii) (172 g, 392 mmol) dissolved in DCM (2000 mL) was added triethanolamine (TEA) (2000 mL). The reaction mixture was warmed to 40° C. and stirred for 16 hours. The reaction mixture was then concentrated in vacuo and purified using silica gel chromatography (DCM:4M NH3 in MeOH 5:1) to give tert-butyl L-alanyl-L-alaninate (iii). LCMS: (ESI, m / z): [M+H] + =217.1.

[0332] [Example 10] Preparation of the intermediate compound 3-(5-cyano-6-methylsulfonyl)nicotinamido)propanoic acid (x) [ka] Step A - Synthesis of Compound v A solution of 5-bromo-6-hydroxypyridine-3-carboxylic acid (iv, 42.8 g, 196 mmol) and cuprous cyanide (35.2 g, 393 mmol) in N-methyl-2-pyrrolidone (NMP) (430 mL) was stirred at 165 °C under a N atmosphere for 2 h. The mixture was concentrated in vacuo, and the crude product was purified using reverse-phase flash chromatography (AQ C18 silica gel, 0% to 20% ACN gradient in water (using 0.5% NH₃·H₂O as a modifier)) to give the crude product. The resulting mixture was filtered, and the filter cake was washed with H₂O. The filtrate was concentrated in vacuo to give 5-cyano-6-hydroxypyridine-3-carboxylic acid (v). LCMS: (ESI, m / z): [MH] - =163.

[0333] Step B—Synthesis of Compound vi A solution of 5-cyano-6-hydroxypyridine-3-carboxylic acid (v) (25.7 g, 157 mmol) and phosphorus oxychloride (130 mL) was stirred at 110° C. for 2 hours. The reaction was monitored by LCMS and then concentrated in vacuo. Water cooled to 10° C. and EtOAc were added, and the solid was filtered off. The mixture was extracted with ethyl acetate, dried, and concentrated in vacuo. The residue was purified using reverse-phase flash chromatography (column AQ silica gel, 0% to 15% acetonitrile / sodium bicarbonate (aqueous)). The pH value of the aqueous layer was adjusted to 2-3 with 1 M HCl, and then the aqueous layer was extracted with ethyl acetate (3×), dried, and concentrated in vacuo to give 6-chloro-5-cyanopyridine-3-carboxylic acid (vi). LCMS: (ESI, m / z): [MH] - =181.

[0334] Step C - Synthesis of Compound vii Dimethylformamide (115 mL) and 6-chloro-5-cyanopyridine-3-carboxylic acid (vi) (7.7 g, 42 mmol) were added to a 500 mL three-necked bottle under nitrogen atmosphere at 25 °C. To the above mixture, (methylsulfanyl)sodium (7.39 g, 105 mmol) was added in portions at 0 °C. The resulting mixture was stirred at 25 °C for 8 h. The reaction was monitored by LCMS. The solution was slowly transferred to H2O (1200 mL) and then extracted with ethyl acetate (1 × 700 mL). The pH value of the aqueous layer was adjusted to 2-3 with 1 M HCl. The solid was collected by filtration to give 5-cyano-6-(methylsulfanyl)pyridine-3-carboxylic acid (vii). LCMS: (ESI, m / z): [MH] - =193.

[0335] Step D - Synthesis of Compound viii A solution of 5-cyano-6-(methylsulfanyl)pyridine-3-carboxylic acid (vii) (7.4 g, 38 mmol) in DCM (185 mL) was treated with m-CPBA (26.3 g, 152 mmol) under a nitrogen atmosphere at 45° C. for 24 hours. The reaction was monitored by LCMS and then quenched with saturated sodium bisulfite at 0° C. and concentrated in vacuo. 2-Methyltetrahydrofuran was added to the residue, and the mixture was then filtered and concentrated in vacuo to give the crude product. The residue was purified using silica gel column chromatography eluting with DCM / MeOH to give 5-cyano-6-(methylsulfonyl)nicotinic acid (viii). LCMS: (ESI, m / z): [M+H] + =227.05.

[0336] Step E - Synthesis of Compound ix 5-Cyano-6-(methylsulfonyl)nicotinic acid (viii) (0.57 g, 2.5 mmol) and HATU (1.0 g, 2.7 mmol) were dissolved in 10 mL DMF and stirred at 25 °C for 30 min. tert-Butyl 3-aminopropanoate (0.42 g, 2.8 mmol) was then added, and the mixture was cooled to 10 °C. DIPEA (0.873 mL, 5.00 mmol) was added dropwise to the reaction mixture at 10 °C. The reaction mixture was stirred at 25 °C for 2 h, then diluted with water and extracted with EtOAc (3x). The organic phase was concentrated in vacuo, and the residue was purified using silica gel column chromatography eluting with 2:1 to 1:1 hexane:EtOAc to give tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoate (ix). 1 H NMR(500MHz,CD3OD)δ 8.49(d,J=2.0Hz,1H),8.07(d,J=2.0Hz,1H),4.12(s,3H),2.92(t,J=6.9Hz,2H),1.88(t,J=6.9Hz,2H),0.74(s,9H).

[0337] Step F - Synthesis of Compound x tert-Butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoate (ix) (0.10 g, 0.28 mmol) was dissolved in 10 mL of 1,4-dioxane. 4 M HCl in 1,4-dioxane (10 mL) was then added to the mixture. The resulting mixture was stirred at 25° C. for 16 hours, and the reaction mixture was filtered. The solid was washed with n-heptane and dried under nitrogen for 5 hours to give 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoic acid (x). 1 H NMR(500MHz,CD3OD)δ 9.22(d,J=1.9Hz,1H),8.80(d,J=1.9Hz,1H),3.66(t,J=5.8Hz,2H),3.44(s,3H),2.67(t,J=6.8Hz,2H).

[0338] [Example 11] Preparation of intermediate compound 4-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiv) [ka]

[0339] Step A - Synthesis of Compound xii A solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (xi) (Fmoc-Ala-Ala-PAB) (13 g, 27 mmol, 1 equiv.) in DMF (130 mL) was added to a 500 mL four-neck round-bottom flask purged and maintained under an inert atmosphere of nitrogen. 2 mol% DIEA (0.534 mmol) and bis(4-nitrophenyl)carbonate (16.3 g, 53.6 mmol, 2 equiv.) were then added to the reaction mixture at 20 °C. The reaction mixture was warmed to 45 °C and stirred for 16 h. The reaction mixture was cooled to room temperature and purified using reverse-phase flash column chromatography (30%-60%, MeCN / water with 0.05% TFA as a modifier) ​​to give (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (xii). LCMS: (ESI, m / z): [M+H] + =653.2.

[0340] Step B - Synthesis of Compound xiii A solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (xii) (8.30 g, 12.7 mmol) in DMF (83 mL) was added to a 250 mL four-neck round-bottom flask purged and maintained with an inert atmosphere of nitrogen. HOBt (340 mg, 2.5 mmol) was then added, and the reaction mixture was stirred at room temperature for 10 minutes. MMAE (9 g, 12.7 mmol) was then added to the reaction mixture, and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was purified using reverse-phase flash column chromatography (20% to 50% MeCN / water with 0.05% TFA as a modifier) ​​to give 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R -3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiii) was obtained. LCMS: (ESI, m / z): [M+H] + =1231.7.

[0341] Step C - Synthesis of Compound xiv 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)propanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxo (isopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiii) (0.092 g, 0.075 mmol) was dissolved in DCM (0.5 mL), and TEA (0.5 mL) was added to the reaction mixture. The reaction mixture was stirred at 40° C. for 16 hours. The reaction mixture containing 4-((S)-2-((S)-2-aminopropanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (xiv) was used directly without further purification or concentration. LC-MS: (ESI, m / z): [M+H] + =1009.8.

[0342] [Example 12] Synthesis of 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanamido)propanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3). [ka]

[0343] 3-(5-cyano-6-(methylsulfonyl)nicotinamido)propanoic acid (x; Example 10) (0.027 g, 0.090 mmol) and HATU (0.037 g, 0.097 mmol) were dissolved in 0.4 mL of DMF and stirred for 10 min. xiv was then added dropwise, followed by DIEA (0.034 ml, 0.19 mmol). The reaction was stirred for 1.5 h and then purified by reverse phase chromatography (Waters The purified product was 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)nicotinamide)propanamide)propanamide)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1 S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3) was obtained. LC-MS: (ESI, m / z): [M+H] + =1288.665.1 1H NMR (600 MHz, CD3CN) δ 9.12 (s, 1H), 8.68 (s, 1H), 8.63 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.4 - 7.06 (m, 9H), 6.66 (d, J = 8.5 Hz, 1H), 6.55 (d, J = 7.75 Hz, 1H), 5.18 (d, J = 12.18 Hz, 1H), 5.04 (d, J = 12.18 Hz, 1H), 4.71 (d, J = 4.16 Hz, 1H), 4.63 (m, 1H), 4.45 (q, J = 7.3 Hz, 1H), 4.32 - 4.00 (m, 4H), 3.9 - 3.73 (m, 3H), 3.67 (m, 2H), 3.58 - 3.38 (m, 3H), 3.36 (s, 3H), 3.35 (s, 3H), 3.28 (s, 3H), 3.18 (m, 1H), 3.01 (s, 3H), 2.86 (br, 3H), 2.63 (m, 1H), 2.52 (m, 1H), 2.46 (br, 2H), 2.17 (br, 2H), 1.99 (m, 1H), 1.9 - 1.6 (m, 4H), 1.59 (br, 1H), 1.48 (d, J = 7.38 Hz, 3H), 1.36 (m, 4H), 1.12 (d, J = 6.81 Hz, 3H), 1.02 (d, J = 6.95 Hz, 3H), 0.98 (m, 4H), 0.92 - 0.67 (m, 15H).

[0344] [Example 13] Conjugation protocol: An antibody (S375C) bearing two engineered Cys residues was decapped, and the interchain disulfides were reduced using tris(2-carboxyethyl)phosphine (TCEP) (20 equivalents, 0.5 M in water adjusted to pH 7.0 with ammonium hydroxide) at 37 °C for 2 h, monitored by HPLC-MS. The reduced antibody was then buffer-exchanged via AKTA™ (desalting column, monitored at 280 nm) into PBS buffer at pH 7.4 or 30 mM ACES (N-(2-acetamido)-2-aminoethanesulfonic acid, N-(carbamoylmethyl)-2-aminoethanesulfonic acid, N-(carbamoylmethyl)taurine) buffer at pH 7.0, and diluted to 10 mg / mL using the same buffer for subsequent steps. A 100 mM solution of dehydroascorbic acid (8.0 equiv.) in water was slowly added, and the solution was mixed at room temperature for 4-10 h or until complete (monitored by reverse-phase liquid chromatography (RP-LC) and sodium dodecyl sulfate capillary electrophoresis (CE-SDS)). A 20 mg / mL solution of linker payload (3.0 equiv.) in DMSO was added, and the conjugation was incubated at room temperature for 2 h (monitored by quadrupole time-of-flight (QTOF) mass spectrometry (MS)). L-cysteine ​​(5 equiv., 50 mM in water) was then added to quench any residual linker payload. The ADCs were purified by AKTA (desalting column, 10 mM histidine buffer pH 6.0, monitored at 280 nm) and characterized by LCMS (Agilent polymeric reversed-phase (PLRP)-S column, 1000 Å, 5 μm, 15–90% MeCN / HO with 0.1% formic acid, column temperature 80 °C) and size-exclusion chromatography (SEC) (Acquity UPLC Protein BEH SEC, 200 Å, 1.7 μm, 100 mM sodium phosphate, 200 mM NaCl, 0.02% azide, 5% isopropyl alcohol (IPA) added to the mobile phase for hydrophobic ADCs). Sartorius Vivacell 70 Centrifugal Concentrators and Amicon® Ultra Centrifugal Filters were used to bring the ADCs to the final desired concentrations.

[0345] Exemplary conjugates were made according to the protocols above to produce the following ADCs:

[0346] αTROP2 conjugated to MP-AA-PABC-MMAE (ADC1) (HC:BSM-YTE-S375C) (LC:BSM-Y53D) αTROP2 conjugated to MP-AA-PABC-MMAE (ADC2) (HC:BSM-YTE-S375C-MMAE) (LC:BSM) αTROP2 conjugated to MP-AA-PABC-MMAE (ADC3) (HC:Sac-S375C-MMAE) (LC:Sac) The ADC was formulated in a solution containing 10 mM histidine, 7.5% sucrose, and 0.02% polysorbate 80 (PS80) at pH 6.0.

[0347] Figure 10 shows a comparison of RP-HPLC performance between ADC2 and ADC3. ADC2 showed better recovery and potency compared to ADC3. Figure 11 shows a mass spectrometry (MS) profile of a composition containing ADC1, with the positions for DAR0, DAR1, DAR2, and DAR3 indicated. Figure 12 shows an HIC profile of a composition containing ADC1, with the positions for DAR0, DAR1, DAR2, and DAR3 indicated. The αTROP2 antibody contains HC:BSM-YTE-S375C-MMAE and LC:BSM-Y53D. The control antibody profile is an unconjugated antibody. Table 20 summarizes the results and shows that the average DAR for ADC1 based on MS was approximately 1.9-2.0.

[0348] [Table 23]

[0349] [Example 14] In vitro cytotoxicity of ADCs containing binding-tuned sacituzumab variants containing an S375C substitution in which the cysteine ​​is conjugated to MP-AA-PABC-MMAE.

[0350] Cytotoxicity data values ​​are the mean IC for each test article. 50 and may include data from different lots of the same construct. All data in this example is from 3-day assays. A control ADC consisting of a non-anti-TROP2 antibody conjugated to MP-AA-PABC-MMAE at the cysteine ​​at position 375 (Control mAb(HC:S375C-MMAE)) was included.

[0351] TROP2+ cells with different TROP2 surface densities (quantified TROP2 density values ​​are in parentheses). Specific bioconjugates were performed on other cells. Human tumors (BxPC3, Calu-3, HCC1806, HCC78, JIMT-1, and NCI-N87) and ATCC-derived primary cells (PCS-301 (primary small airway) and PCS-200 (primary keratinocytes)) were seeded at 1500 or 300 cells, respectively, on day 0 in growth medium (Table 21) on white tissue culture (TC)-treated 384-well microplates (Corning, catalog no. 3570). On day 1, 10x intermediate assay plates (Waters plates, catalog no. 186002632) were prepared using a BRAVO liquid handler. Serial dilutions were performed using ADC formulation buffer (10 mM pH 6.5 histidine, 9% sucrose). Media (without cells) was used as Max_E, and cells were used as Min_E. 5 μL of 10x ADC was then added from the middle plate to the assay plate using a Bravo liquid handler at a very slow speed to avoid disturbing the cell monolayer. The plate was then incubated at 37°C for 4 days. On day 5, 20 μL of CellTiter-Glo 2.0 Reagent (Promega Corporation, Madison, WI, catalog number G9242) was added to 50 μL of cell-containing media using a Standard Cassette Combi. The contents were mixed on an orbital shaker for 2–3 minutes to induce cell lysis. The plate was incubated at room temperature (RT) for 5 minutes to stabilize the luminescence signal. Luminescence was recorded and EC was determined using an integration time of 0.25–1 second per well and the guidelines. 50 Values ​​were calculated. Activity data were normalized as percent effect according to the formula %E=((Response-Min_E) / (Max_E-Min_E)*100. Cytotoxicity results are shown in Table 22.

[0352] [Table 24]

[0353] [Table 25] TIFF2026505934000073.tif159154

[0354] [Table 26] TIFF2026505934000075.tif173153

[0355] [Example 15] The in vitro cytotoxicity of wild-type sacituzumab and its Y53D mutant against different framework or backbone constructs containing the S375C substitution, in which the cysteine ​​was conjugated to MP-AA-PABC-MMAE, was evaluated in cells with different TROP2 surface densities. A control ADC consisting of a non-anti-TROP2 antibody conjugated to MP-AA-PABC-MMAE at the cysteine ​​at position 375 (Control mAb(HC:S375C-MMAE)) was included.

[0356] Cytotoxicity data are shown in Table 23. Values ​​reported are the mean IC for each test article. 50 and may include data from different lots of the same construct. All data in this table is from a 4-day assay using a variety of TROP2+ cells with different TROP2 surface densities. Certain ADCs were tested on other cells. [Table 27] [Table 28]

[0357] [Example 16] In vivo antitumor efficacy of several binding-tuned sacituzumab variants conjugated to the MMAE linker payload MP-AA-PABC-MMAE in the BxPC3 mouse model.

[0358] Experimental protocol: 7-8 week-old female BALB / c nude mice were purchased from Gempharmatech Co., Ltd. (La Jolla, CA). Mice were anesthetized with isoflurane inhalant and inoculated with 10 × 10 PBS:Matrigel (50:50) in a 0.1 mL volume. 6 A single-cell suspension of BxPC3 pancreatic cancer cells (viability ≥ 95%) was inoculated subcutaneously into the right lower flank. Mice were weighed and tumors were measured twice a week and calculated using the formula V = 0.5[a * b 2 Tumor volume was calculated using the formula: where a and b are the long and short diameters of the tumor, respectively. A computer-generated randomization procedure was used to minimize tumor volume variation between groups, and 110 mice were assigned to 11 groups of 10 mice each. When tumors were 200 mm 3 Treatment began when the tumors reached an average size of 100 mg / kg. PBS and MMAE conjugates were dosed intravenously (IV) at 2 mg / kg according to Table 25.

[0359] [Table 29]

[0360] FIG. 13 shows that binding-tuned sacituzumab variants conjugated to MP-AA-PABC-MMAE were effective in reducing tumor volume after treatment dosing.

[0361] [Example 17] Dose-response study with αTROP2 (HC:BSM-YTE-S375C-MMAE) (LC:BSM-Y53D) in the BxPC3 mouse model. A control ADC consisting of a non-anti-TROP2 antibody conjugated to MP-AA-PABC-MMAE at the cysteine ​​at position 375 (Control mAb (HC:S375C-MMAE)) was included.

[0362] Experimental protocol: 7-8 week-old female BALB / c nude mice were purchased from Gempharmtech Co., Ltd. Mice were anesthetized with isoflurane inhalant and inoculated with 10 × 10 PBS:Matrigel (50:50) in a volume of 0.1 mL.6 A single-cell suspension of BxPC3 pancreatic cancer cells (viability ≥ 95%) was inoculated subcutaneously into the right lower flank. Mice were weighed and tumors were measured twice a week and calculated using the formula V = 0.5[a * b 2 Tumor volume was calculated using the formula: where a and b are the long and short diameters of the tumor, respectively. Seventy mice were assigned to seven groups of 10 mice each using a computer-generated randomization procedure to minimize tumor volume variation between groups. When tumors were 200 mm 3 Treatment began when the cells reached an average size of 100 μg / ml. PBS, control mAb, and bioconjugates were dosed intravenously (IV) according to Table 26.

[0363] [Table 30]

[0364] Figure 14 shows that αTROP2(HC:BSM-YTE-S375C-MMAE)(LC:BSM-Y53D) was effective in reducing tumor volume after treatment administration.

[0365] [Example 18] The αTROP2(HC:BSM-YTE-S375C-MMAE)(LC:BSM-Y53D) antibody in rat PK assays demonstrated linker stability with little payload shedding from the antibody. In these experiments, αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) was conjugated to MP-AA-PABC-MMAE at the cysteine ​​at position 375.

[0366] The αTROP2 MMAE ADC was administered intravenously at 5 mg / kg to male Wistar Hannover rats. Plasma concentrations of payload-conjugated anti-TROP2 antibody (cAb), total antibody (tAb), and released payload were measured up to 14 days post-dose. PK parameters were estimated by non-compartmental analysis using Phoenix WinNonlin (version 6.3, Certara). Released MMAE concentrations were determined using an LC / MS-MS method with a lower limit of quantitation (LLOQ) of 0.055 ng / mL for MMAE. Plasma cAb and tAb concentrations were determined by a ligand-binding assay using the anti-TROP2 MMAE antibody conjugate and an anti-human IgG framework antibody.

[0367] Plasma tAb, cAb, and released payload concentrations over time for αTROP2 (HC:BSM-YTE-S375C-MMAE) (LC:BSM-Y53D) are plotted in Figure 15. For the ADC, the measured tAb and cAb PK curves were superimposable, with no detectable unconjugated MMAE in plasma, indicating linker stability in vivo. The terminal half-life was 9.9 days.

[0368] Table 27 provides the sequences referred to in this disclosure. [Table 31] TIFF2026505934000081.tif234153TIFF2026505934000082.tif235154TIFF2026505934000083.tif234154TIFF2026505934000084.tif233154TIFF2026505934000085.tif233154TIFF2026505934000086.tif234155TIFF2026505934000087.tif234154TIFF2026505934000088.tif235154TIFF2026505934000089.tif230154TIFF2026505934000090.tif234154TIFF2026505934000091.tif232154TIFF2026505934000092.tif234153TIFF2026505934000093.tif233154TIFF2026505934000094.tif236153TIFF2026505934000095.tif233153TIFF2026505934000096.tif234154TIFF2026505934000097.tif235155TIFF2026505934000098.tif235153TIFF2026505934000099.tif232153TIFF2026505934000100.tif233153TIFF2026505934000101.tif232153TIFF2026505934000102.tif233154TIFF2026505934000103.tif234154TIFF2026505934000104.tif232154TIFF2026505934000105.tif237153TIFF2026505934000106.tif235154TIFF2026505934000107.tif234154TIFF2026505934000108.tif233155TIFF2026505934000109.tif236152TIFF2026505934000110.tif233154TIFF2026505934000111.tif236154TIFF2026505934000112.tif236153

[0369] While the present invention has been described herein with reference to illustrated embodiments, it should be understood that the invention is not limited thereto. Those skilled in the art and with access to the teachings herein will recognize additional modifications and embodiments within its scope. Various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims. Therefore, the present invention is limited only by the language of this specification and the claims appended hereto.

[0370] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. An antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that specifically binds to human TROP2 conjugated to a linker-monomethyllauristatin E (linker-MMAE) payload, wherein the antibody comprises two heavy chains, each heavy chain comprising a variable domain and a constant domain, the variable domains comprising complementarity determining regions (CDRs) H1, CDRH2 and CDRH3; and two light chains, each light chain comprising a variable domain and a constant domain, the variable domains comprising CDRL1, CDRL2 and CDRL3; wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences of NYGMN (SEQ ID NO: 4), WINTYTGEPTYTDDFKG (SEQ ID NO: 5), GGFGSSYWYFDV (SEQ ID NO: 6), KASQDVSIAVA (SEQ ID NO: 7), SASDRYT (SEQ ID NO: 10), and QQHYITPLT (SEQ ID NO: 9), respectively.

2. 2. The ADC of claim 1, wherein the antibody or antigen-binding fragment thereof exhibits reduced binding to low TROP2-expressing cells compared to high TROP2-expressing cells and has reduced hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

3. The ADC of claim 1 , wherein the antibody or antigen-binding fragment thereof is humanized.

4. The antigen-binding fragment of the antibody may be a Fab fragment, a Fab' fragment, or an F(ab') ２ The ADC of claim 1, which is a fragment.

5. 2. The ADC of claim 1 , wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 or 14, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or 16.

6. 6. The ADC of claim 5, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3, or the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 14 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

16.

7. 2. The ADC of claim 1 , wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, 17, or 18.

8. The antibody (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain comprising the amino acid sequence of SEQ ID NO:

18.

8. The ADC of claim 7, comprising:

9. 2. The ADC of claim 1 , wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58, 59, or 60.

10. The antibody (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 59; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain comprising the amino acid sequence of SEQ ID NO:

60.

10. The ADC of claim 9, comprising:

11. 2. The ADC of claim 1, wherein the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the constant domain of the heavy chain and positions 165 and 168 of the constant domain of the light chain, wherein the position numbering of the heavy chain constant domain is according to Eu numbering, and the position numbering of the light chain constant domain is according to consecutive numbering of the entire light chain sequence.

12. 12. The ADC of claim 11, wherein the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

13. 13. The ADC of claim 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, 20, 61, or 62.

14. 14. The ADC of any one of claims 11 to 13, wherein the linker-MMAE payload is conjugated to the cysteine ​​or non-standard amino acid.

15. The antibody has a thiol (SH) group of the formula: MP-AA-PABC-MMAE: 【Chemistry 1】 ； CM2P-AA-PABC-MMAE: 【Chemistry 2】 CM3P-AA-PABC-MMAE: 【Transformation 3】 2. The ADC of claim 1, comprising a cysteine ​​residue conjugated to a linker-MMAE payload comprising:

16. The ADC has the formula: 【Chemistry 4】 where Ab is an anti-TROP2 antibody, and p is an integer from 1 to 8, and S is derived from the side chain of a cysteine ​​residue of the antibody.

2. The ADC of claim 1, comprising:

17. The ADC formula: 【Transformation 5】 wherein Ab is an anti-TROP2 antibody comprising a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue; wherein the anti-TROP2 antibody comprising the engineered cysteine ​​residue is (A): (a) αTROP2 (HC:Sac-E152C) (LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:64 and two light chains having the amino acid sequence set forth in SEQ ID NO:13; (b) αTROP2 (HC: Sac-E153C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 65 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (c) αTROP2 (HC: Sac-E171C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 66 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (d) αTROP2 (HC: Sac-E172C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 67 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (e) αTROP2 (HC: Sac-E173C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 68 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (f) αTROP2 (HC: Sac-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 69 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (g) αTROP2 (HC: Sac-YTE-E152C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 71 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (h) αTROP2 (HC: Sac-YTE-E153C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 72 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (i) αTROP2 (HC: Sac-YTE-E171C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 73 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (j) αTROP2 (HC: Sac-YTE-E172C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 74 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (k) αTROP2 (HC: Sac-YTE-E173C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 75 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (l) αTROP2 (HC: Sac-YTE-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 76 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (m) αTROP2 (HC:Sac) (LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:58 and two light chains having the amino acid sequence set forth in SEQ ID NO:50; (n) αTROP2 (HC:Sac) (LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:58 and two light chains having the amino acid sequence set forth in SEQ ID NO:51; (o) αTROP2 (HC: Sac-YTE) (LC: Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 63 and two light chains having the amino acid sequences set forth in SEQ ID NO: 50; (p) αTROP2 (HC: Sac-YTE) (LC: Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 63 and two light chains having the amino acid sequence set forth in SEQ ID NO: 51; (q) αTROP2 (HC: BSM-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 83 and two light chains having the amino acid sequence set forth in SEQ ID NO: 21; (r) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:78 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (s) αTROP2 (HC:BSM-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:79 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (t) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:80 and two light chains having the amino acid sequence set forth in SEQ ID NO:22; (u) αTROP2 (HC: BSM-E172C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 81 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (v) αTROP2 (HC: BSM-E173C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 82 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (w) αTROP2 (HC: BSM-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 83 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (x) αTROP2 (HC:BSM) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:54; (y) αTROP2 (HC:BSM) (LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:55; (z) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:60 and two light chains having the amino acid sequences set forth in SEQ ID NO:54; (aa) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:58 and two light chains having the amino acid sequences set forth in SEQ ID NO:57; (bb) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:85 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (cc) αTROP2 (HC: BSM-YTE-E153C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 86 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (dd) αTROP2 (HC: BSM-YTE-E171C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 87 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (ee) αTROP2 (HC: BSM-YTE-E172C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 88 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (ff) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:89 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; and (gg) αTROP2 (HC: BSM-YTE-S375C) (LC: BSM-Y53D) comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 90 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; is selected from the group consisting of wherein S is derived from the side chain of the engineered cysteine ​​residue and p is an integer selected from 1 or 2; or (B): (a) αTROP2 (HC: Sac-E152C-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 70 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (b) αTROP2 (HC: Sac-YTE-E152C-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 77 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (c) αTROP2 (HC: BSM-E152C-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 84 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; and (d) αTROP2 (HC: BSM-YTE-E152C-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 91 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; wherein S is derived from the side chain of said engineered cysteine ​​residue, and wherein p is an integer selected from 1, 2, 3, or 4.

2. The ADC of claim 1, comprising:

18. The ADC formula: 【Transformation 6】 wherein Ab is an anti-TROP2 antibody comprising a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue; wherein the anti-TROP2 antibody comprising the engineered cysteine ​​residue is (A): (a) αTROP2 (HC: Sac-E152C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 24 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (b) αTROP2 (HC: Sac-E153C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 25 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (c) αTROP2 (HC: Sac-E171C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 26 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (d) αTROP2 (HC: Sac-E172C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 27 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (e) αTROP2 (HC: Sac-E173C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 28 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (f) αTROP2 (HC: Sac-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 29 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (g) αTROP2 (HC: Sac-YTE-E152C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 31 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (h) αTROP2 (HC: Sac-YTE-E153C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 32 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (i) αTROP2 (HC: Sac-YTE-E171C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 33 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (j) αTROP2 (HC: Sac-YTE-E172C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 34 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (k) αTROP2 (HC: Sac-YTE-E173C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 35 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (l) αTROP2 (HC: Sac-YTE-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 36 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (m) αTROP2 (HC:Sac) (LC:Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 11 and two light chains having the amino acid sequence set forth in SEQ ID NO: 52; (n) αTROP2 (HC:Sac) (LC:Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 11 and two light chains having the amino acid sequence set forth in SEQ ID NO: 53; (o) αTROP2 (HC: Sac-YTE) (LC: Sac-Y53D-E165C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 23 and two light chains having the amino acid sequence set forth in SEQ ID NO: 52; (p) αTROP2 (HC: Sac-YTE) (LC: Sac-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 23 and two light chains having the amino acid sequence set forth in SEQ ID NO: 53; (q) αTROP2 (HC: BSM-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 19 and two light chains having the amino acid sequence set forth in SEQ ID NO: 21; (r) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:38 and two light chains having the amino acid sequence set forth in SEQ ID NO:22; (s) αTROP2 (HC:BSM-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:39 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (t) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:40 and two light chains having the amino acid sequence set forth in SEQ ID NO:22; (u) αTROP2 (HC: BSM-E172C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 41 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (v) αTROP2 (HC: BSM-E173C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 42 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (w) αTROP2 (HC: BSM-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 19 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (x) αTROP2 (HC:BSM) (LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 17 and two light chains having the amino acid sequence set forth in SEQ ID NO: 55; (y) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E165C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 18 and two light chains having the amino acid sequence set forth in SEQ ID NO: 54; (z) αTROP2 (HC:BSM-YTE) (LC:BSM-Y53D-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 55; (aa) αTROP2 (HC: BSM-YTE-E152C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 44 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (bb) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:45 and two light chains having the amino acid sequences set forth in SEQ ID NO:22; (cc) αTROP2 (HC: BSM-YTE-E171C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 46 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (dd) αTROP2 (HC: BSM-YTE-E172C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 47 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; (ee) αTROP2 (HC: BSM-YTE-E173C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 48 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; and (ff) αTROP2 (HC: BSM-YTE-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 20 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; wherein S is derived from the side chain of the engineered cysteine ​​residue and p is an integer selected from 1 or 2; or (B): (a) αTROP2 (HC: Sac-E152C-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 30 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (b) αTROP2 (HC: Sac-YTE-E152C-S375C) (LC: Sac-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 37 and two light chains having the amino acid sequence set forth in SEQ ID NO: 13; (c) αTROP2 (HC: BSM-E152C-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 43 and two light chains having the amino acid sequences set forth in SEQ ID NO: 22; and (d) αTROP2 (HC: BSM-YTE-E152C-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 49 and two light chains having the amino acid sequence set forth in SEQ ID NO: 22; wherein S is derived from the side chain of said engineered cysteine ​​residue, and wherein p is an integer selected from 1, 2, 3, or 4.

2. The ADC of claim 1, comprising:

19. The ADC has the formula: 【Transformation 7】 wherein Ab is an anti-Trop2 antibody comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:20 and two light chains having the amino acid sequences set forth in SEQ ID NO:22, where p is 1 or 2, and where S is derived from the side chain of an engineered cysteine ​​residue at position 375 of the constant domain of the heavy chain, said positions being defined according to EU numbering.

2. The ADC of claim 1, comprising:

20. The ADC has the formula: 【Transformation 8】 wherein Ab is an anti-TROP2 antibody, where p is 1 or 2, and where S is derived from the side chain of an engineered cysteine ​​residue at position 375 of the constant domain of the heavy chain, said positions being defined according to EU numbering.

2. The ADC of claim 1, comprising:

21. A composition comprising the ADC of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. 22. The composition of claim 21, wherein the predominant ADC species in the composition comprises (i) an antibody whose heavy chain C-terminus lacks a lysine residue, (ii) an antibody whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate, or (iii) an antibody whose heavy chain C-terminus lacks a lysine residue and whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate.

23. 1. A method for treating cancer in an individual in need thereof, comprising: The method comprises administering to the individual a therapeutically effective amount of the ADC of any one of claims 1 to 20 or the composition of claim 21 or claim 22 to treat the cancer, wherein the cancer is a cancer that overexpresses TROP2.

24. 24. The method of claim 23, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

25. Use of the ADC of any one of claims 1 to 20 or the composition of claim 21 or 22 for the manufacture of a medicament for treating cancer that overexpresses TROP2.

26. 26. The use of claim 25, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

27. 23. The ADC of any one of claims 1 to 20 or the composition of claim 21 or claim 22 for the treatment of cancer that overexpresses TROP2.

28. 28. The ADC of claim 27, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

29. A combination therapy for treating cancer, comprising the ADC of any one of claims 1 to 20 or the composition of claim 21 or 22, and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2.

30. 30. The combination therapy of claim 29, wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

31. 31. The combination therapy of claim 30, wherein the therapeutic antibody is a checkpoint inhibitor.

32. The combination therapy of claim 31 , wherein the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

33. 30. The combination therapy of claim 29, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

34. 1. An antibody-drug conjugate (ADC) comprising an antibody that specifically binds to human TROP2 conjugated to a linker-monomethyllauristatin E (linker-MMAE) payload, wherein the antibody comprises two heavy chains, each heavy chain comprising a variable domain and a constant domain, wherein the variable domain comprises the amino acid sequence of SEQ ID NO: 14, and two light chains, each light chain comprising a variable domain and a constant domain, wherein the variable domain comprises the amino acid sequence of SEQ ID NO:

15.

35. 35. The ADC of claim 34, wherein the antibody exhibits reduced hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

36. 35. The ADC of claim 34, wherein the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the constant domain of the heavy chain and positions 165 and 168 of the constant domain of the light chain, wherein the numbering of the positions of the heavy chain constant domain is according to Eu numbering, and the numbering of the positions of the light chain constant domain is according to consecutive numbering of the entire light chain sequence.

37. 37. The ADC of claim 36, wherein the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

38. 35. The ADC of claim 34, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, 18, 19, 20, 59, 60 or 61 and a light chain comprising the amino acid sequence of SEQ ID NO:

21.

39. 37. The ADC of claim 36, wherein the linker-MMAE payload is conjugated to the cysteine ​​or non-standard amino acid.

40. The antibody has a thiol (SH) group of the formula: MP-AA-PABC-MMAE: 【Chemistry 9】 ； CM2P-AA-PABC-MMAE: 【Chemistry 10】 CM3P-AA-PABC-MMAE: 【Chemistry 11】 35. The ADC of claim 34, comprising a cysteine ​​residue conjugated to a linker-MMAE payload comprising:

41. The ADC has the formula: 【Chemistry 12】 where Ab is an antibody and p is an integer from 1 to 8, and S is derived from the side chain of a cysteine ​​residue of the antibody.

35. The ADC of claim 34, comprising:

42. The ADC formula: 【Chemistry 13】 wherein Ab is an antibody, wherein said antibody comprises a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue, wherein said antibody comprising said engineered cysteine ​​residue is (A): (a) αTROP2 (HC:BSM-E152C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:78 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (b) αTROP2 (HC:BSM-E153C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:79 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (c) αTROP2 (HC:BSM-E171C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 80 and two light chains having the amino acid sequence set forth in SEQ ID NO: 21; (d) αTROP2 (HC:BSM-E172C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:81 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (e) αTROP2 (HC:BSM-E173C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 82 and two light chains having the amino acid sequence set forth in SEQ ID NO: 21; (f) αTROP2 (HC:BSM) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:52; (g) αTROP2 (HC:BSM) (LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:59 and two light chains having the amino acid sequences set forth in SEQ ID NO:53; (h) αTROP2 (HC:BSM-YTE) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:60 and two light chains having the amino acid sequences set forth in SEQ ID NO:52; (i) αTROP2 (HC:BSM-YTE) (LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:60 and two light chains having the amino acid sequences set forth in SEQ ID NO:53; (j) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:85 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (k) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 86 and two light chains having the amino acid sequence set forth in SEQ ID NO: 21; (l) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:87 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (m) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:88 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (n) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:89 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; and (o) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:90 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; wherein S is derived from the side chain of the engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4; or (B): (a) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:84 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; and (b) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:91 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; wherein S is derived from the side chain of said engineered cysteine ​​residue, and wherein p is an integer selected from 1, 2, 3, or 4.

35. The ADC of claim 34, comprising:

43. The ADC formula: 【Chemistry 14】 wherein Ab is an antibody, wherein said antibody comprises a heavy chain engineered cysteine ​​residue or a light chain engineered cysteine ​​residue, wherein said antibody comprising said engineered cysteine ​​residue is (A): (a) αTROP2 (HC:BSM-E152C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:38 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (b) αTROP2 (HC:BSM-E153C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:39 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (c) αTROP2 (HC:BSM-E171C) (LC:BSM), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO:40 and two light chains having the amino acid sequences set forth in SEQ ID NO:21; (d) αTROP2 (HC:BSM-E172C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:41 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (e) αTROP2 (HC:BSM-E173C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:42 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (f) αTROP2 (HC:BSM) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 17 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (g) αTROP2 (HC:BSM) (LC:BSM-E168C), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO: 17 and two light chains having the amino acid sequence set forth in SEQ ID NO: 53; (h) αTROP2 (HC:BSM-YTE) (LC:BSM-E165C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 52; (i) αTROP2 (HC:BSM-YTE) (LC:BSM-E168C), comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 18 and two light chains having the amino acid sequences set forth in SEQ ID NO: 53; (j) αTROP2 (HC:BSM-YTE-E152C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:44 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (k) αTROP2 (HC:BSM-YTE-E153C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:45 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (l) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:46 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (m) αTROP2 (HC:BSM-YTE-E172C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:47 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; (n) αTROP2 (HC:BSM-YTE-E173C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:48 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; and (o) αTROP2 (HC:BSM-YTE-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:20 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; wherein S is derived from the side chain of the engineered cysteine ​​residue and p is an integer selected from 1 or 2; or (B): (a) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:43 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; and (b) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequence set forth in SEQ ID NO:49 and two light chains having the amino acid sequence set forth in SEQ ID NO:21; wherein S is derived from the side chain of the engineered cysteine ​​residue and p is an integer selected from 1, 2, 3, or 4.

35. The ADC of claim 34, comprising:

44. The ADC has the formula 【Chemistry 15】 where Ab is an antibody, where p is 1 or 2, and where S is derived from the side chain of a cysteine ​​residue of the antibody.

35. The ADC of claim 34, comprising:

45. A composition comprising the ADC of any one of claims 34 to 44 and a pharmaceutically acceptable carrier.

46. 46. ​​The composition of claim 45, wherein the predominant ADC species in the composition comprises (i) an antibody whose heavy chain C-terminus lacks a lysine residue, (ii) an antibody whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate, or (iii) an antibody whose heavy chain C-terminus lacks a lysine residue and whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamate.

47. 1. A method for treating cancer in an individual in need thereof, comprising: The method comprises administering to the individual a therapeutically effective amount of the ADC of any one of claims 34 to 44 or the composition of claim 45 or claim 46 to treat the cancer, wherein the cancer is a cancer that overexpresses TROP2.

48. 48. The method of claim 47, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

49. Use of the ADC of any one of claims 34 to 44 or the composition of claim 45 or 46 for the manufacture of a medicament for treating cancer that overexpresses TROP2.

50. 50. The use of claim 49, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

51. 47. The ADC of any one of claims 34 to 44 or the composition of claim 45 or claim 46 for treating cancer that overexpresses TROP2.

52. 52. The ADC of claim 51 , wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

53. A combination therapy for treating cancer, comprising the ADC of any one of claims 34 to 44 or the composition of claim 45 or claim 46, and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2.

54. 54. The combination therapy of claim 53, wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

55. 55. The combination therapy of claim 54, wherein the therapeutic antibody is a checkpoint inhibitor.

56. 56. The combination therapy of claim 55, wherein the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

57. 54. The combination therapy of claim 53, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

58. An antibody or antigen-binding fragment thereof that specifically binds to human TROP2, an antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain comprising CDRL1, CDRL2, and CDRL3, wherein said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences NYGMN (SEQ ID NO: 4), WINTYTGEPTYTDDFKG (SEQ ID NO: 5), GGFGSSYWYFDV (SEQ ID NO: 6), KASQDVSIAVA (SEQ ID NO: 7), SASDRYT (SEQ ID NO: 10), and QQHYITPLT (SEQ ID NO: 9), respectively.

59. 59. The antibody or antigen-binding fragment thereof of claim 58, wherein the antibody or antigen-binding fragment thereof exhibits reduced binding to low TROP2-expressing cells compared to high TROP2-expressing cells and has reduced hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

60. 60. The antibody or antigen-binding fragment thereof of claim 58 or claim 59, wherein the antibody or antigen-binding fragment thereof is humanized.

61. The antigen-binding fragment is a Fab fragment, a Fab' fragment, or an F(ab') ２ The antibody or antigen-binding fragment thereof according to any one of claims 58 to 60, which is a fragment.

62. 62. The antibody or antigen-binding fragment thereof according to any one of claims 58 to 61, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 or 14, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3 or 16.

63. The antibody or antigen-binding fragment thereof of claim 62, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 3, or the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 14 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

16.

64. 59. The antibody or antigen-binding fragment thereof of claim 58, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, 17, or 18.

65. The antibody (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain comprising the amino acid sequence of SEQ ID NO:

18.

65. The antibody or antigen-binding fragment thereof of claim 64, comprising:

66. 59. The antibody or antigen-binding fragment thereof of claim 58, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58, 59, or 60.

67. The antibody (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 22 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 59; or (c) a light chain comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain comprising the amino acid sequence of SEQ ID NO:

60.

67. The antibody or antigen-binding fragment thereof of claim 66, comprising:

68. 59. The antibody or antigen-binding fragment thereof of claim 58, wherein the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain and positions 165 and 168 of the light chain constant domain, wherein the numbering of the heavy chain constant domain positions is according to Eu numbering, and the numbering of the light chain constant domain positions is according to consecutive numbering of the entire light chain sequence.

69. 69. The antibody or antigen-binding fragment thereof of claim 68, wherein the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

70. 70. The antibody or antigen-binding fragment thereof of claim 69, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, 20, 61, or 62.

71. 59. The antibody or antigen-binding fragment thereof of claim 58, wherein the antibody is conjugated to a payload.

72. 69. The antibody or antigen-binding fragment thereof of claim 68, wherein the cysteine ​​or non-standard amino acid is conjugated to a payload.

73. 73. The antibody or antigen-binding fragment thereof of claim 71 or 72, wherein the payload is a therapeutic moiety, a detectable label, a radionuclide or a protecting group.

74. 74. The antibody or antigen-binding fragment thereof of claim 73, wherein the therapeutic moiety is a cytotoxic moiety, an anti-inflammatory moiety, a peptide, a nucleic acid molecule, or a nucleic acid analog.

75. The cytotoxic moiety may be selected from the group consisting of taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (such as etoposide or etoposide phosphate), vinblastine, vincristine, vindesine, taxanes (such as taxol), taxoteretic acid, butyric acid, N8-acetylspermidine, and the like.

75. The antibody or antigen-binding fragment thereof of claim 74, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of maytansin, camptothecin, calicheamicin, esperamicin, ene-diyne, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, maytansinoids (DM1, DM2, DM3, DM4, etc.), auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and monomethyl auristatin D (MMAD)), 7-ethyl-10-hydroxy-camptothecin (SN-38), anthracyclines, alkylcyclines, and derivatives thereof.

76. 75. The antibody or antigen-binding fragment thereof of claim 74, wherein the cytotoxic moiety is an inhibitor of topoisomerase I, topoisomerase II, or microtubule polymerization.

77. 59. A method for treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 58, or a composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, to treat the cancer, wherein the cancer is a TROP2-overexpressing cancer.

78. 78. The method of claim 77, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

79. 60. Use of the antibody or antigen-binding fragment thereof described in claim 58 for the manufacture of a medicament for treating cancer that overexpresses TROP2.

80. 80. The use of claim 79, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

81. 59. The antibody or antigen-binding fragment thereof described in claim 58 for treating cancer that overexpresses TROP2.

82. 82. The antibody or antigen-binding fragment thereof of claim 81, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

83. 60. A combination therapy for treating cancer comprising the antibody or antigen-binding fragment thereof of claim 58 and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2.

84. 84. The combination therapy of claim 83, wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

85. 85. The combination therapy of claim 84, wherein the therapeutic antibody is a checkpoint inhibitor.

86. 85. The combination therapy of claim 84, wherein the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

87. 84. The combination therapy of claim 83, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

88. An antibody or antigen-binding fragment thereof that specifically binds to human TROP2, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

15.

89. 89. The antibody or antigen-binding fragment thereof of claim 88, wherein the antibody or antigen-binding fragment thereof exhibits reduced hydrophobicity compared to sacituzumab as determined by hydrophobic interaction chromatography (HIC).

90. The antigen-binding fragment is a Fab fragment, a Fab' fragment, or an F(ab') ２ 90. An antibody or antigen-binding fragment thereof according to claim 88 or claim 89, which is a fragment.

91. The antibody or antigen-binding fragment thereof according to any one of claims 88 to 90, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 21 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 17 or 18.

92. The antibody or antigen-binding fragment thereof according to any one of claims 88 to 90, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 59 or 60.

93. 93. The antibody or antigen-binding fragment thereof according to any one of claims 88 to 92, wherein the antibody further comprises a cysteine ​​or non-canonical amino acid substitution at one or more positions selected from the group consisting of positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain and positions 165 and 168 of the light chain constant domain, wherein the numbering of the heavy chain constant domain positions is according to Eu numbering, and the numbering of the light chain constant domain positions is according to consecutive numbering of the entire light chain sequence.

94. 94. The antibody or antigen-binding fragment thereof of claim 93, wherein the antibody comprises a cysteine ​​or non-canonical amino acid substitution at position 375 of the constant domain of the heavy chain.

95. 95. The antibody or antigen-binding fragment thereof of claim 94, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, 20, 61, or 62.

96. 89. The antibody or antigen-binding fragment thereof of claim 88, wherein the antibody is conjugated to a payload.

97. 94. The antibody or antigen-binding fragment thereof of claim 93, wherein the cysteine ​​or non-standard amino acid is conjugated to a payload.

98. 98. The antibody or antigen-binding fragment thereof of claim 96 or 97, wherein the payload is a therapeutic moiety, a detectable label, a radionuclide or a protecting group.

99. The antibody or antigen-binding fragment thereof of claim 98, wherein the therapeutic moiety is a cytotoxic moiety, an anti-inflammatory moiety, a peptide, a nucleic acid molecule, or a nucleic acid analog.

100. The cytotoxic moiety may be selected from the group consisting of taxol, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurocidin, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin, podophyllotoxin derivatives (such as etoposide or etoposide phosphate), vinblastine, vincristine, vindesine, taxanes (such as taxol), taxoteretic acid, butyric acid, N8-acetylspermidine, and the like.

100. The antibody or antigen-binding fragment thereof of claim 99, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of maytansin, camptothecin, calicheamicin, esperamicin, ene-diyne, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, hemiasterin, maytansinoids (DM1, DM2, DM3, DM4, etc.), auristatins (including monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and monomethyl auristatin D (MMAD)), 7-ethyl-10-hydroxy-camptothecin (SN-38), anthracyclines, alkylcyclines, and derivatives thereof.

101. 100. The antibody or antigen-binding fragment thereof of claim 99, wherein the cytotoxic moiety is an inhibitor of topoisomerase I, topoisomerase II, or a microtubule polymerization inhibitor.

102. 90. A method for treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment thereof of claim 88, or a composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, to treat the cancer, wherein the cancer is a TROP2-overexpressing cancer.

103. 103. The method of claim 102, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

104. 90. Use of the antibody or antigen-binding fragment thereof described in claim 88 for the manufacture of a medicament for treating cancer that overexpresses TROP2.

105. 105. The use of claim 104, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

106. The antibody or antigen-binding fragment thereof described in claim 88 for treating cancer that overexpresses TROP2.

107. The antibody or antigen-binding fragment thereof of claim 106, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

108. 90. A combination therapy for treating cancer comprising the antibody or antigen-binding fragment thereof of claim 88 and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2.

109. 109. The combination therapy of claim 108, wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

110. 110. The combination therapy of claim 109, wherein the therapeutic antibody is a checkpoint inhibitor.

111. The combination therapy of claim 109, wherein the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

112. 109. The combination therapy of claim 108, wherein the cancer is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, chronic lymphocytic lymphoma (CLL), Raji-Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, stomach cancer, renal cancer, salivary gland duct carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

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