TROP2 binder and its conjugate

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 of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 497,994, filed on 24 April 2023, the entirety of which is incorporated herein by reference.

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

[0003] The present invention relates to a TROP2 conjugate and its variants. In a specific embodiment, the present invention relates to a TROP2 conjugate and its conjugate, which is an antibody that preferentially binds to high-expression TROP2 cells rather than low-expression TROP2 cells, and includes a TROP2 conjugate conjugated in a payload. [Background technology]

[0004] Proteins that play a role in the growth, differentiation, invasion, and / or metastasis of breast cancer may influence the biological progression of tumors and thus provide important prognostic information. One such candidate is TROP2 (GA733-1, EGP-1), a 45 kDa monomeric transmembrane glycoprotein belonging to the TACSTD gene family, specifically TACSTD2, which is expressed in human epithelial cells at various differentiation stages. Overexpression of TROP2 has been demonstrated to be necessary and sufficient to stimulate tumor growth and is associated with a poor overall prognosis. TROP2 expression is associated with a 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 who underwent surgery for curative purposes. Similarly, in gastric cancer, 56% of patients may exhibit TROP2 overexpression on their tumors, which also correlated with shorter disease-free survival rates and a 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 refractory cancers, TROP2 is an attractive target for therapeutic intervention. Nevertheless, although TROP2 is expressed in some normal tissues, it is usually in much smaller amounts compared to neoplastic tissue 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 studied for the treatment of cancer.

[0007] International Publication No. 9714796 of the patent application describes BR110, a monoclonal antibody known to bind to TROP2 on the cell surface and internalize within the cell. International Publication No. 2003074566 of the patent application, U.S. Patent Publication No. 2004001825, U.S. Patent Publication No. 2007212350, and U.S. Patent 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 International Publication No. 2007095748, and AR52A301.5 is disclosed in International Publication No. 2007095749, both of which are antibodies that specifically bind to TROP2-expressing cancer cells.

[0008] International Publication No. 2008 / 144891 of the patent application teaches a humanized version of AR47A 6.4.2 as an anti-TROP2 monoclonal antibody for treating tumors. International Publication No. 2011155579 of the patent application teaches a monoclonal antibody or antibody fragment thereof that binds to the extracellular domain of human TROP2 with high affinity and exhibits high ADCC activity and high antitumor activity. International Publication No. 2013077458 of the patent application teaches an anti-human TROP2 antibody with antitumor activity, in particular a humanized antibody containing Huk5-70-2, in particular an anti-human TROP2 antibody with antitumor activity in vivo. International Publication No. 2013068946 of the patent application teaches an antibody that specifically binds to TROP2.

[0009] A promising application of antibodies for targeted tumor therapy involves the conjugation of numerous (2–8) highly toxic payloads into antibodies, 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 in healthy tissues. After binding to the receptor, ADCs are thought to internalize in tumor cells and then release toxic payloads upon degradation of the antibody and / or linker in lysosomes.

[0010] ADCs targeting TROP2 are known in the art and are in various stages of clinical development. DS-1062a is a humanized antibody hTINA-derived ADC conjugated to the camptothecin analog exatecan via a protease-sensitive cleavable linker disclosed in International Publication No. 2015098099 of a patent application, and is currently under 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 because the ADC exhibited 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 had received at least two prior treatments for metastatic disease (Bardia et al., N.Engl.J.Med.380:741-51 (2019)). SG is an antibody-drug conjugate (ADC) consisting of approximately eight SN-38-linked humanized anti-TROP2 monoclonal antibodies (mAbs) and hRS7, which are potent inhibitors of the active metabolite of irinotecan and topoisomerase 1 (Thomas et al., Clin. Cancer Res.25:6581-9 (2019)). In particular, topoisomerase I inhibitors had not been used for metastatic triple-negative breast cancer (TNBC) until SG, and SG effectively constitutes a new cytotoxic agent for treating a disease that still heavily relies on chemotherapy. However, the effectiveness of SG has been hampered by its toxicity.

[0012] The SG-targeting epitopes in TROP2 may further limit their effectiveness. The hRS7 mAb has been shown to bind to the same epitopes 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)). Therefore, RS7 was added to the list of 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 were shown to bind to the 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 issue of a lack of cancer specificity (Trerotola et al., Biochim. Biophys. Acta 1805:119-20(2010)).The Rinat-Pfizer RN926 anti-Trop-2 mAb, developed with 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; International Publication No. 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 progressive solid tumors. Doses of 3.60, 4.2, and 4.8 mg / kg were found to be unacceptable due to skin rash, mucosal lesions, and neutropenia. PF-06664178 showed moderate antitumor activity and was ultimately discontinued (King et al., Invest. New Drugs 36:836-47 (2018)). Therefore, exposure of normal tissue to anti-TROP2 ADCs with high-potency payloads can result in uncontrollable toxicity.

[0013] Furthermore, SG has a short half-life in patients (Ocean et al., Cancer 123:3843-54 (2017)), requiring frequent administration, which can induce side effects such as neutropenia and diarrhea, and is suggested to be due to the release of SN38 as a free drug in circulation (Santi et al., Ann. Transl. Med. 9:1113 (2021)). Considering the above, cell targeting and improvement of the 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)

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Summary of the Invention

[0016] The present invention relates to the following TROP2 binders: (i) cells with higher TROP2 expression (TROP2 低 cells) than cells with low TROP2 expression (TROP2高 (ii) a binding-modified anti-TROP2 antibody derived from the antibody hRS7 (sacituzumab) that preferentially binds to cells and exhibits decreased hydrophobicity compared to hRS7, but retains antitumor benefits in preclinical tumor models comparable to those of hRS7; (ii) an anti-TROP2 antibody that is a re-humanized derivative of the antibody hRS7 exhibiting decreased hydrophobicity and decreased immunogenicity compared to hRS7; and (iii) a binding-modified re-humanized anti-TROP2 antibody that combines the advantages of the binding-modified anti-TROP2 antibody and the re-humanized anti-TROP2 antibody; or an antigen-binding fragment thereof.

[0017] The present invention further provides an ADC comprising the TROP2 conjugate of the present invention conjugated into a payload. The TROP2 conjugate and ADC of the present invention are TROP2-expressing cells, particularly TROP2 高 It is useful for treating, imaging, diagnosing, preventing proliferation, containing, and reducing cells and TROP2-expressing tumors.

[0018] In one embodiment, the present invention provides an antibody or antigen-binding fragment that specifically binds to human TROP2, wherein the antibody or antigen-binding fragment 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 each 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). 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 an F(ab')2 fragment.

[0019] In further embodiments, the present invention provides an antibody or its antigen-binding fragment that exhibits reduced binding to low-TROP2-expressing cells compared to high-TROP2-expressing cells and, as determined by hydrophobic interaction chromatography (HIC), has reduced hydrophobicity compared to sacituzumab.

[0020] In further embodiments of the antibody or its antigen-binding fragment, 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 its antigen-binding fragment, 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 a further embodiment of the antibody or its antigen-binding fragment, the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain containing the amino acid sequence of SEQ ID NO: 11, 17, or 18.

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

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

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

[0026] In further embodiments of the antibody or its antigen-binding fragment, the antibody further comprises cysteine ​​or non-standard amino acid substitutions 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 position numbering of the heavy chain constant domain follows Eu numbering and the position numbering of the light chain constant domain follows sequential numbering of the entire light chain sequence.

[0027] In further embodiments of the antibody or its antigen-binding fragment, the antibody includes a cysteine ​​or non-standard amino acid substitution at position 375 of the constant domain of the heavy chain.

[0028] In embodiments of the antibody or its antigen-binding fragment, the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NOs. 19, 20, 61, or 62.

[0029] In further embodiments of the antibody or its antigen-binding fragment, the antibody is conjugated to a payload. In further embodiments of the antibody or its antigen-binding fragment, cysteine ​​or a non-standard amino acid is conjugated to the payload. In further embodiments of the antibody or its antigen-binding fragment, the payload is a therapeutic moiety, a detectable label, a radionuclide, or a protecting group. In further embodiments of the antibody or its antigen-binding fragment, 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 its antigen-binding fragment, the cytotoxic moiety may be taxol, methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leulosin, leulosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tarisomycin, podophyllotoxin, podophyllotoxin derivatives (e.g., etoposide or phosphate etoposide), vinblastine, vincristine, vindesine, taxane (e.g., taxol), taxote These include aretinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamycin, ene-diyne, duocalmycin A, duocalmycin SA, calicheamicin, camptothecin, hemiasterin, meitansinoids (DM1, DM2, DM3, DM4, etc.), auristatin (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.

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

[0031] In a further embodiment, the present invention provides the use of the aforementioned antibodies.

[0032] In one embodiment, the present invention provides a method for treating cancer in an individual in need of cancer treatment, comprising administering to the individual an antibody or antigen-binding fragment thereof that specifically binds to a therapeutically effective amount of human TROP2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain comprising CDRH1, CDRH2 and CDRH3 and a light chain variable domain comprising CDRL1, CDRL2 and CDRL3, each CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 comprising the amino acid sequences of SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 10 and SEQ ID NOs. 9, respectively, for treating cancer, 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (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, the antibody or antigen-binding fragment comprising 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 each comprise the amino acid sequences of SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 10 and SEQ ID NOs. 9, respectively, for use in the manufacture of pharmaceuticals for treating cancers that overexpress TROP2.

[0035] In further embodiments of use, 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (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, the antibody or antigen-binding fragment comprising 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 each comprise the amino acid sequences of SEQ ID NOs. 4, SEQ ID NOs. 5, SEQ ID NOs. 6, SEQ ID NOs. 7, SEQ ID NOs. 10 and SEQ ID NOs. 9, respectively, for treating cancers that overexpress TROP2.

[0037] In further embodiments, 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (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 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 each 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, 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 a further embodiment 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 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, kidney cancer, salivary ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

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

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

[0043] In further embodiments, the antigen-binding fragment of the antibody is a Fab fragment, a Fab' fragment, or an 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 containing the amino acid sequence of SEQ ID NO: 21 and a heavy chain containing the amino acid sequence of SEQ ID NO: 59 or 60.

[0046] In further embodiments, the antibody further comprises cysteine ​​or non-standard amino acid substitutions 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 position numbering of the heavy chain constant domain follows Eu numbering and the position numbering of the light chain constant domain follows sequential numbering of the entire light chain sequence.

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

[0048] In further embodiments, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NOs: 19, 20, 61, or 62.

[0049] In further embodiments, an antibody is conjugated to the payload. In further embodiments, cysteine ​​or a 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 may include taxol, methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leulosin, leulosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminocycline, aminopterin, tarisomycin, podophyllotoxin, podophyllotoxin derivatives (e.g., etoposide or phosphate etoposide), vinblastine, vincristine, vindesine, taxane (e.g., taxol), and taxoteretinoic acid. These include butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamycin, ene-diyne, duocalmycin A, duocalmycin SA, calicheamicin, camptothecin, hemiasterin, meitansinoids (DM1, DM2, DM3, DM4, etc.), auristatin (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 further embodiments, the cytotoxic moiety is topoisomerase I, topoisomerase II, or a microtubule polymerization inhibitor.

[0051] The present invention further provides a method for treating cancer in an individual requiring cancer treatment, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment 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, wherein the cancer is a cancer that overexpresses TROP2.

[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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

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

[0054] In further embodiments of use, 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (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 containing the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 15, for the treatment of cancers that overexpress TROP2.

[0056] In further embodiments, 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (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, 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 a further embodiment 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 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, kidney cancer, salivary ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0062] In a further embodiment, the present invention provides an ADC (MMAE-conjugated 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, the variable domain comprising complementarity-determining regions (CDR) 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 each 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).

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

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

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

[0066] In a further embodiment 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 a further embodiment of the MMAE-conjugated ADC, the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain containing 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 containing the amino acid sequence of SEQ ID NO: 13 and a heavy chain containing the amino acid sequence of SEQ ID NO: 11; (b) a light chain containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain containing the amino acid sequence of SEQ ID NO: 17; or (c) a light chain containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain containing the amino acid sequence of SEQ ID NO: 18.

[0070] In a further embodiment of the MMAE-conjugated ADC, the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO: 13 or 22 and a heavy chain containing 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 containing the amino acid sequence of SEQ ID NO: 13 and a heavy chain containing the amino acid sequence of SEQ ID NO: 58; (b) a light chain containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain containing the amino acid sequence of SEQ ID NO: 59; or (c) a light chain containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain containing the amino acid sequence of SEQ ID NO: 60.

[0072] In a further embodiment of the MMAE-conjugated ADC, the antibody further comprises cysteine ​​or non-standard amino acid substitutions 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 position numbering of the heavy chain constant domain follows Eu numbering, and the position numbering of the light chain constant domain follows sequential numbering of the entire light chain sequence.

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

[0074] In a further embodiment of the MMAE-conjugated ADC, the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NOs: 19, 20, 61, or 62.

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

[0076] In a further embodiment of the MMAE-conjugated ADC, the antibody has an SH group of formula: MP-AA-PABC-MMAE: [ka] CM2P-AA-PABC-MMAE: [ka] CM3P-AA-PABC-MMAE: [ka] It contains cysteine ​​residues conjugated into a linker-MMAE payload.

[0077] In a further embodiment of the MMAE-conjugated ADC, the ADC is given by 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 is given by the formula: [ka] (In the formula, Ab is an anti-TROP2 antibody containing a heavy-chain manipulated cysteine ​​residue or a light-chain manipulated cysteine ​​residue, where the anti-TROP2 antibody containing the manipulated cysteine ​​residue is, (A): (a) αTROP2(HC:Sac-E152C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 64 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (b) αTROP2(HC:Sac-E153C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 65 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (c) αTROP2(HC:Sac-E171C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 66 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (d) αTROP2(HC:Sac-E172C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 67 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (e) αTROP2(HC:Sac-E173C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 68 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (f) αTROP2(HC:Sac-S375C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 69 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (g) αTROP2(HC:Sac-YTE-E152C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 71 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (h) αTROP2(HC:Sac-YTE-E153C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 72 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (i) αTROP2(HC:Sac-YTE-E171C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 73 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (j) αTROP2(HC:Sac-YTE-E172C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 74 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (k) αTROP2(HC:Sac-YTE-E173C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 75 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (l) αTROP2(HC:Sac-YTE-S375C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 76 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 58 and two light chains having the amino acid sequence shown in SEQ ID NO: 50; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 58 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; (o) αTROP2(HC:Sac-YTE)(LC:Sac-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 63 and two light chains having the amino acid sequence shown in SEQ ID NO: 50; (p) αTROP2(HC:Sac-YTE)(LC:Sac-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 63 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; (q) αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 83 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (r) αTROP2(HC:BSM-E152C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 78 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (s) αTROP2(HC:BSM-E153C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 79 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (t) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 80 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (u) αTROP2 (HC:BSM-E172C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 81 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (v) αTROP2(HC:BSM-E173C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 82 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (w) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 83 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (x) αTROP2(HC:BSM)(LC:BSM-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 54; (y) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 55; (z) αTROP2(HC:BSM-YTE)(LC:BSM-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 60 and two light chains having the amino acid sequence shown in SEQ ID NO: 54; (aa) αTROP2(HC:BSM-YTE)(LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 58 and two light chains having the amino acid sequence shown in SEQ ID NO: 57; (bb) αTROP2 (HC:BSM-YTE-E152C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 85 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (cc) αTROP2 (HC:BSM-YTE-E153C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 86 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (dd) αTROP2 (HC:BSM-YTE-E171C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 87 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (ee) αTROP2 (HC:BSM-YTE-E172C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 88 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (ff) αTROP2(HC:BSM-YTE-E173C)(LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 89 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; and (gg) αTROP2 (HC:BSM-YTE-S375C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 shown in SEQ ID NO: 70 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (b) αTROP2(HC:Sac-YTE-E152C-S375C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 77 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 84 and two light chains having the amino acid sequence shown 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 shown in SEQ ID NO: 91 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 is given by the formula: [ka] (In the formula, Ab is an anti-TROP2 antibody containing a heavy-chain manipulated cysteine ​​residue or a light-chain manipulated cysteine ​​residue, where the anti-TROP2 antibody containing the manipulated cysteine ​​residue is, (A): (a) αTROP2(HC:Sac-E152C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 24 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (b) αTROP2(HC:Sac-E153C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 25 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (c) αTROP2(HC:Sac-E171C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 26 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (d) αTROP2(HC:Sac-E172C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 27 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (e) αTROP2(HC:Sac-E173C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 28 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (f) αTROP2(HC:Sac-S375C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 29 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (g) αTROP2(HC:Sac-YTE-E152C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 31 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (h) αTROP2(HC:Sac-YTE-E153C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 32 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (i) αTROP2(HC:Sac-YTE-E171C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 33 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (j) αTROP2(HC:Sac-YTE-E172C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 34 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (k) αTROP2(HC:Sac-YTE-E173C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 35 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (l) αTROP2(HC:Sac-YTE-S375C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 36 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 11 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 11 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (o) αTROP2(HC:Sac-YTE)(LC:Sac-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 23 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (p) αTROP2(HC:Sac-YTE)(LC:Sac-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 23 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (q) αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 19 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (r) αTROP2(HC:BSM-E152C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 38 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (s) αTROP2(HC:BSM-E153C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 39 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (t) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 40 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (u) αTROP2(HC:BSM-E172C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 41 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (v) αTROP2(HC:BSM-E173C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 42 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (w) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 19 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (x) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 17 and two light chains having the amino acid sequence shown in SEQ ID NO: 55; (y) αTROP2(HC:BSM-YTE)(LC:BSM-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 18 and two light chains having the amino acid sequence shown in SEQ ID NO: 54; (z) αTROP2(HC:BSM-YTE)(LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 18 and two light chains having the amino acid sequence shown in SEQ ID NO: 55; (aa) αTROP2(HC:BSM-YTE-E152C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 44 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (bb) αTROP2(HC:BSM-YTE-E153C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 45 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (cc) αTROP2 (HC:BSM-YTE-E171C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 46 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (dd) αTROP2 (HC:BSM-YTE-E172C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 47 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (ee) αTROP2(HC:BSM-YTE-E173C)(LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 48 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; and (ff) αTROP2 (HC:BSM-YTE-S375C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 shown in SEQ ID NO: 30 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (b) αTROP2(HC:Sac-YTE-E152C-S375C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 37 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 43 and two light chains having the amino acid sequence shown 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 shown in SEQ ID NO: 49 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 is given by the formula: [ka] (wherein Ab is an anti-Trop2 antibody comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 and two light chains having the amino acid sequence shown in SEQ ID NO: 22, p is 1 or 2, and S is derived from the side chain of a cysteine ​​residue at position 375 of the constant domain of the heavy chain, as defined according to Eu numbering).

[0081] The present invention further includes the formula: [ka] The present invention provides an ADC comprising (wherein Ab is an anti-Trop2 antibody comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 and two light chains having the amino acid sequence shown in SEQ ID NO: 22, p is 1 or 2, and S is derived from the side chain of a 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] (In the formula, Ab is the 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 dominant ADC species in the composition includes (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.

[0084] The present invention further provides a method for treating cancer in an individual requiring treatment for cancer, 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, wherein the cancer is a cancer that overexpresses TROP2. 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0085] The present invention further provides the use of either the MMAE-conjugated ADC described above or one of the first compositions described above for the manufacture of a pharmaceutical product for treating cancers that overexpress TROP2. In further embodiments of 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0086] The present invention further provides either the MMAE-conjugate ADC described above or one of the first compositions described above for treating cancers that overexpress 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (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 MMAE-conjugated ADCs described above or the first composition described above, and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2. 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 a further embodiment 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 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, kidney cancer, salivary ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0088] In a further embodiment, the present 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, the variable domain comprising 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 compared to sacituzumab, as determined by hydrophobic interaction chromatography (HIC).

[0090] In a further embodiment of the second MMAE-conjugated ADC, the antibody further comprises cysteine ​​or non-standard amino acid substitutions 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 position numbering of the heavy chain constant domain follows Eu numbering and the position numbering of the light chain constant domain follows sequential numbering of the entire light chain sequence.

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

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

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

[0094] In a further embodiment of the second MMAE-conjugated ADC, the antibody has an SH group of formula: MP-AA-PABC-MMAE: [ka] ; CM2P-AA-PABC-MMAE: [ka] CM3P-AA-PABC-MMAE: [ka] It contains cysteine ​​residues conjugated into a linker-MMAE payload.

[0095] In a further embodiment of the second MMAE-conjugated ADC, the ADC is given by the formula: [ka] (In the formula, Ab is an antibody of a 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 is given by the formula: [ka] (In the formula, Ab is an antibody of a second MMAE-conjugated ADC that specifically binds to human TROP2, and the antibody contains a heavy-chain manipulated cysteine ​​residue or a light-chain manipulated cysteine ​​residue, where the antibody containing the manipulated cysteine ​​residue is, (A): (a) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 78 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (b) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 79 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (c) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 80 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (d) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 81 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (e) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 82 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (f) αTROP2(HC:BSM)(LC:BSM-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (g) αTROP2(HC:BSM)(LC:BSM-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (h) αTROP2(HC:BSM-YTE)(LC:BSM-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 60 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (i) αTROP2(HC:BSM-YTE)(LC:BSM-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 60 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (j) αTROP2(HC:BSM-YTE-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 85 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (k) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 86 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (l) αTROP2(HC:BSM-YTE-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 87 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (m) αTROP2(HC:BSM-YTE-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 88 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (n) αTROP2(HC:BSM-YTE-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 89 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; and (o) αTROP2(HC:BSM-YTE-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 shown in SEQ ID NO: 84 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; and (b) αTROP2(HC:BSM-YTE-E152C-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 91 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 is given by the formula: [ka] (In the formula, Ab is an antibody of a second MMAE-conjugated ADC that specifically binds to human TROP2, and the antibody contains a heavy-chain manipulated cysteine ​​residue or a light-chain manipulated cysteine ​​residue, where the antibody containing the manipulated cysteine ​​residue is, (A): (a) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 38 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (b) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 39 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (c) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 40 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (d) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 41 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (e) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 42 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (f) αTROP2(HC:BSM)(LC:BSM-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 17 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (g) αTROP2(HC:BSM)(LC:BSM-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 17 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (h) αTROP2(HC:BSM-YTE)(LC:BSM-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 18 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (i) αTROP2(HC:BSM-YTE)(LC:BSM-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 18 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (j) αTROP2(HC:BSM-YTE-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 44 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (k) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 45 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (l) αTROP2(HC:BSM-YTE-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 46 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (m) αTROP2(HC:BSM-YTE-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 47 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (n) αTROP2(HC:BSM-YTE-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 48 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; and (o) αTROP2(HC:BSM-YTE-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 shown in SEQ ID NO: 43 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; and (b) αTROP2(HC:BSM-YTE-E152C-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 49 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; Selected from the group consisting of, where S is derived from the side chain of the manipulated 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 is given by the formula: [ka] (wherein Ab is an antibody of a 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 above-described second MMAE-conjugated ADCs and a pharmaceutically acceptable carrier. In further embodiments of the composition, the dominant ADC species in the composition includes (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.

[0100] The present invention further provides a method for treating cancer in an individual requiring treatment for cancer, comprising administering to the individual a therapeutically effective amount of any one of the aforementioned second MMAE-conjugate ADCs or the aforementioned second composition, wherein the cancer is a cancer that overexpresses TROP2. 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0101] The present invention further provides the use of either the second MMAE-conjugate ADC described above or one of the second composition described above for the manufacture of a pharmaceutical product for treating cancers that overexpress TROP2. In further embodiments of 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 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0102] The present invention further provides either the above-described second MMAE-conjugated ADC or one of the above-described second composition for the treatment of cancers overexpressing 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 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 ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0103] The present invention further provides a combination therapy for treating cancer comprising either the second MMAE-conjugated ADC described above or the second composition described above, and a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2. 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 a further embodiment 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 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, kidney cancer, salivary ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

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

[0106] [Figure 1]Figure 1 shows an ELISA demonstrating that Y53D substitution of the CDR2 in the sacituzumab light chain leads to the production of anti-TROP2 antibodies that preferentially bind 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] Figure 2 shows a comparison of the performance of sacituzumab (hRS7 clinical sequence) and sacituzumab BSM (hRS7 BSM sequence) in reverse-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 reverse-phase high-performance liquid chromatography (RP-HPLC) at 70°C. AU on the Y axis represents absorbance units. [Figure 3] Figure 3 shows a comparison of the hydrophobicity of αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody and αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) antibody, and αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y105S) antibody and 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 pharmacokinetics (PK) of several framework antibody variants in rhesus monkeys after intravenous (IV) bolus administration of 3 mg / kilogram (mpk). The antibodies were sacituzumab (αTROP2), αTROP2 (HC:Sac-S375C) (LC:Sac), and αTROP2 (HC:BSM-S375C) (LC:BSM). [Figure 6]Figure 6 shows the rhesus monkey PK of light chain Y53D and heavy chain Y105S antibody affinity variants after 1 mpk IV bolus administration. The antibodies used were sacituzumab (αTROP2), αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D), and αTROP2 (HC:BSM-S375C-Y105S) (LC:BSM). [Figure 7] Figure 7 shows the antibody affinity variant rhesus monkey PK after a single 20 mpk IV administration. The antibodies used were αTROP2(HC:BSM-YTE-S375C)(LC:BSM) and αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D). [Figure 8] Figure 8 shows the antibody affinity variant rhesus monkey PK after a single 20 mpk IV administration. The antibodies used 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 profiles of the αTROP2 heavy and light chain amino acid sequences (HC:BSM-S375C) (LC:BSM-Y53D) compared with the immunogenicity risk profiles of the sacituzumab heavy and light chains. [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 the composition containing αTROP2-MP-AA-PABC-MMAEADC, with the positions for DAR0, DAR1, DAR2, and DAR3 indicated. The αTROP2 antibodies included HC:BSM-YTE-S375C-MMAE and LC:BSM-Y53D. [Figure 12]Figure 12 shows the hydrophobic chromatography (HIC) profile of a composition containing αTROP2-MP-AA-PABC-MMAEADC, with the positions for DAR0, DAR1, DAR2, and DAR3 indicated. The αTROP2 antibodies include HC:BSM-YTE-S375C-MMAE and LC:BSM-Y53D. The control antibody profile is for an unconjugated antibody. [Figure 13] Figure 13 shows a comparison of the antitumor effects of several binding-modified sacituzumab mutants 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 effects of αTROP2(HC:BSM-YTE-S375C-MMAE)(LC:BSM-Y53D)ADC at four doses in the 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 the rat pharmacokinetics 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). The CDR as defined by Kabat is underlined, and BSM amino acid substitutions are shown in bold. The numbering and sequential numbering of the VH according to Kabat are shown. The sequential numbering of the VL is also shown. [Modes for carrying out the invention]

[0107] definition To make the present invention more easily understandable, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined elsewhere in this specification, all other technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which the present invention pertains.

[0108] As used herein, including in the attached claims, the singular forms of words such as “a,” “an,” and “the” include their corresponding plural references unless the context clearly indicates otherwise.

[0109] As used herein, the term "TROP2" refers to tumor-associated calcium signaling molecule 2 (TACSTD2) or trophoblast cell surface antigen 2, also known as epithelial glycoprotein-1 antigen (EGP-1). TROP2 is a protein encoded in humans by the TACSTD2 gene. 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 transmits intracellular calcium signals and acts as a cell surface receptor. TROP2 expression was first reported in trophoblast cells (placenta) and fetal tissues (e.g., lungs). Later, its expression was also described in normal laminar 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. Abnormal overexpression of TROP2 has been described in several solid tumors, including colorectal cancer, renal cancer, lung cancer, bladder cancer, and breast cancer. TROP2 expression has also been described in several rare and aggressive malignancies, such as salivary duct, undifferentiated thyroid, uterine / ovarian, and neuroendocrine prostate cancers.

[0110] When used herein, the antigen and antigen-binding polypeptide (K D The term "affinity," expressed by the equilibrium constant of dissociation with K, is a measure of the binding strength between the antigenic determinant and the antigen-binding site on the antibody (or fragment thereof), D The smaller the value, the stronger the binding strength between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity is 1 / K. D The affinity constant (K A) can also be represented as. The affinity can be determined by known methods according to the specific antigen of interest. For example, K D can be determined by surface plasmon resonance (SPR; Biacore (trademark)). 10 -6 values less than K D are considered to indicate binding. Specific binding of an antibody or a fragment thereof to an antigen or an antigenic determinant can be determined by, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays, equilibrium dialysis, equilibrium binding, gel filtration, enzyme-linked immunosorbent assay (ELISA), SPR or spectroscopy (e.g., use of fluorescence assays) and any suitable known methods including 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 a fragment of an antibody and its associated antigen. Avidity relates to both the affinity between the 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, and it simply increases the association rate constant with the multiplicity of the reaction. For example, a typical IgG antibody is bivalent to a particular target, and each arm of the antibody contains a Fab moiety that can bind to the target independently. Following the first association, the other Fab moiety can bind to an adjacent copy of the target in an intramolecular reaction called ring closure. Since ring closure occurs intramolecularly, it is independent of concentration. Instead, it depends on the structure of the antibody and antigen, and together they determine 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 therefore depends on the cyclization equilibrium and effective concentration. In principle, the binding activity of the divalent interaction could be predicted from the effective concentration of cyclization (Bobrovnik, J.Mol.Recognit.20:253-262 (2007)). The effective concentration and binding activity have been previously studied using either a model system (Mack et al., ibid.) or a theoretical model (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 "administer" and "treat" refer to the contact of an exogenous pharmaceutical, treatment, diagnostic agent, or composition comprising a human TROP2 binder or ADC disclosed herein with an animal, human, subject, cell, tissue, organ, or biological fluid when applied to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of the reagent with the cell as well as contact of the reagent with a fluid in which the cell is in contact. "Administer" and "treat" also mean, for example, in vitro and ex vivo treatment of a cell by a reagent, diagnostic, binding compound or another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., 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, and the L-form is the amino acid in naturally occurring proteins, polypeptides and peptides. Amino acids and their codename are shown in Table 1 below.

[0114]

Table 1

[0115] As used herein, the term "antibody" or "immunoglobulin" as used herein refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC is composed of 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 LIt consists of the HC / LC constant domain. In certain naturally occurring IgG, IgD, IgE, IgM, and IgA antibodies, the heavy chain constant region consists of three domains: CH1, CH2, and CH3. Generally, the basic antibody structural unit 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 has one V H : 1 V L Includes pairs. One V H : 1 V L The pair is sometimes referred to as "Fab." Therefore, each antibody tetramer contains two Fabs, one in each arm of the Y-shaped antibody.

[0116] The LC constant domain consists of one domain CL. Human V H The following seven family members: V H 1. V H 2, V H 3, V H 4. V H 5, V H 6, and V H Includes 7, Human V L The following 16 family members: V 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 This includes 10. Each of these family members can be further divided into specific subtypes. V H and V LThis can be further subdivided into hypervariable regions called complementary determination regions (CDRs), which contain scattered, more conserved regions called framework regions (FRs). H and V L It consists of three CDR regions and four FR regions arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The numbering of 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 domain of an antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (C1q) of the classical complement system. Typically, the amino acid numbering in the heavy chain constant domain begins with number 118, following the Eu numbering scheme. The Eu numbering scheme is based on the human IgG1(Eu) amino acid sequence having a constant domain that begins at position 118(Eu) in the IgG1 amino acid sequence described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63:78-85 (1969), and for the IgG1, IgG2, IgG3, and IgG4 constant domains, see Beranger et al., (opposite).

[0118] The variable regions of the heavy and light chains contain binding domains, including CDRs that interact with the antigen. Several methods are available in the art for defining the CDR sequences of antibody variable domains (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 region of the antibody by sequence); the Chothia numbering scheme is based on the position of the structural loop region (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) See 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 CRMartin'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 derived from antibody light and heavy chains, as well as T cell receptor chains from different species, counting residues sequentially from 1 to 128 based on germline V sequence alignment (Giudicelli et al., Nucleic Acids Res. 25:206-11 (1997); Lefranc, Immunol Today 18:509 (1997); Lefranc et al., Dev Comp See Immunol. 27:55-77 (2003).

[0119] CDRs in antibody sequences containing amino acids that specifically interact with the amino acids containing the epitope in the antigen to which the antibody binds may be defined using the following general rules, disclosed to Prof. Andrew CRMartin's Group at www.bioinf.org.uk and reproduced in Table 2 below. There are rare cases in which these generally consistent features do not occur. However, Cys residues are the most conserved feature.

[0120] [Table 2]

[0121] V H The entire amino acid sequence is generally numbered according to Kabat, and the three CDRs within the variable region can be defined according to any one of the aforementioned numbering schemes. In certain embodiments, V H The numbering of amino acid positions within the sequence may start from amino acid position 1 and continue continuously to the end of the sequence, or it may continue continuously according to Kabat. Figure 16 shows the V of αTROP2 BSM with continuous numbering according to Kabat. H This indicates that unless otherwise specified, V in this specification H and V L The amino acid positions in are defined according to sequential numbering.

[0122] The numbering of amino acid positions in the heavy chain constant domain may begin at amino acid position 1 and continue consecutively to the end of the sequence, or it may continue consecutively according to Eu numbering. The IgG1 heavy chain constant domain amino acid sequence has 330 amino acids that are numbered consecutively from 1 to 330. The corresponding sequence numbered according to Eu begins at position number 118 and ends at position number 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 terms “Fc domain” or “Fc” refer to a crystalline fragment domain or region obtained from an antibody containing the CH2 and CH3 domains of the antibody. In an antibody, the two Fc domains are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. Fc domains can be obtained by digesting the antibody with the protease papain. Typically, the amino acids in the Fc domain are numbered according to the Eu numbering rules (see Edelmann et al., Biochem. 63:78-85 (1969)).

[0124] As used herein, the term “antigen” 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 retain the ability to specifically bind to an antigen bound by a full-length antibody, and / or the ability to compete with the full-length antibody for specific binding to the antigen. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv regions, and scFv.

[0126] As used herein, “specifically binds” means that, with respect to the target antigen, all or part of the binder preferentially associates with the target antigen and does not preferentially associate with other molecules, particularly those found in human blood or serum. The binders described herein are typically 10 -7 ~10 -11 Dissociation constants less than or equal to M (K D It specifically binds to the target antigen with high affinity, as reflected in the approximately 10 -6 Any K greater than M D Generally, this is considered to exhibit nonspecific binding. As used herein, a binder that "specifically binds" or "specifically binds" to a target antigen refers to a binder that binds to the target antigen with high affinity, which is 10 -7 K below M DIn particular, in the embodiment 10 -8 M or less, or 5×10 -9 M or less, or 10 -8 M~10 -11 K below M D This means that the antibody has binding to a non-target antigen, but does not bind in a measurable manner, such as by cell ELISA or surface plasmon resonance assay (SPR) using a 10 μg / mL antibody. This term does not exclude antibodies that bind to the target homolog. For example, an antibody that specifically binds to human TROP2 may also bind to human TROP2 homologs such as rhesus monkey TROP2 and rat TROP2, as long as it is specific to the TROP2 homolog.

[0127] As used herein, the term "Fab fragment" refers to the CH1 and V of one antibody light chain and one antibody heavy chain. H This refers to antigen-binding agents containing [specific components]. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules. A "Fab fragment" may be a product of papain cleavage of an antibody.

[0128] As used herein, the term “Fab' fragment” means one antibody light chain and V, such that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule. H This refers to an antigen-binding agent that includes a portion or fragment of an antibody heavy chain, including 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 the region between the CH1 domain and the CH2 domain of two antibody light chains and two heavy chains, such that an interchain disulfide bond is formed between them. H It refers to an antigen-binding agent containing two heavy chains, including the CH1 domain. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The "F(ab')2 fragment" can be a product of pepsin cleavage of an antibody.

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

[0131] As used herein, the term "ScFv" or "single-stranded variable fragment" refers to V molecules fused or linked together by short linker peptides of 10 to approximately 25 amino acids. H and V L This refers to a fusion protein containing V. The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. H The N-terminus of V L It can be linked to the C-terminus of and vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker.

[0132] As used herein, the term "diabody" refers to the same polypeptide chain (V H -V L or V L -V H ) Light chain variable domain (V L ) linked to a heavy chain variable domain (V H This refers to an antigen conjugate containing a small antibody fragment having two antigen-binding regions, including ). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding regions. Diabodies are described more fully, for example, in European Patent No. 404,097; International Publication No. 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants, see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

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

[0134] As used herein, the term "binding agent" refers to an antibody or an 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 of which is conjugated to a specific site on the antibody or binding agent via a linker. The antibody is typically a monoclonal antibody specific for a cancer antigen and can deliver the payload to cells expressing the cancer antigen on the extracellular surface of the cell.

[0136] As used herein, the term “DAR” or “drug-antibody ratio” refers to the average number of linker / payload portions bound to the antibody present in the composition. For compositions comprising the antibody-drug conjugates of the present disclosure, the DAR of the composition is the average of the DARs (all linker-payload portions of the individual antibody-drug conjugate molecules present in the composition), and this average is expressed as a decimal. Thus, in some embodiments of compositions comprising the antibody-drug conjugates of the present disclosure, the DAR of the composition is a decimal number 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 compositions comprising the antibody-drug conjugates of the present disclosure, the DAR of the composition is a decimal number 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 the antibody-drug conjugate of the present 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 the antibody-drug conjugate of the present 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 The values ​​are 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. Where used above, the term “composition” is understood to include pharmaceutical compositions. The average DAR can be determined by various conventional methods such as UV spectroscopy, mass spectrometry, ELISA assay, radiometric assay, 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, a transmembrane domain, and an intracellular T cell receptor activation signaling domain that specifically binds to an antigen or target. Engagement of the extracellular domain of a CAR with the target antigen on the surface of a target cell results in the clustering of CARs, delivering activating stimuli to CAR-containing cells. 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 independently of major histocompatibility (MHC).

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

[0139] As used herein, the term “hinge region,” as used in relation to CARs, refers to the portion of a CAR protein that connects two adjacent domains, such as the extracellular domain and the transmembrane domain.

[0140] As used herein, the term “transmembrane domain” refers to the portion of the CAR that extends across the cell membrane 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 the CAR located inside the cell membrane that is capable of transmitting effector signals.

[0142] As used herein, the term “isolated” antibody or antigen-binding fragment does not include, at least partially, other biological molecules from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth media. Isolated antibodies or antigen-binding fragments may also not include, at least partially, expression system components such as biological molecules from the 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, buffer, or salt, or to a component of a pharmaceutical formulation containing the antibody or fragment.

[0143] As used herein, the term “monoclonal antibody” refers to a substantially homogeneous population of antibodies, i.e., the antibody molecules constituting the population have identical amino acid sequences, except for any naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a number of different antibodies, each having different amino acid sequences in its variable domains, which are often specific to different epitopes. The modifier “monoclonal” indicates a characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production 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 by the recombinant DNA method (see, e.g., U.S. Patent 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 the coding and / or regulatory sequences necessary for its expression. For example, “gene” refers to mRNA, functional RNA, or nucleic acid fragment expressing a particular protein, including regulatory sequences. “Genes” also include, for example, non-expressed DNA segments that form recognition sequences for other proteins. “Genes” can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters. Genes include both naturally occurring nucleotide sequences encoding the molecule of interest and synthetically derived nucleotide sequences encoding complementary DNA (cDNA) obtained from the molecule of interest, for example, messenger RNA (mRNA) nucleotide sequences.

[0145] As used herein, the term “polynucleotide” as discussed herein forms part of the present invention. “Polynucleotide,” “nucleic acid,” or “nucleic acid molecule” includes single-stranded or double-stranded DNA and RNA. For example, in one embodiment of the present invention, a polynucleotide encoding an immunoglobulin chain or component of the antibody display system may be adjacent to a native regulatory (expression control) sequence, or may be associated with a heterogeneous sequence including a promoter, an internal ribosome entry site (IRES) and other ribosome binding site sequences, an enhancer, a response element, a suppressor, a signal sequence, a polyadenylation sequence, an intron, or 5' and 3' non-coding regions.

[0146] For example, a polynucleotide encoding an immunoglobulin chain or component of the antibody or ADC of the present invention may operably associate with a promoter. In one embodiment of the present invention, a “promoter” or “promoter sequence” is a DNA regulatory region that can bind to an intracellular RNA polymerase (e.g., directly or via a protein or substance bound to another promoter) and initiate transcription of the coding sequence. A promoter sequence generally binds to a transcription start site at its 3' end, extends upstream (5' direction), and contains the minimum number of bases or elements required to initiate transcription at any level. Within the promoter sequence, a transcription start site (conveniently defined, for example, by mapping by nuclease S1), as well as a protein-binding domain (consensus sequence) involved in RNA polymerase binding may be found. The promoter may operably associate with other expression regulatory sequences or nucleic acids of the present invention, including enhancer and repressor sequences.Promoters that may be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (US Patent 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-term repeat sequence of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980)), the herpesthymidine 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)); and the β-lactamase promoter (Villa-Komaroff et al.) Prokaryotic expression vectors such as al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978), or the tac promoter (see also “Useful proteins from recombinant bacteria” in DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983); 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) that can transform a host and promote the expression and / or replication of an introduced sequence, and that can introduce a DNA or RNA sequence into a host cell. Polynucleotides encoding immunoglobulin chains or components of the antibody or ADC of the present invention may be present in the vector in one embodiment of the present invention.

[0148] Where used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable, and all such designations include offspring. Therefore, the terms “transformed” and “transformed cell” include primary target cells and cultures derived therefrom, regardless of the number of times they have been transferred. It is also understood that, due to intentional or accidental mutations, not all offspring of parental cells will have exactly the same DNA content. This includes mutant offspring that have the same function or biological activity as those screened in the initially transformed cells. Where a clear designation is intended, it will be evident from the context.

[0149] As used herein, the terms “regulatory sequence” or “control sequence” refer to DNA sequences necessary for the expression of an operablely linked coding sequence in a particular host organism. Examples of regulatory sequences suitable for expression in eukaryotes include promoters, operator or enhancer sequences, transcription termination sequences, and polyadenylation sequences for the expression of protein-coding messenger RNA, as well as ribosome binding sites to facilitate the translation of messenger RNA.

[0150] As used herein, a nucleic acid is “operably ligated” if it has a functional relationship with another nucleic acid sequence, such as a regulatory sequence. For example, DNA for a pre-sequence or secretion leader is operably ligated to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably ligated to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably ligated to a coding sequence if it is positioned to facilitate translation. Generally, “operably ligated” means that the ligated DNA sequences are adjacent, and in the case of a secretion leader, it means adjacent and in the reading phase. Enhancers, however, do not need to be adjacent. Ligation is achieved by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0151] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties arising therefrom, which serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, may be said to code for a protein or other product of that gene or cDNA. Unless otherwise specified, “nucleotide sequences that code for an amino acid sequence” include all nucleotide sequences that code for the same amino acid sequence, even if they are degenerate versions of each other. Nucleotide sequences that code for proteins and RNA may contain introns.

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

[0153] As used herein, the term “TROP2 conjugate” refers to the anti-TROP2 antibody and its antigen-binding fragment of the present invention. Specifically, this term excludes other anti-TROP2 antibodies, such as sacituzumab.

[0154] As used herein, the terms “to treat” or “to treat” mean administering a therapeutic portion, such as a composition containing either the anti-TROP2 binder or ADC of the present invention, topically, subcutaneously, intramuscularly, intradermally, intravenously, or systemically to an individual in need. The amount of therapeutic portion effective in treating cancer or proliferative disorder in an individual may vary depending on factors such as the individual’s injury or disease state, age and / or weight, and the ability of the therapeutic agent to induce a desired response in the individual. Whether the therapeutic objective has been achieved can be assessed by personal and / or any clinical measurements commonly used by a physician or other skilled healthcare provider to assess the severity or progression of treatment. Thus, these terms indicate that beneficial results have been or will be given to an individual human or animal in need. Treatment may be therapeutic or prophylactic.

[0155] As used herein, the term “treatment” as applied to human or veterinary subjects refers to therapeutic or prophylactic treatments, as well as diagnostic applications. “Treatment” as applied to human or veterinary subjects includes the contact of the TOP2 binder or ADC of the present invention with a human or animal subject.

[0156] As used herein, the term “therapeutic dose” refers to the amount of a particular substance sufficient to achieve the desired effect in the individual being treated. For example, this may be the amount required 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 organism comprising administering a first therapeutic agent and a second therapeutic agent to the organism sequentially or simultaneously. Generally, the first and second therapeutic agents are administered to the organism individually, not as a mixture. However, embodiments may exist in which the first and second therapeutic agents are mixed before administration.

[0158] As used herein, the term “solvate” means the physical association of an ADC disclosed herein with one or more solvent molecules. This physical association includes varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, a solvate may be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. “Solvate” encompasses both the solution phase and the isolateable solvate. Non-limiting examples of solvates include ethanolates, methanelates, etc. “Hydrate” is a solvate in which the solvent molecule is water.

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

[0160] As used herein, the term “pharmaceutically acceptable salt” includes acid addition salts and basic salts.

[0161] Examples of acid addition salts include acetate, ammonium, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphor, camphor sulfonate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate (also known as mesylate), naphthalenesulfonate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, and toluenesulfonate (also known as tosylate). Furthermore, acids generally considered suitable for forming pharmaceutically useful salts from basic pharmaceutical compounds can be found in, for example, P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2 nd This has been discussed in 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). These disclosures are incorporated herein by reference. In one embodiment, the salt is an ammonium salt or a diammonium salt.

[0162] Examples of basic salts include alkali metal salts such as ammonium salts, 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, and choline, and salts with amino acids such as arginine and lysine. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl chloride, bromide, and iodide, ethyl, and butyl), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, and dibutyl sulfate), long-chain halides (e.g., decyl chloride, bromide, and iodide, lauryl, and stearyl), and aralkyl halides (e.g., benzyl bromide and phenethyl bromide).

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

[0164] Introduction: This invention relates to low TROP2-expressing cells (TROP2 低 Cells with high TROP2 expression (TROP2 高 The present invention provides a TROP2 conjugate that preferentially binds to TROP2 cells, and a conjugate containing the TROP2 conjugate conjugated into a payload. In certain embodiments, the TROP2 conjugate is the anti-TROP2 antibody of the present invention, which is conjugated into the payload to provide the 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 the treatment, imaging, diagnosis, prevention of proliferation, and containment and reduction of TROP2-expressing cells, particularly TROP2-expressing tumors.

[0165] The ADC of the present invention may be used to treat disorders involving cells that overexpress TROP2 on their 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), head and neck squamous cell carcinoma (SCCHN), colon cancer, neuroendocrine cancer, prostate cancer, sarcoma, gastric cancer, esophageal cancer, and cervical cancer.

[0166] TROP2 binder The present invention can be determined by a cell-based enzyme-linked immunosorbent assay (ELISA), as TROP2 低 TROP2 is better than cells 高 The present invention provides a TROP2 conjugate (an anti-TROP2 antibody and its antigen-binding fragment) that preferentially binds to cells and exhibits reduced hydrophobicity compared to sacituzumab, as can be 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 registered under U.S. Patent No. 9745380 (SEQ ID NO: 14 (V H ) and 13(V L The antibody hRS7, which contains the antibody, is disclosed in [reference]. The antibody-drug conjugate (ADC) sacituzumab govitecan-hziy is currently marketed under the trade name TRODELVY.

[0167] Generally, TROP2 is overexpressed in various cancers, including colorectal cancer, pancreatic cancer, gastric cancer, oral squamous cell carcinoma, ovarian cancer, bladder cancer, and breast cancer, compared to its 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)). These studies have shown that cancers with high TROP2 expression have a poor prognosis. 高 The preferential binding of the TROP2 conjugate of the present invention to cells reduces the risk of off-target binding and thus limits undesirable adverse events (AEs) during treatment planning. This selectivity is particularly advantageous for use in cancer treatment regimes targeting cancers that overexpress TROP2. Therefore, an ADC containing the anti-TROP2 antibody of the present invention, conjugated to a therapeutic moiety such as a cytotoxin, e.g., a topoisomerase I or II inhibitor or a microtubule aggregation inhibitor, is particularly useful in treatment regimes targeting cancers that overexpress TROP2.

[0168] In the present invention, when tyrosine is introduced into the aspartic acid amino acid substitution (Y53D amino acid substitution) at position 53 of the light chain of sacituzumab, the binding activity to TROP2 is reduced, and therefore TROP2 低 TROP2 is better than cells 高It incorporates the discovery that a modified sacituzumab (αTROP2(HC:Sac)(LC:Sac-Y53D) antibody) having preferential or selective binding to cells is provided. Further, 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 can be determined by hydrophobic interaction chromatography (HIC). The decrease in hydrophobicity can provide a TROP2 binder that reduces the tendency to aggregate and enables the preparation of a concentrated aqueous solution of an antibody with reduced viscosity.

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

[0170] Thus, the binding-modulated TROP2 binder of the invention comprises (i) a heavy chain variable domain (V H ) comprising heavy chain complementarity-determining regions (HC-CDR) 1, 2, 3 shown in the amino acid sequence of SEQ ID NO: 1 (CDRs are defined according to Kabat, ABM, IMGT, Chothia or Contact); and (ii) a light chain variable domain (V[[ID=1九]] L ) comprising light chain complementarity-determining regions (LC-CDR) 1, 2, 3 shown in the amino acid sequence of SEQ ID NO: 3 (CDRs are defined according to Kabat, ABM, IMGT, Chothia or Contact).

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

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

[0173] In a particular embodiment, V H It is linked to the constant domain of human IgG1 containing the amino acid sequence shown in SEQ ID NO: 92, or to a variant thereof containing the S375C substitution and having the amino acid sequence shown in SEQ ID NO: 103.

[0174] In further embodiments, the constant domain of human IgG1 includes substitutions of amino acids Tyr(Y), Thr(T), and Glu(E) (M252Y, S254T, T256E substitutions) (numbering follows Eu) (positions corresponding to consecutive numbers are 256, 258, and 260, respectively) of amino acids at positions 252, 254, and 256 of the constant domain of the heavy chain, to provide a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 93, which includes a "YTE" substitution, or a variant thereof having the amino acid sequence shown in SEQ ID NO: 104, which includes an S375C substitution.

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

[0176] In further embodiments, the human IgG1 heavy chain constant domain includes a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 95, or an S375C substitution, and includes the L234A L235A D265S substitution (numbering follows EU) to provide a variant thereof having the amino acid sequence shown in SEQ ID NO: 106.

[0177] In further embodiments, the human IgG1 heavy chain constant domain includes a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 96, or an S375C substitution, and includes the L234A L235A P329G substitution (numbering follows EU) to provide a variant thereof having the amino acid sequence shown in SEQ ID NO: 107.

[0178] In further embodiments, the human IgG1 heavy chain constant domain includes a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 97, or an S375C substitution, and includes an L235E substitution (numbering follows Eu) to provide a variant thereof having the amino acid sequence shown in SEQ ID NO: 108.

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

[0180] In further embodiments, the human IgG1 heavy chain constant domain includes a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 99, or an S375C substitution, and includes a D265A N297G substitution (numbering follows EU) to provide a variant thereof having the amino acid sequence shown in SEQ ID NO: 110.

[0181] In further embodiments, 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 shown in SEQ ID NO: 100, or a variant thereof having the amino acid sequence shown in SEQ ID NO: 111, comprising the S375C substitution (numbering follows Eu).

[0182] In further embodiments, the human IgG1 heavy chain constant domain includes a heavy chain constant domain having the amino acid sequence shown in SEQ ID NO: 101, or an S375C substitution, and includes the N297A / D356E / L358M substitution (numbering follows EU) to provide a variant thereof having the amino acid sequence shown in SEQ ID NO: 112.

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

[0184] The present invention further provides a TROP2 conjugate, a re-humanized variant of sacituzumab, which has a more human-like sequence than sacituzumab, has a lower predicted epitope content compared to sacituzumab, and whose re-humanization process unexpectedly produces an antibody with reduced hydrophobicity compared to sacituzumab, as can be determined by hydrophobic interaction chromatography (HIC), and which has a lower predicted epitope content compared to sacituzumab, as can be determined in silico using, for example, an immunogenicity prediction program.

[0185] These re-humanized TROP2 conjugates have an amino acid sequence that, compared to the amino acid sequence of the sacituzumab light chain shown in SEQ ID NO: 12, has an amino acid substitution S20T, D60S, V85T and A100P (positions are defined by sequential numbering), and (ii) Compared to the amino acid sequence of the heavy chain of sacituzumab having the amino acid sequence shown in Sequence ID No. 11,The amino acid sequence of sacituzumab is modified to include the amino acid substitutions Q5L, K38R, A69S, T78Q, D89E, F95Y, S115T, R218K, E360D, and M362L in the heavy chain (positions are defined by sequential numbering) (the same positions in the VH defined by Kabat numbering are Q5L, K38R, A68S, T77Q, D85E, F91Y, and S107T, and in the heavy chain constant domain by Eu numbering, R214K, E256D, and M359L). When these mutations are present together on the heavy chain or light chain, they are referred to herein as "BSMs (Best Single Mutations)". In exemplary embodiments, the re-humanized sacituzumab is αTROP2(HC:BSM)(LCBSM), which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 17 or 61 and a light chain containing the amino acid sequence of SEQ ID NO: 21. Re-humanized sacituzumab αTROP2(HC:BSM)(LC:BSM) exhibits a higher degree of humanization and lower hydrophobicity compared to sacituzumab, as determined by hydrophobic interaction chromatography (HIC).

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

[0187] In further embodiments, the αTROP2(HC:BSM-YTE)(LC:BSM) conjugate comprises a light chain further containing a Y53D amino acid substitution that provides the TROP2 conjugate αTROP2(HC:BSM-YTE)(LC:BSM-Y53D). These TROP2 conjugates exhibit (i) preferential binding to cells expressing large amounts of TROP2, such as those found in TROP2-expressing cancer cells, rather than cells expressing small amounts of TROP2, such as those found in non-cancer cells; (ii) reduced hydrophobicity compared to sacituzumab; and (iii) reduced potential immunogenicity compared to sacituzumab.

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

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

[0190] Specific examples of cytotoxic components include, but are not limited to, taxol, methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leulosin, leulosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminocycline, aminopterin, tarisomycin, podophyllotoxin, podophyllotoxin derivatives (etoposide or phosphate etoposide, etc.), vinblastine, vincristine, vindesine, taxane (Taxol, etc.), and taxoteretinoic acid. Examples include butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamycin, ene-diyne, duocalmycin A, duocalmycin SA, calicheamicin, camptothecin, hemiasterin, meitansinoids (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 portion can be linked to a linker via any suitable method known in the art.

[0191] The payload used in this invention can be bound to an anti-TROP2 antibody via a linker. Various linkers for ADCs are known in the art. The linker useful in this invention is not particularly limited, as long as it includes 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 a haloacetyl-functionalized linker. Examples of linkers include, but are not limited to, linkers having the following structures; 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 Sequence ID No. 185. iii.-MC-vc-, iv.-MP-("MP": Maleimidopropanil (maleimide-C3)), v.-MP-GGGG-("-GGGG-": tetrapeptide-Gly-Gly-Gly-Gly)GGGG is disclosed as Sequence 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 the payload.

[0192] In further embodiments, the payload provides a 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 containing 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 linker payload described above, the payload may include: Taxol, methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leulosin, leulosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tarisomycin, podophyllotoxin, podophyllotoxin derivatives (etoposide or phosphate eto These include poside (etc.), vinblastine, vincristine, vindesine, taxanes (taxol, etc.), taxoteretinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamycin, ene-diyne, duocalmycin A, duocalmycin 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. Examples of 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 may form salts or solvates, which are also within the scope of this disclosure. An exemplary linker payload further comprises the compound of formula I, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof: [ka] (In the formula, R 1 teeth, [ka] [ka] The wavy line indicates the covalent bond site, R 2 This is the cytotoxic part, R 3 and R 4 C 1~3 (This represents an alkyl or naturally occurring or unnatural amino acid side chain, where n is an integer between 1 and 4).

[0195] In a particular embodiment, R 2 This is selected from anthracyclines, auristatins, camptothecin, duocalmycin, etoposide, meitansinoids, pyrrolobenzodiazepine dimers, DNA sulcus binders, taxanes, engine, antitubulin, and vinca alkaloids. In further embodiments, R 2This includes auristatin T, auristatin E, auristatin F phenylenediamine, benzoyl-auristatin E ester, 5-benzoylvalerate-AE ester, monomethyl auristatin F, lipophilic MMAF, MMAE, lexitropsin, duocalmycin, paclitaxel and docetaxel, T67 (Tularik), vincristine, vinblastine, vindesine, vinorelbine, nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), tubulicin M, The following are selected: alkylcyclines, melphalan, methotrexate, mitomycin C, etoposide, CC-1065 analogues, calicheamicin, maytansine, drastatin 10 analogues, rhizoxin, palytoxin, baccatin derivatives, taxane analogues (e.g., epotilon A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycin, semadotin, maytansinoids, combretastatin, discodermoids, tesirin, and eleusrobin.

[0196] In a particular embodiment, R 3 and R 4 C 1~3 Either the alkyl group is independently selected, or both are CH3. An exemplary linker payload is given by formula II: [ka] (In the formula, R 1 and R 2 This may include (as described herein). In certain embodiments, R 2 This is an auristatin selected from auristatin E, auristatin F phenylenediamine, benzoyl auristatin E ester, 5-benzoylvalerate-AE ester, MMAF, MMAE, or pyrrolobenzodiazepine dimers.

[0197] Examples of linker payloads containing 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] An exemplary linker payload further includes 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 exemplary linker-payload described above is conjugated to the anti-TROP2 antibody of the present invention via cysteine ​​residues provided by selected interchain disulfide bonds opened by reduction of the anti-TROP2 antibody to provide the ADC of the present invention. In some embodiments, the ADC may contain 1, 2, 3, 4, 5, 6, 7, or 8 payloads conjugated thereto. For compositions or mixtures of ADCs, the mixture or composition may have a drug-to-antibody (DAR) ratio in the range of 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 1 to 2. This ratio may refer to the average ratio in a population, such as the average DAR 2 for a population of ADCs. In certain embodiments, the conjugate mainly contains payloads conjugated to the Fab domain, and possibly contains all four payloads conjugated to the Fab domain.

[0200] In further embodiments, compositions or mixtures comprising or consisting of the ADCs of the present invention are provided herein, 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, a mixture of ADCs has a DAR of about 4, and the majority of the ADCs in the mixture have four payload molecules linked thereto. In other words, the dominant species of ADC in the mixture or composition contains four payload molecules.

[0201] In some embodiments, the anti-TROP2 antibody of the present invention is genetically engineered to include 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] Accordingly, the anti-TROP2 antibody of the present invention may further include one or more substitutions of cysteine ​​residues or non-standard amino acid residues in its heavy chain or light chain, 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) in the heavy chain constant domain, and positions 165 and 168 (numbering beginning with N-terminal amino acid 1) in the light chain constant domain. In certain embodiments, cysteine ​​may be substituted at one or more of the positions 152, 153, 171, 172, 173 and 375 (numbering according to the Eu numbering scheme) in the heavy chain constant domain, and positions 165 and 168 (numbering beginning with N-terminal amino acid 1) in the light chain constant domain. In certain embodiments, the anti-TROP2 antibody includes the S375C amino acid substitution. In certain embodiments, the antibody includes S375C amino acid substitution and E152C amino acid substitution. In certain embodiments, the antibody includes S375C amino acid substitution and S168C amino acid substitution. A non-exclusive list of exemplary embodiments of the anti-TROP2 antibody of the present invention, which includes one or more substitutions of amino acids in a cysteine ​​that can be conjugated to the aforementioned payload, is shown in Tables 3-8.

[0203] [Table 3]

[0204] [Table 4]

[0205] [Table 5]

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8]

[0209] In exemplary embodiments, the ADC comprises the anti-TROP2 antibody of the present 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 (In the formula, Ab is the anti-TROP2 antibody of the present invention).

[0210] In certain embodiments of the aforementioned ADC, the payload may include taxol, methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leulosin, leulosidine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminocycline, aminopterin, tarisomycin, podophyllotoxin, podophyllotoxin derivatives (such as etoposide or phosphate etoposide). ), vinblastine, vincristine, vindesine, taxane (taxol, etc.), taxoteretinoic acid, butyric acid, N8-acetylspermidine, camptothecin, calicheamicin, esperamycin, ene-diyne, duocalmycin A, duocalmycin SA, calicheamicin, camptothecin, hemiasterin, DM1, DM2, DM3, DM4, MMAE, MMAF, MMAD, 7SN-38, anthracyclines, alkylcyclines, or derivatives thereof. Examples of 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 a linker payload having the formula shown in Formula III or an anti-TROP2 antibody of the present invention conjugated to such a stereoisomer: [ka] (In the formula, R 1 teeth, [ka] Selected from, [ka] A single wavy line represents -(CH2) n It shows the covalent bonding site to, [ka] The double dash indicates the covalent bonding site to sulfur in the cysteine ​​residue of Ab, and R 2 This is the cytotoxic part, R 3 and R 4 C 1~3 (where n represents an alkyl or naturally occurring or unnatural amino acid side chain, n is an integer from 1 to 4, Ab is the anti-TROP2 binder of the present invention, and p is a positive rational number from 1 to 24, including fractions and decimals). In further embodiments, R 2 R is an alistatin drug selected from auristatin E, auristatin F phenylenediamine, benzoyl auristatin E ester, 5-benzoylvalerate-AE ester, MMAF, MMAE, or pyrrolobenzodiazepine dimers. 3 and R 4 Both are CH3.

[0212] In a further embodiment, the present invention further provides an ADC comprising the anti-TROP2 antibody of the present invention conjugated to a linker-MMAE payload, wherein the ADC is of formula: [ka] (wherein Ab is the anti-Trop2 antibody of the present invention, S is the sulfur atom of a cysteine ​​residue present in the heavy or light chain of 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 Ab). In further embodiments, 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 present invention provides an ADC comprising the anti-TROP2 antibody of the present invention having an engineered cysteine ​​residue conjugated to a linker-MMAE payload, wherein the ADC is of formula: [ka] (In the formula, Ab is the anti-Trop2 antibody of the present invention comprising two heavy chains and two light chains, wherein the heavy chain or light chain comprises engineered cysteine, where the heavy chain and light chain are, (A) (a) αTROP2(HC:Sac-E152C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 64 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (b) αTROP2(HC:Sac-E153C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 65 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (c) αTROP2(HC:Sac-E171C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 66 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (d) αTROP2(HC:Sac-E172C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 67 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (e) αTROP2(HC:Sac-E173C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 68 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (f) αTROP2(HC:Sac-S375C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 69 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (g) αTROP2(HC:Sac-YTE-E152C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 71 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (h) αTROP2(HC:Sac-YTE-E153C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 72 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (i) αTROP2(HC:Sac-YTE-E171C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 73 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (j) αTROP2(HC:Sac-YTE-E172C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 74 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (k) αTROP2(HC:Sac-YTE-E173C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 75 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (l) αTROP2(HC:Sac-YTE-S375C)(LC:Sac-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 76 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (m) αTROP2(HC:Sac)(LC:Sac-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 58 and two light chains having the amino acid sequence shown in SEQ ID NO: 50; (n) αTROP2(HC:Sac)(LC:Sac-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 58 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; (o) αTROP2(HC:Sac-YTE)(LC:Sac-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 63 and two light chains having the amino acid sequence shown in SEQ ID NO: 50; (p) αTROP2(HC:Sac-YTE)(LC:Sac-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 63 and two light chains having the amino acid sequence shown in SEQ ID NO: 51; (q) αTROP2(HC:BSM-E152C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 78 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (r) αTROP2(HC:BSM-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 79 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (s) αTROP2(HC:BSM-E171C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 80 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (t) αTROP2(HC:BSM-E172C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 81 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (u) αTROP2(HC:BSM-E173C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 82 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (v) αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 83 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (w) αTROP2 (HC:BSM-E152C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 78 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (x) αTROP2(HC:BSM-E153C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 79 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (y) αTROP2 (HC:BSM-E171C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 80 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (z) αTROP2(HC:BSM-E172C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 81 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (aa) αTROP2(HC:BSM-E173C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 82 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (bb) αTROP2 (HC:BSM-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 83 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (cc) αTROP2(HC:BSM)(LC:BSM-E165C) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (dd) αTROP2(HC:BSM)(LC:BSM-E168C) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (ee) αTROP2(HC:BSM)(LC:BSM-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 54; (ff) αTROP2(HC:BSM)(LC:BSM-Y53D-E168C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 59 and two light chains having the amino acid sequence shown in SEQ ID NO: 55; (gg) αTROP2(HC:BSM-YTE)(LC:BSM-E165C) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 60 and two light chains having the amino acid sequence shown in SEQ ID NO: 52; (hh) αTROP2(HC:BSM-YTE)(LC:BSM-E168C) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 60 and two light chains having the amino acid sequence shown in SEQ ID NO: 53; (ii) αTROP2(HC:BSM-YTE)(LC:BSM-Y53D-E165C) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 60 and two light chains having the amino acid sequence shown in SEQ ID NO: 54; (jj) αTROP2(HC:BSM-YTE)(LC:BSM-Y53D-E168C) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 58 and two light chains having the amino acid sequence shown in SEQ ID NO: 57; (kk) αTROP2(HC:BSM-YTE-E152C)(LC:BSM) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 85 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (ll) αTROP2(HC:BSM-YTE-E153C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 86 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (mm) αTROP2 (HC:BSM-YTE-E171C) (LC:BSM) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 87 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (nn) αTROP2(HC:BSM-YTE-E172C)(LC:BSM) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 88 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (oo) αTROP2(HC:BSM-YTE-E173C)(LC:BSM) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 89 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (pp) αTROP2(HC:BSM-YTE-S375C)(LC:BSM) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (qq) αTROP2 (HC:BSM-YTE-E152C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 85 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (rr) αTROP2 (HC:BSM-YTE-E153C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 86 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (ss) αTROP2 (HC:BSM-YTE-E171C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 87 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (tt) αTROP2 (HC:BSM-YTE-E172C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 88 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (uu) αTROP2(HC:BSM-YTE-E173C)(LC:BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 89 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; and (vv) αTROP2 (HC:BSM-YTE-S375C)(LC:BSM-Y53D) comprises two heavy chains having the amino acid sequence shown in SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; Selected from the group consisting of, where S is the sulfur atom of the manipulated cysteine ​​conjugated to the 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 sequence shown in SEQ ID NO: 70 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (b) αTROP2(HC:Sac-YTE-E152C-S375C)(LC:Sac-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 77 and two light chains having the amino acid sequence shown in SEQ ID NO: 13; (c) αTROP2 (HC:BSM-E152C-S375C) (LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 84 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; (d) αTROP2 (HC:BSM-E152C-S375C)(LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 84 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (e) αTROP2(HC:BSM-YTE-E152C-S375C)(LC:BSM), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 91 and two light chains having the amino acid sequence shown in SEQ ID NO: 21; and (f) αTROP2 (HC:BSM-YTE-E152C-S375C) (LC:BSM-Y53D) comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 91 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (Selected from the group consisting of, where S is the sulfur atom of the manipulated cysteine ​​conjugated to the linker-MMAE payload, and p is an integer selected from 1, 2, 3, or 4) That is the case.

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

[0215] In a further embodiment, the present invention further provides an ADC comprising the anti-TROP2 antibody of the present invention having an manipulated cysteine ​​residue at position 375 of the heavy chain constant domain conjugated to the linker payload, wherein the ADC is of formula: [ka] (wherein Ab is the anti-TROP2 antibody of the present invention comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 and two light chains having the amino acid sequence shown in SEQ ID NO: 22, where p is 1 or 2, 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 ADC of the present invention, when the maleimide residue of the MP-AA-PABC-MMAE linker payload is conjugated to a cysteine ​​residue in the antibody, it undergoes a ring-opening reaction, thereby resulting in a more stable link between the antibody and the maleimide group. [ka]

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

[0218] In further embodiments, pharmaceutical compositions are provided herein that include one or a mixture thereof of the above-described ADCs and a pharmaceutically acceptable carrier. In certain embodiments of the pharmaceutical composition, the dominant species of ADC comprises an antibody whose heavy chain contains C-terminal lysine. In certain embodiments of the pharmaceutical composition, the dominant species of ADC comprises an antibody whose heavy chain lacks C-terminal lysine. In certain embodiments of the pharmaceutical composition, the dominant species of ADC comprises an antibody whose heavy chain lacks C-terminal glycine-lysine dipeptide. In certain embodiments of the pharmaceutical composition, the dominant species of ADC comprises an antibody whose heavy chain N-terminal amino acid is glutamine. In certain embodiments of the pharmaceutical composition, the dominant species of ADC comprises an antibody whose heavy chain N-terminal amino acid is glutamic acid. In certain embodiments of the pharmaceutical composition, the dominant species of ADC comprises an antibody whose heavy chain N-terminal amino acid is glutamine cyclized to pyroglutamate. In certain embodiments of the pharmaceutical composition, the dominant species of ADC includes antibodies in which the heavy chain N-terminal amino acid is glutamic acid cyclized to pyroglutamate. In certain embodiments of the pharmaceutical composition, the dominant species of ADC includes antibodies in which the heavy chain N-terminal amino acid is pyroglutamate. In certain embodiments of the pharmaceutical composition, the dominant species of ADC includes antibodies in which the heavy chain N-terminal amino acid is pyroglutamate and the heavy chain C-terminus lacks lysine. In certain embodiments of the pharmaceutical composition, the dominant species of ADC includes antibodies in which the heavy chain N-terminal amino acid is pyroglutamate and the heavy chain C-terminus lacks glycinelysine dipeptide.

[0219] The present 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, V disclosed herein L and V H V L and V H The domains are expressed as an ScFv fusion protein linked together by a peptide linker. The peptide linker is V H -V L Without compromising the fidelity of the pairing site and antigen-binding site, the carboxyl terminus of one variable domain is joined to the amino terminus of the other variable domain. Therefore, ScFv is V L The C-terminus is V by the peptide linker H A fusion protein linked to the N-terminus of, or V H The C-terminus is V by the peptide linker L It may contain a fusion protein linked to the N-terminus. The peptide linker for linking the variable domain can vary in length from 10 to 25 amino acids and is typically, though not always, a glycine (G) and serine (S) with the structure G4S (SEQ ID NO: 187), e.g., (G4S) n It is composed of hydrophilic amino acids such as (SEQ ID NO: 188) (wherein n is 1, 2, 3, 4, or 5). Although shorter peptide linkers (0-4 amino acids) are also used, ScFv with shorter linkers can form polymers. In general, (G4S)3 peptides containing three repeating G4S units (disclosed as SEQ ID NO: 189, "(G4S)3") 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] The exemplary ScFv fusion protein has structure V L -(G4S) n -V H or V H -(G4S) n -V L including, V HThe domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6. L The domain includes CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, CDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 9, where the CDR sequences are defined by the Kabat numbering scheme. In certain embodiments, n is 1, 2, 3, 4, or 5.

[0222] The exemplary ScFv fusion protein has structure V L -(G4S) n -V H or V H -(G4S) n -V L including, V H It contains the amino acid sequence shown in Sequence ID No. 1, V L The sequence contains the amino acid sequence shown in Sequence ID No. 3, where n is 1, 2, 3, 4, or 5.

[0223] The exemplary ScFv fusion protein has structure V L -(G4S) n -V H or V H -(G4S) n -V L including, V H It contains the amino acid sequence shown in SEQ ID NO: 14, V L The amino acid sequence is shown in Sequence ID No. 15, where n is 1, 2, 3, 4, or 5.

[0224] The exemplary ScFv fusion protein has structure V L -(G4S) n -V H or V H -(G4S) n -V L including, V H It contains the amino acid sequence shown in SEQ ID NO: 14, V L The amino acid sequence is shown in Sequence ID No. 16, where n is 1, 2, 3, 4, or 5.

[0225] The ScFv disclosed herein may be provided in a bispecific format comprising a CD3 conjugate (ScFv) linked by a peptide linker to ScFv that binds to TROP2 disclosed herein. When these molecules, called bispecific T cell engagers (BiTE®), bind to CD3 on T cells and TROP2 expressed on the cell surface, they deliver T cells to the tumor site.

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

[0227] In further embodiments, the ScFv disclosed herein may be ligated or inserted at different positions on an intact IgG molecule to confer a double epitope binding. For example, a bispecific antibody comprising two heterodimeric heavy chain constant domains may be provided, wherein the N-terminus of one heavy chain constant domain is fused to the C-terminus of the ScFv disclosed herein, and the N-terminus of the other heavy chain constant domain is fused to the C-terminus of an ScFv targeting an antigen other than TROP2 or a Fab' targeting an antigen other than TROP2.

[0228] Nucleic acid molecule encoding the TROP2 binder of the present invention The present invention further provides a nucleic acid molecule encoding the TROP2 binder of the present invention. In a particular embodiment, the TROP2 binder is encoded by the first nucleic acid molecule V H and V encoded by the second nucleic acid molecule L This includes, in certain embodiments, the TROP2 binder is an antibody in which the heavy chain is encoded by a first nucleic acid molecule and the light chain is encoded by a second nucleic acid molecule.

[0229] In certain embodiments, heavy chain and light chain (or V H and VL ) are the heavy chain and light chain (or V H and V L The N-terminus of the ScFv fusion protein is fused to a leader peptide at its N-terminus to be expressed as a fusion protein that facilitates the transport of the TROP2 binder through the secretory pathway. In certain embodiments, the N-terminus of the ScFv fusion protein is fused to a leader or signal peptide at its N-terminus to facilitate the transport of ScFv through the secretory pathway. Examples of leader / signal peptides that may be used include those containing the amino acid sequences shown in SEQ ID NO: 56 or SEQ ID NO: 57. Thus, in certain embodiments, the aforementioned nucleic acid molecule may include a polynucleotide encoding a leader peptide ligated to the 5' end of a nucleic acid molecule encoding an anti-TROP2 binder.

[0230] The nucleic acid molecules disclosed herein may include one or more substitutions that optimize one or more codons to enhance the 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] Method for producing the TROP2 binder of the present invention The present invention relates to a recombinant method for producing the TROP2 binder of the present invention, wherein (i) the V of the TROP2 binder H and V L (i) an expression vector comprising (one or more) nucleic acid molecules encoding the heavy and light chains of an anti-TROP2 conjugate, or (ii) introducing two expression vectors comprising nucleic acid molecules into a host cell, one of which vectors comprising the TROP2 conjugate V H Alternatively, the vector may contain a nucleic acid molecule encoding the heavy chain of an anti-TROP2 binder, while the other vector may contain the V of the TROP2 binder. L The method includes a nucleic acid molecule that encodes the light chain of a TROP2 binder. H , V LNucleic acid molecules or polynucleotides encoding heavy or light chains are operably ligated to promoters and other transcriptional and translational regulatory sequences. Host cells are cultured under conditions and for a period suitable for the expression of the nucleic acid molecule, and the TROP2 conjugate is subsequently isolated from the host cells and / or the culture medium in which the host cells grew. See, for example, International Publication Nos. 2004041862, 2006122786, 2008020079, 2008142164, or 2009068627. The expression vector may be a plasmid or a viral vector. The present invention also relates to such nucleic acid molecules (V) encoding the TROP2 conjugate. H and V L A host cell containing nucleic acid molecules encoding heavy chains and light chains (V) or its components (V H or heavy chain only or V L This relates to host cells (or host cells containing nucleic acid molecules that encode only light chains).

[0232] Eukaryotic and prokaryotic host cells, including mammalian cells, are well known in the art as hosts for the expression of TROP2 conjugates 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, cattle, horse, and hamster cells. In particular, cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines (e.g., armyworm (Spodoptera frugiperda) or golden ringworm (Trichoplusia ni)), amphibian cells, bacterial cells, plant cells, and fungal cells. Examples of fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Saccharomyces cerevisiae, and Trichoderma reesei. The present invention further comprises any host cell comprising the TROP2 conjugate of the present invention, or comprising one or more nucleic acid molecules encoding such a TROP2 conjugate, or comprising an expression vector comprising one or more nucleic acid molecules encoding such a TROP2 conjugate.

[0233] Furthermore, the expression of TROP2 conjugates from manufactured cell lines can be enhanced using several known techniques. For example, glutamine synthetase gene expression systems (GS systems) are a common approach for enhancing expression under specific conditions. GS systems are described in whole or in part in relation to European Patent Nos. 0216846B1, 0256055B1, 0323997B1, and 0338841B1. Thus, in one embodiment of the present invention, mammalian host cells lack the glutamine synthetase gene and grow in the absence of glutamine in the culture medium, but a nucleic acid molecule encoding an immunoglobulin chain contains the glutamine synthetase gene, compensating for the gene deficiency in the host cells. Such host cells comprising the TROP2 conjugates or (one or more) nucleic acid molecules or (one or more) expression vectors discussed herein, as well as expression methods discussed herein for producing TROP2 conjugates using such host cells, are part of the present invention.

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

[0235] Generally, glycoproteins produced in specific cell lines or transgenic animals have glycosylation patterns characteristic of the glycoproteins produced in those cell lines or transgenic animals. Therefore, the specific glycosylation pattern of a TROP2 conjugate depends on the specific cell line or transgenic animal used to produce the TROP2 conjugate. TROP2 conjugates containing only non-fucosylated N-glycans are part of the present invention and may be advantageous because non-fucosylated antibodies have typically been shown to exhibit potent efficacy over their fucosylated counterparts both in vitro and in vivo (e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patents 6,946,292 and 7,214,775). These TROP2 conjugates with non-fucosylated 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 a TROP2 binder comprising an immunoglobulin (CHON-linked glycan) produced in Chinese hamster ovary cells or an N-linked glycan typically attached to engineered yeast cells (engineered yeast N-linked glycan), such as Pichia pastoris. For example, in one embodiment of the present invention, the TROP2 binder comprises 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 binder comprises engineered yeast N-linked glycans, i.e., G0 and / or G1 and / or G2, and may further comprise Man5. In one embodiment of the present invention, the TROP2 binder comprises CHO N-linked glycans, i.e., G0-F, G1-F and G2-F, and may further comprise G0 and / or G1 and / or G2 and / or Man5. In one embodiment of the present invention, approximately 80% to 95% (e.g., approximately 80-90%, 85%, 90%, or 95%) of the total N-linked glycans on the TROP2 binder 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 a strain described in U.S. Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also publication number 2013066765 of the international patent application.

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

[0238] The mode of administration of the TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 binder or ADC of the present invention, may be any suitable route, such as parenteral administration, such as intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, pulmonary, transmucosal (oral, intranasal, vaginal, rectal), or other means recognized by those skilled in the art, as well as those well known in the art.

[0239] The TROP2 binder or ADC of the present invention may be administered to an individual (e.g., a patient) by any suitable route, such as parenteral, intramuscular, subcutaneous, or intraperitoneal via intravenous (iv) infusion or bolus injection. The iv infusion may be performed 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 the TROP2 binder or ADC of the present invention, or a pharmaceutical composition containing the TROP2 binder or ADC of the present 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. Repeated courses of treatment are possible, as are chronic administration. Repeated administrations may be at the same dose or different doses.

[0241] The TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 binder or ADC of the present invention, may be administered as maintenance therapy, for example, once a week for a period of six months or more.

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

[0243] The TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 binder or ADC of the present invention, can be lyophilized for storage and reconstituted in a suitable carrier before 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 The combination therapy of the present invention, comprising the TROP2 conjugate or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 conjugate or ADC of the present invention and another therapeutic agent (e.g., small molecule or antibody), may be used for the treatment of any proliferative disorder, particularly cancer. In certain embodiments, the combination therapy of the present invention is It may be used to treat breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0245] Combination therapy and chemotherapy comprising the TROP2 binder or ADC of the present invention The combination therapy of the present invention, comprising the TROP2 conjugate or ADC of the present invention, or the pharmaceutical composition comprising the TROP2 conjugate or ADC of the present invention, may be administered to an individual with cancer in combination with chemotherapy. An individual may receive chemotherapy concurrently with receiving the combination therapy of the present invention. An individual may receive the combination therapy of the present invention after completing chemotherapy. An individual may be administered chemotherapy after completing the combination therapy. The combination therapy of the present invention may also be administered to an individual with recurrent or metastatic cancer with disease progression or recurrence, who is receiving or has completed chemotherapy.

[0246] Chemotherapy may include chemotherapeutic agents selected from the following group: (i) Alkylating agents including, but not limited to, difunctional alkylating agents, cyclophosphamide, mechloretamine, chlorambucil, and melphalan; (ii) Monofunctional alkylating agents, including but not limited to dacarbazine, nitrosourea, and temozolomide (oral dacarbazine); (iii) Anthracyclines or alkylcyclines; (iv) Cytoskeletal disruptors (taxanes), including but not limited to paclitaxel, docetaxel, abraxane, and taxotere; (v) Epothyron, including but not limited to isebepyron and uchideron; (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 bismodegib; (x) Nucleotide analogues and precursor analogues, including but not limited to azacitidine, azathioprine, fluoropyrimidine (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 drugs, including but not limited to carboplatin, cisplatin, and oxaliplatin; (xii) Retinoids, including but not limited to tretinoin, ali-tretinoin, and bexarotene; and (xiii) Vinca alkaloids and derivatives, including but not limited to vinblastine, vincristine, vindesine, and vinorelbine.

[0247] The selection of the dose of a chemotherapeutic agent for chemotherapy depends on several factors, including the serum or tissue turnover rate of the drug, the level of symptoms, the immunogenicity of the drug, 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 it provides an acceptable level of side effects. Therefore, the dosage and frequency of administration of each additional therapeutic agent depend in part on the specific agent, the severity of the cancer being treated, and the patient's characteristics. Guidelines are available for selecting appropriate doses of antibodies, cytokines, and small molecules. 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 dose regimen may be done by a clinician using, for example, parameters or factors known or suspected in the art that may affect or be expected to affect the treatment, and which depend on, for example, the individual's medical history (e.g., previous treatments), the type and stage of cancer being treated, and biomarkers of the response to one or more therapeutic agents in combination therapy.

[0249] Accordingly, the present invention intends to describe embodiments of the combination therapy of the present invention that further include a chemotherapy step comprising platinum-containing chemotherapy, for example, pemetrexed and platinum chemotherapy or carboplatin and paclitaxel or nab-paclitaxel. In certain embodiments, the combination therapy with the chemotherapy step may be used to treat at least NSCLC and HNSCC.

[0250] Combination therapies, further combined with chemotherapy steps, may be used to treat any proliferative disorder, particularly cancer. In certain embodiments, the combination therapies of the present invention may be used to treat breast cancer (e.g., triple-negative breast cancer), cervical cancer, colorectal cancer, esophageal cancer, lung cancer, non-Hodgkin 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, kidney cancer, salivary ductal cancer, anaplastic thyroid cancer, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

[0251] Combination therapy including a TROP2 conjugate or ADC and a therapeutic antibody The TROP2 conjugate or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 conjugate or ADC of the present invention, may be administered in combination with one or more therapeutic antibodies for the treatment of cancer or proliferative disorders. An individual may receive treatment with therapeutic antibodies concurrently with receiving the combination therapy of the present invention. An individual may receive the combination therapy of the present invention after completing treatment with therapeutic antibodies. An individual may be administered treatment with therapeutic antibodies after completing the combination therapy. The combination therapy of the present invention may also be administered to individuals who have recurrent or metastatic cancer with disease progression or recurrence and who are receiving or have completed chemotherapy. In certain embodiments, the therapeutic agent targets programmed death 1 receptor or ligand, PD-1 and PD-L1, respectively.

[0252] Examples of anti-PD-1 antibodies that may be used in combination therapy with the TROP2 conjugate or ADC of the present invention as 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, the exemplary anti-PD-1 antibody is pembrolizumab (KEYTRUDA). In certain embodiments, the exemplary anti-PD-1 antibody is nivolumab (OPDIVO). In certain embodiments, the exemplary anti-PD-1 antibody is cemiprimab (LIBTAYO). In certain embodiments, the exemplary anti-PD-L1 antibody is durvalumab (IMFINZI). In certain embodiments, the exemplary anti-PD-L1 antibody is atezolizumab (TECENTRIQ). In certain embodiments, the exemplary anti-PD-L1 antibody is avelumab (BAVENCIO).

[0253] Injector for administering TROP2 binder or ADC The present invention also provides an injection device comprising either the TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising either the anti-TROP2 binder or ADC of the present invention. An injection device is a device for introducing a substance into a patient's body via a parenteral route, e.g., intramuscular, subcutaneous, or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an autoinjector) comprising, for example, a cylinder or barrel for holding the fluid to be injected (e.g., comprising either the TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising either the TROP2 binder or ADC of the present invention), a needle for puncturing the skin and / or blood vessels for injecting the fluid, and a plunger for pushing the fluid out of the cylinder through the needle hole. In one embodiment of the present invention, the injection device comprising either the TROP2 binder or ADC of the present invention, or the pharmaceutical composition comprising either the TROP2 binder or ADC of the present invention, is an intravenous (IV) injection device. Such a device includes a composition comprising the TROP2 binder or ADC, or a pharmaceutical composition in a cannula or trocar / needle that can be attached to a tube that can be attached to a bag or reservoir for holding a fluid (e.g., physiological saline; or Ringer's lactate solution containing NaCl, sodium lactate, KCl, CaCl2, and possibly glucose) introduced into the body of a subject through a cannula or trocar / needle.

[0254] In embodiments of the present invention, the TROP2 binder or ADC, or a pharmaceutical composition comprising the TROP2 binder or ADC, can be introduced into a device after a trocar and cannula are inserted into a target vein and the trocar is removed from the inserted cannula. The IV device may be inserted, for example, into a peripheral vein (e.g., in the hand or arm); into the superior or inferior vena cava or into the right atrium of the heart (e.g., central IV); or into a subclavian, internal jugular, or femoral vein and advance 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 autosynergy, a jet injector, or an external injection pump. The jet injector uses a high-pressure narrow jet of liquid to penetrate the epidermis and introduce the TROP2 binder or ADC, or a pharmaceutical composition comprising the TROP2 binder or ADC, into the patient's body. An external infusion pump is a medical device for delivering a controlled amount of the TROP2 binder or ADC of the present invention, or a pharmaceutical composition containing the TROP2 binder or ADC of the present invention, into a patient's body. The external infusion pump may be electrically or mechanically powered. Various pumps operate in various ways; for example, a syringe pump holds fluid in a syringe reservoir, and a movable piston controls fluid delivery; an elastomer pump holds fluid in an expandable balloon reservoir, and pressure from the elastic wall of the balloon drives fluid delivery; a peristaltic pump uses a pair of rollers to clamp a flexible tube of a certain length, pushing the fluid forward; and a multi-channel pump can deliver fluid from multiple reservoirs at multiple speeds.

[0255] TROP2 binder or ADC or the composition of the present invention or a kit containing the composition. As discussed herein, kits are further provided that include one or more components comprising, but not limited to, the TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 binder or ADC of the present invention in combination with one or more further components comprising, but not limited to, a further therapeutic agent. The TROP2 binder or ADC of the present invention, or a pharmaceutical composition comprising the TROP2 binder or ADC of the present invention and / or a therapeutic agent, can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in the pharmaceutical composition.

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

[0257] In another embodiment, the kit includes a combination of the present invention comprising the TROP2 binder or ADC of the present invention, or a pharmaceutical composition which may be formulated together as a pharmaceutical composition in a single common container by combining the TROP2 binder or ADC of the present invention with one or more therapeutic agents.

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

[0259] The kit may include a package insert containing information about the pharmaceutical compositions and dosage forms contained within the kit. Generally, such information helps patients and physicians to use the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the package insert may provide the following information regarding the combination of the present invention: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and uses, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, supply method, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0260] The examples describe the discovery of a new class of TROP2 binders and ADCs of the present invention comprising the new class of TROP2 binders.

[0261] General method Ultra-fast size exclusion chromatography (UP-SEC) To measure aggregation by UP-SEC, 5 μg of purified antibody was injected at 0.5 mL / min into an Acquity BEH200 SEC, 1.7 μm, 4.6 × 150 mm size exclusion column (Waters Corporation, Milford, Massachusetts) equilibrated with 100 mM sodium phosphate, 200 mM sodium chloride, and 0.02% sodium azide, pH 7, using a Waters H-Class ultrafast chromatography (UPLC) column. Chromatograms were collected at both 215 nm and 280 nm wavelengths, and an integrated trace of absorption at 280 nm (wavelength) 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 at a 1 / 1 (v / v) ratio with a solution of 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 / isopropanol (95:5 v / v). The prepared sample is then filtered through a 0.22 μm polyvinylidene fluoride (PVDF) membrane and loaded onto a Thermo Fisher Scientific, Inc. (Waltham, Massachusetts, USA) MAbPac® HIC-butyl high-performance liquid chromatography (HPLC) system according to the manufacturer's manual, equilibrated with 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 / isopropanol (95:5 v / v) (mobile phase A). 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, then the data is exported and analyzed using Empower software. The retention time of each sample is compared to a baseline and is characteristic of the antibody's hydrophobicity, with longer elution times correlating with higher hydrophobicity.

[0263] Reverse-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 × 30 mm column (Applied Biosystems, Thermo Fisher Corporation) and equilibrated with 30% acetonitrile and 0.2% trifluoroacetic acid at 70°C and 2 mL / min. The sample was eluted in a linear gradient from 30–58% acetonitrile in 0.2% trifluoroacetic acid over 5 minutes. Chromatograms were collected at both 215 nm and 280 nm wavelengths, and integration of the A280 nm trace was performed using Chemstation Rev.B.04.01 (Agilent Technologies, Inc., Santa Cra, California).

[0264] Tonset / Tm / Tagg measurement by nano-differential scanning fluorescence (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 the NanoTemper Prometheus NT.48 instrument (NanoTemper Technologies, Inc., South San Francisco, California). 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 temperature gradient of 1 °C / min from 20 °C to 94.8 °C. Up to 48 samples can be analyzed in parallel, and their stability can be evaluated for 3 seconds at each temperature.

[0265] The melting point (Tm) onset (°C) and Tm (°C) values ​​indicate the structural stability of the sample and are obtained by monitoring endogenous tryptophan and tyrosine fluorescence at emission wavelengths of 330 nm and 350 nm. To generate an unfolding curve, the ratio of fluorescence intensity (F350 nm / F330 nm) is plotted against temperature or time. The thermal stability of the sample is described by the thermal unfolding transition midpoint Tm (°C) at which half of the protein population remains unfolded. Tm corresponds to the inflection point of the unfolding curve and is determined via the derivative of the curve.

[0266] The aggregation point tag (°C) represents the colloidal stability of the sample and is obtained by monitoring the back reflection of near-ultraviolet (UV) light using a back-reflection optical system. The back-reflection optical system utilizes near-UV light scattering by protein aggregates, and therefore only unscattered light reaches the detector. Thus, a reduction in back-reflection light is a direct measure of aggregation in the sample.

[0267] Non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS NR) 5 μL of each sample was mixed at 1 mg / mL in a 96-well plate with 35 μL of loading buffer (HT Protein Express Sample Buffer) (Perkin Elmer, Waltham, Massachusetts) containing either 50 mM iodoacetamide or 50 mM dithiothreitol. The plate was incubated at 70°C for 20 minutes, and 75 μL of water was added to each well. Each sample was analyzed using a LabChip GXII (Perkin Elmer) with an HT Protein Express tip (Perkin Elmer). Electrophoresis maps were collected by measuring the fluorescence of the samples over time and integrated using LabChip GX software V 4.1.1619.0 SP1 (Perkin Elmer).

[0268] Affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) The AC-SINS assay measures protein self-interaction by capturing antibodies on the surface of gold colloids that exhibit surface resonance vibrations of a frequency in visible light. When immobilized antibodies self-interact, the colloid aggregates and changes its vibrational frequency to absorb at longer wavelengths. Gold nanoparticles are incubated overnight with an 80 / 20 (v / v) capture antibody / uncapture antibody mixture. The coated gold nanoparticles are then spun down and resuspended in conjugation buffer (20 mM sodium acetate pH 5.5 and PBS 1× pH 7.4) to a final volume of 50 μL. The sample is diluted to 0.05 mg / mL in the conjugation buffer, and 45 μL of each dilution is loaded into a 384-well plate. 5 μL of the pre-prepared gold nanoparticles are then added to each well of the plate containing the antibody and buffer control. The plate is then covered with an aluminum lid and incubated at room temperature for 2 hours, and then rapidly spun down at 3000 rpm before reading the absorbance spectrum of each well from 450 nm to 650 nm using a plate reader. The spectrum of each sample is recorded and analyzed for the redshift of the maximum absorption peak compared to the buffer and antibody control. The redshift and its intensity indicate the self-interaction tendency of the tested antibody sample.

[0269] Evaluation of aggregation and self-interactions using dynamic light scattering (DLS) All DLS studies were performed at 25°C on undiluted samples in glass-bottomed 96-well plates using a DynaPro Plate Reader II (Wyatt Technology Corporation, Santa Barbara, California). Acquisitions were averaged over 20 sessions per well (5 seconds each), 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 The concentration is determined by DLS. The high-concentration sample is diluted with the target buffer (20 mM sodium acetate pH 5.5 and 10 mM histidine hydrogen chloride pH 6.5) to obtain a concentration of 20 mg / mL, filtered through a 0.22 μm filter, and diluted with the filter buffer (having the desired pH and ionic strength) to obtain low-concentration samples (2, 5, 10, 15, 20 mg / mL), which are then added to a microplate. D This is determined by a linear fit of the measured (mutual)diffusion coefficient as a function of concentration.

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

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

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

[0274] Oxidation by 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, replace the buffer with 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 UV and 1200 k-lux visible light) at 25°C, and store at -80°C until analysis.

[0276] Evaluation of isoelectric point and charge variants by capillary isoelectric focusing (cIEF) To determine the isoelectric point (pI) by cIEF, the sample is diluted to 0.2 mg / mL in a buffer containing 0.35% methylcellulose, 3M 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, California), and 0.5% pI marker 9.77. The sample is run on iCE3 (ProteinSimple) at 1500V for 1 minute, followed by 3000V for 8 minutes, using FC-coated capillary focusing. Data is 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 minutes, and alkylated with 5 μL of 1 M iodoacetamide in the dark for 45 minutes. Before digestion, the samples were buffered with 50 mM ammonium bicarbonate using a 7 kDa molecular weight cutoff ZEBA cartridge. The samples were digested with 2 μg of trypsin and chymotrypsin at 37°C for 2 hours. The digestion was quenched by adding 3 μL of 5 M hydrochloride to each sample. Data were acquired using a Dionex / QE plus MS with a linear gradient over 50 minutes from 2–36% acetonitrile in 0.1% formic acid. The samples are analyzed using PEAKS DB (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) for database searching, as well as PepFinder (Thermo Fisher Scientific Corporation) and manual validation for rate of change assessment.

[0278] Analysis by surface plasmon resonance (SPR) The binding kinetics of the antibody to the target are determined by SPR on a BIAcore T200 or BIAcore 4000 (GE Healthcare). 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) are used for immobilization and reagent dilution. All binding rates are measured at 25°C.

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

[0280] The dual-reference titration data were fitted to a 1:1 Langmuir-coupled model, and the association rate constant k was determined using BIAcore T200 evaluation software version 2.0 or BIAcore 4000 evaluation software version 1.1 (GE Healthcare). a (M-1 s-1) and dissociation rate constant k d Determine (s-1). Set the equilibrium dissociation constant to K D (M=k) d / k a It was calculated as follows.

[0281] Evaluation of subvisible particles after 10 days of incubation at 50°C using flow cytometry. The Guava EasyCyte 5HT™ flow cytometer (GFC) used in this test will be purchased from EMD Millipore Corp (Villerica, Massachusetts). Briefly, the plate supporting the protein sample (160 μL of 1 mg / mL protein solution) will be degassed by standing it at 5°C overnight to minimize potential interference from microbubbles that may be trapped in the solution during sample preparation. Data will be collected for 250 seconds, allowing for a 60 μL sample volume to be analyzed, unless the particle count reaches the instrument's 200,000 count limit 250 seconds prior and the instrument automatically terminates data acquisition and moves to the next sample. The number of particles measured is 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 expansion factor and the measured volume. Instrument performance will be verified using the Easy Check Kit (EMD Millipore Corporation (St. Louis, Missouri)) before sample analysis. The sample plates are gently mixed manually before loading into the GFC instrument, and the assay is performed without using a GFC mixer to avoid potentially altering the protein aggregate population and generating bubbles.

[0282] [Example 1] Sacituzumab CDR and framework-saturated mutagenesis 57 V H and 43 V L The locations were selected within the CDR and framework regions of the sacituzumab (hRS7) sequence. For each location, each of the 17 amino acids was extracted at 976 V. H +731 V L All sequence mutations of the 1707-point mutant were tested (excluding M, C, and W). Yeast cells expressing the sacituzumab mutant (mAb) were sorted with incremental 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 40 single-point (20 V) L +20 V HThe parameters were precisely measured using [tool name]. Mutants with reduced affinity were identified and evaluated for binding using surface plasmon resonance single-cycle dynamics. 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 the S375C substitution (Hm_S375C, having the amino acid sequence shown in SEQ ID NO: 162), and all Hm muteins contain a sacituzumab light chain (LC, having the amino acid sequence shown in SEQ ID NO: 12). The amino acid sequences containing the antibodies disclosed in Table 9 can be found in Table 27.

[0283] [Table 9] TIFF0007848412000046.tif87167

[0284] [Example 2] Comparison of ELISA binding affinity on the cell surface of sacituzumab mutants between the BxPC3 cell line and the MDA-MB-231 cell line.

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

[0286] [Table 10]

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

[0288] [Table 11] TIFF0007848412000049.tif31155

[0289] Surface plasmon resonance (SPR) analysis of the Y53D substitution of CDR2 in 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 antibody's Fab bound (monovalent mode). See Table 12.

[0290] [Table 12]

[0291] Figure 1 shows that Y53D substitution in the CDR2 of the sacituzumab light chain is associated with low TROP2 expression in MDA-MB-231-KWL cells (TROP2 低 BxPC3 cells (TROP2) express more TROP2 than cells. 高This demonstrates the production of a binding-modulated anti-TROP2 antibody that preferentially binds to cells.

[0292] [Example 3] Characteristics of various mutant development. The developmental characteristics of various mutants were described in the same manner as in Bailly et al., Predicting Antibody Developability Profiles Through Early Stage Discovery Screening, MABS 12(1):e1743053(2020)(doi:10.1080 / 19420862.2020.17430530), which is incorporated herein by reference in its entirety. The developmental characteristics of various mutants were described. The various methods used can be found herein under the general methods.

[0293] The results of 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 sacituzumab heavy chains (HC having the amino acid sequence shown in SEQ ID NO: 11), and all Hm muteins contain sacituzumab light chains (LC having the amino acid sequence shown in SEQ ID NO: 12). The amino acid sequences containing the antibodies disclosed in Tables 13, 14, and 15 can be found in Table 27.

[0294] [Table 13] TIFF0007848412000052.tif92154

[0295] [Table 14] TIFF0007848412000054.tif114153

[0296] [Table 15]

[0297] [Table 16]

[0298] [Table 17]

[0299] [Table 18]

[0300] In the table, K DApp Because it reflects the average of measurements from different chip loadings, the “obvious” K D RU (Response Unit) refers to the loading onto the chip (8RU and 28.8RU mean that measurements were taken at lower antigen densities (8RU) and higher antigen densities (28.8RU)). BC (Binding Capacity) is a measure of the number of molecules that remain on the chip relative to the standard (lower k D This is a qualitative measure (usually equal to a lower BC due to the off-rate).

[0301] [Example 4] Sacituzumab rehumanization and identification of best single mutations (BSMs). The amino acid sequence of sacituzumab was evaluated using the software package BIOVIA Discovery Studio with the "Predict Humanizing Mutations" tool. Conformation was set to exclude substitutions in the Vernier zone, 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 mutant in the framework region predicted to be stabilized. Overall, BSM mutations were calculated to provide a stabilization of -9.03 kcal / mol using Harvard Macromolecular Mechanics (CHARMM) energy theory chemistry against the clinical sacituzumab sequence. The obtained sacituzumab BSM sequence exhibits higher identity to the human germline compared to the clinical sequence (81% identity for both strands), with 83% identity to IGHV7-4-1*02 and 84% identity to IGKV1-13*02.

[0302] The performance of sacituzumab and sacituzumab BSM in reverse-phase high-performance liquid chromatography (RP-HPLC) was comparable, as shown in Figures 2 and 2-1. RP-HPLC was performed at 70°C using POROS® resin, as described in general methods.

[0303] Hydrophobicity determination was performed for various sacituzumab mutants using HIC, which was performed using a butyl HIC column as described in the general method.

[0304] Figure 3 shows a comparison of the hydrophobicity of αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody and αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) antibody, and αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y105S) antibody and αTROP2(HC:BSM-S375C)(LC:BSM-Y105S) antibody. YTE substitution in the heavy chain resulted in little increase in hydrophobicity.

[0305] Figure 4 shows a comparison of the hydrophobicity of αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody and αTROP2(HC:BSM-S375C)(LC:BSM) antibody. The peak elution of the αTROP2(HC:BSM-YTE-S375C)(LC:BSM-Y53D) antibody was 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] αTROP2 mAb mutants were administered intravenously to biologically naive male rhesus monkeys, and their pharmacokinetics (PK) were evaluated. The study plan 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 ligand-binding assay using anti-human IgG framework antibodies. PK parameters were estimated by non-compartmental analysis using Phoenix WinNonlin (version 6.3, Certara).

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

[0308] [Table 19]

[0309] [Table 20]

[0310] [Table 21]

[0311] [Table 22]

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

[0313] First, mutant amino acid sequences were screened for MHC class II epitope content using the EpiMatrix tool. The 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). Next, the ClustiMer algorithm was used to identify regions of high epitope density within the protein sequences. ClustiMer searches for consecutive segments of 15–30 amino acids with high binding across common HLA-DR alleles. Subsequently, the epitope clusters identified by ClustiMer were evaluated using the Janus Matrix algorithm to determine epitope autonomy and assess the potential for immune tolerance to a given epitope. Using the Janus Matrix Algorithm (Moise et al., Hum.Vaccin.Immunother.9:1577-1586 (2013)), we identified epitopes that share T cell receptor (TCR) plane conservation (positions 2, 3, 5, 7, 8) with epitopes restricted by the same alleles found in the human proteome. Epitopes with identical TCR-facing residues that are also predicted to bind to the same MHC allele are more likely to induce cross-reactive T cells. Furthermore, the Janus-Matrix algorithm identifies epitopes (called Tregitopes) known to correspond to immunosuppressive regulatory T cell responses that are thought to reduce the immunogenic performance of constructs. Applying two Janus Matrix homology score thresholds for cross-conservation with human (self) proteins (cross-conserved HLA allele-specific epitopes averaged over sequence length) we identified epitopes that are likely to be tolerable or actively regulatory.

[0314] Of the TROP2 variant sequences analyzed, αTROP2(HC:BSM-S375C)(LC:BSM-Y53D) exhibited the lowest predicted immunogenicity, demonstrating a total Tregitope-modulated protein immunogenicity score of (-31.72), which is comparable to the immunogenicity profile of known non-immunogenic antibodies tested in clinical settings. Antibody analysis tools predicted a 1.85% ADA response in the clinical population due to its relatively low predicted effector epitope content and high predicted tregitope content, and predicted 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 a lower 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 messy non-self epitopes found in wild-type sacituzumab and introduces two highly HLA-binding Tregitopes, 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-pyrrole-1-yl)propanamide)propanamide)propanamide)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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 at 0°C 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}propanamide]propanoic acid I1-a (100 g, 261 mmol) in dichloromethane (DCM):MeOH (2:1) (3000 mL). The reaction mixture was stirred at room temperature for 18 hours and monitored by LC-MS. The solvent was evaporated under vacuum, and the residue was diluted with methyl tert-butyl ether (TBME) (2000 mL) and stirred for 30 minutes. The solid was recovered by filtration, washed with TBME (1000 mL), and dried in vacuum to obtain 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 at room temperature 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). The mixture was stirred at room temperature for 18 hours. The reaction was monitored by LC-MS. The solvent was evaporated, the residue was diluted with ethyl acetate (SiO) (500 mL), and stirred for 30 minutes. The solid was collected by filtration, washed with SiO (300 mL), and dried under vacuum to obtain (2S)-2-[(2S)-2-aminopropanamide]-N-[4-(hydroxymethyl)phenyl]propanamide (I1-c). LC-MS:(ES,m / z):[M+H] + =266.

[0318] Synthesis of compound I1-d in step C At room temperature, a mixture of 2,5-dioxopyrrolidine-1-yl 3-(2,5-dioxopyrrole-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 for 16 hours. The reaction mixture was added to H2O (600 mL) while stirring. The solid was recovered by filtration, washed with water (500 mL), and dried under vacuum to obtain (2S)-2-[3-(2,5-dioxopyrrole-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 at room temperature to a stirred mixture of bis(4-nitrophenyl) carbonate (60.3 g, 198 mmol) and (2S)-2-[3-(2,5-dioxopyrrole-1-yl)propanamide]-N-[(1S)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}ethyl]propanamide (I1-d) (55 g, 32 mmol) in dimethylformamide (DMF) (550 mL). The mixture was stirred for 16 hours and monitored by liquid chromatography-mass spectrometry (LCMS). The reaction solution was added to H2O (400 mL) while stirring. The mixture was filtered, and the filter cake was washed with water (1 × 100 mL). The solid was recovered by filtration and purified using reverse-phase chromatography (dynamic axial chromatography column C-18, eluted with 15%-65% ACN / water (containing 0.1% ammonium acetate (NH4OAc) as a modifier)). The resulting mixture was concentrated under vacuum, and the solid was dried under vacuum at 45°C to obtain {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrole-1-yl)propanamide]propanamide]propanamide]phenyl}methyl-4-nitrophenyl carbonate (I1-e). LC-MS:(ES,m / z):[M+H] + = 582.

[0320] Synthesis in process E-1 In a 60 mL round-bottom flask under N2 conditions, {4-[(2S)-2-[(2S)-2-[3-(2,5-dioxopyrrole-1-yl)propanamide]propanamide]propanamide]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 minutes, and then MMAE (4.00 g, 5.57 mmol) was added at 25°C. The reaction mixture was stirred at 25°C for 16 hours. The reaction product was then purified using reverse-phase column chromatography (AQ C18 with 0.1% ammonium acetate as a modifier, 30%-60% acetonitrile (MeCN) / water). MeCN was concentrated under vacuum, the remaining aqueous mixture was extracted with ethyl acetate (300 ml x 3), and the combined organic matter was concentrated under vacuum. Four batches were performed in parallel, combined, dissolved in 200 mL of MeCN, 400 mL of water was added, the solution was frozen, and then freeze-dried to obtain 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)propanamide)propanamide)propanamide)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R ,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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 have been reported.

[0321] [Example 8] Synthesis of 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)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)pyrrolidine-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 organic matter was concentrated under vacuum to obtain 5-cyano-6-(methylthio)picolinic acid (I-2b). LC-MS: (ES, m / z): [M + H] + =195.

[0323] Step B - Synthesis of compound I-2c 5-cyano-6-(methylthio)picolinic acid (I-2b) (66 g, 0.34 mol) was dissolved in tetrahydrofuran (THF) (3000 mL), to which (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (155 g, 0.41 mol) was added. The resulting mixture was stirred at 25°C for 30 minutes. Tert-butyl 3-aminopropanoate HCl salt (67.7 g, 0.37 mol) was added to the mixture, and then it was cooled to 10°C. N,N-diisopropylethylamine (DIEA) (175 g, 1.35 mol) was added dropwise over 1 hour at 10°C. The reaction mixture was stirred at 25°C for 16 hours, concentrated under vacuum, and diluted with ethyl acetate. The resulting solution was washed three times with water, and the combined organic matter was concentrated under vacuum. The residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether 1:2) to obtain tert-butyl 3-(5-cyano-6-(methylthio)picolinamide)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)picolinamide)propanoate (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 hours. The resulting reaction mixture was diluted with DCM and poured into ice water. The organic phase was washed four times with 10% sodium bicarbonate aqueous solution, the combined organic phase was dried, and concentrated under vacuum to obtain tert-butyl 3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoate (I-2d). LC-MS: (ES, m / z): [M + Na] + =376

[0325] Step D - Synthesis of Compound I-2e To a solution of tert-butyl 3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoate (I-2d) (90 g, 0.25 ol) dissolved in 1,4-dioxane (1000 mL), 4 M HCl in 1,4-dioxane (2500 mL) was added. The resulting mixture was stirred at 25°C for 16 hours and then filtered. The solid was washed with n-heptane and dried under nitrogen to obtain 3-(5-cyano-6-(methylsulfonyl)picolinamide)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, 3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoic acid (I-2e) (69 g, 0.23 mol) and HATU (106 g, 0.28 mol) were added. 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 overnight at 20°C. The reaction mixture was then concentrated under vacuum, and the resulting residue was diluted with ethyl acetate. The combined organic matter was washed three times with water, dried, and concentrated under vacuum. The resulting residue was purified using silica gel chromatography with DCM:MeOH (2:1) to obtain tert-butyl(3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoyl)-L-alanyl-L-alaninate (I-2f). LC-MS:(ES,m / z):[M+Na] + = 518

[0327] Synthesis of compound I-2g in step F 4M HCl in 1,4-dioxane (1800 mL) was added to tert-butyl(3-(5-cyano-6-(methylsulfonyl)picolinamide)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 under vacuum. The resulting solid was washed with MTBE and filtered. The solid was air-dried overnight to obtain (3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoyl)-L-alanyl-L-alanine (I-2 g). LC-MS: (ES, m / z): [M + Na] + =462

[0328] Step G - Synthesis of compound I-2h In a 500 mL four-necked round-bottom flask, purged with nitrogen and maintained under an inert nitrogen atmosphere, (3-(5-cyano-6-(methylsulfonyl)picolinamide)propanoyl)-L-alanyl-L-alanine (I-2 g) (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 obtain 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)benzyl(4-nitrophenyl)carbonate (I-2h). LC-MS:(ES,m / z):[M+Na] + =732

[0329] Synthesis of step H-compound 2 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)benzyl(4-nitrophenyl)carbonate (I-2h) (5.6 g, 7.9 mmol) and 1H-benzo[d][1,2,3]triazole-1-ol (0.213 g, 1.58 mmol) were mixed with DMF (56.0 mL). MMAE (5.67 g, 7.89 mmol) was added to the reaction mixture at 20°C, and the reaction mixture was stirred at 40°C for 16 hours. The resulting mixture was subjected to Prep-HPLC (10-95% MeCN / water, 0.05% Purified using TFA, 4-((S)-2-((S)-2-(3-(5-cyano-6-(methylsulfonyl)picolinamide)propanamide)propanamide)propanamide)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)pyrrolidine-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 the intermediate tert-butyl L-alanyl-L-alaninate (iii) [ka] Step A - Synthesis of compound ii (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine(i) (140 g, 450 mmol) dissolved in DCM (4500 mL) was mixed with PyBOP (281 g, 540 mmol, 1.2 equivalents) 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 mixture over 1 hour, and the mixture was stirred overnight at 20°C. The reaction mixture was concentrated under vacuum, diluted with ethyl acetate, and washed with aqueous sodium carbonate (1 M), potassium bicarbonate (1 M), water, and brine. The organic phase was then concentrated under vacuum and evaporated to 25% of the original solvent volume. MTBE (1400 mL) was added dropwise, and the mixture was stirred for 5 hours. The suspension was filtered, and the solid was washed with MTBE to obtain 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 172 g, 392 mmol of tert-butyl(((9H-fluoren-9-yl)methoxy)carbonyl-L-alanyl-L-alaninate (ii) (2000 mL) dissolved in DCM (2000 mL) was mixed with triethanolamine (TEA) (2000 mL). The reaction mixture was heated to 40 °C and stirred for 16 hours. The reaction mixture was then concentrated under vacuum and purified using silica gel chromatography (DCM: 4 M NH35:1 in MeOH) to obtain 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)nicotinamide)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 for 2 hours under an N2 atmosphere. The mixture was concentrated under vacuum, and the crude product was purified using reverse-phase flash chromatography (AQ C18 silica gel, ACN 0%~20% gradient in water (using 0.5% NH3·H2O as a modifier)) to obtain the crude product. The resulting mixture was filtered, and the filtrate was washed with H2O. The filtrate was concentrated under vacuum to obtain 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 LC-MS, and then concentrated under vacuum. Water cooled to 10°C and ethyl acetate were added, and the solid was filtered off. The mixture was extracted with ethyl acetate, dried, and concentrated under vacuum. The residue was purified using reverse-phase flash chromatography (AQ silica gel column, 0%-15% acetonitrile / sodium bicarbonate (aqueous solution)). The pH 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 under vacuum to obtain 6-chloro-5-cyanopyridine-3-carboxylic acid (vi). LC-MS: (ESI, m / z): [MH] - =181.

[0334] Synthesis of step C-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 a nitrogen atmosphere at 25°C. To the mixture, sodium (methylsulfanyl) (7.39 g, 105 mmol) was added in fractions at 0°C. The resulting mixture was stirred at 25°C for 8 hours. The reaction was monitored by LC-MS. This solution was slowly transferred to H2O (1200 mL) and then extracted with ethyl acetate (1 × 700 mL). The pH of the aqueous layer was adjusted to 2-3 with 1 M HCl. The solid was recovered by filtration to obtain 5-cyano-6-(methylsulfanyl)pyridine-3-carboxylic acid (vii). LC-MS: (ESI, m / z): [MH] - =193.

[0335] Step D - Synthesis of compound viiii 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 LC-MS, then quenched with saturated sodium bisulfite at 0°C and concentrated under vacuum. 2-methyltetrahydrofuran was added to the residue, and the mixture was filtered and concentrated under vacuum to obtain the crude product. The residue was purified by silica gel column chromatography eluted with DCM / MeOH to obtain 5-cyano-6-(methylsulfonyl)nicotinic acid (viii). LC-MS: (ESI, m / z): [M+H] + =227.05.

[0336] Synthesis of compound ix in step E 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 of DMF and stirred at 25°C for 30 minutes. Then, tert-butyl 3-aminopropanoate (0.42 g, 2.8 mmol) was 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 hours, then diluted with water and extracted with RINKAN (3×). The organic phase was concentrated under vacuum, and the residue was purified by silica gel column chromatography eluting with 2:1 to 1:1 hexane:RINKAN to obtain tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamide)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] Synthesis of compound x in step F tert-butyl 3-(5-cyano-6-(methylsulfonyl)nicotinamide)propanoate (ix) (0.10 g, 0.28 mmol) was dissolved in 10 mL of 1,4-dioxane. Then, 4 M HCl (10 mL) was added to the mixture in 1,4-dioxane. 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 obtain 3-(5-cyano-6-(methylsulfonyl)nicotinamide)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 the intermediate compound 4-((S)-2-((S)-2-aminopropanamide)propanamide)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)pyrrolidine-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 equivalent) in DMF (130 mL) was added to a 500 mL four-necked round-bottom flask that had been purged and maintained under an inert nitrogen atmosphere. Then, 2 mol% DIEA (0.534 mmol) and bis(4-nitrophenyl) carbonate (16.3 g, 53.6 mmol, 2 equivalents) were added to the reaction mixture at 20 °C. The reaction mixture was heated to 45 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature and purified using reverse-phase flash column chromatography (30%-60%, MeCN / water containing 0.05% TFA as a modifier) ​​to obtain (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-necked round-bottom flask that had been purged and maintained under an inert atmosphere of nitrogen. Then, HOBt (340 mg, 2.5 mmol) was added, and the reaction mixture was stirred at room temperature for 10 minutes. Then, MMAE (9 g, 12.7 mmol) was 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%-50% MeCN / water containing 0.05% TFA as a modifier) ​​to obtain 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)propanamide)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R) -3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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] Synthesis of compound xiv in step C 4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propanamide)propanamide)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-oxy Sopropyl)pyrrolidine-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-aminopropanamide)propanamide)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)pyrrolidine-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 as is 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)nicotinamide)propanamide)propanamide)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)pyrrolidine-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)nicotinamide)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 minutes. Then, xiv was added dropwise, followed by DIEA (0.034 ml, 0.19 mmol). The reaction mixture was stirred for 1.5 hours, and then reversed-phase chromatography (Waters The sample was purified using a CSH-C18 column, 19 × 250 mm × 5 μm, with 35-70% acetonitrile aqueous solution (containing 0.1% formic acid as a modifier). 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-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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: The antibody (S375C) with two manipulated Cys residues was decapped, and the interchain disulfide was reduced at 37°C for 2 hours using tris(2-carboxyethyl)phosphine (TCEP) (20 equivalents, 0.5 M in water adjusted to pH 7.0 with ammonium hydroxide), and monitored by HPLC-MS. The reduced antibody was then buffer-exchanged via AKTA™ (desalting column, monitoring at 280 nm) to PBS buffer at pH 7.4 or 30 mM ACES (N-(2-acetamide)-2-aminoethanesulfonic acid, N-(carbamoylmethyl)-2-aminoethanesulfonic acid, N-(carbamoylmethyl)taurine) buffer at pH 7.0, and diluted to 10 mg / mL for subsequent steps using the same buffer. A 100 mM solution of dehydroascorbic acid (8.0 equivalents) in water was slowly added, and the solution was mixed at room temperature for 4–10 hours or until complete (monitored by reversed-phase liquid chromatography (RP-LC) and sodium dodecyl sulfate capillary electrophoresis (CE-SDS)). A 20 mg / mL solution of linker payload (3.0 equivalents) in DMSO was added, and the conjugation was incubated at room temperature for 2 hours (monitored by quadrupole time-of-flight (QTOF) mass spectrometry (MS)), after which the residual linker payload was quenched by adding L-cysteine ​​(5 equivalents, 50 mM in water). ADCs were purified by AKTA (desalting column, 10 mM histidine buffer pH 6.0, monitored at 280 nm) and characterized by LCMS (Agilent polymer reversed-phase (PLRP)-S column, 1000 Å, 5 μm, 15-90% MeCN / H2O 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). The ADCs were then adjusted to the final desired concentration using Sartorius Vivacell 70 Centrifugal Concentrators and Amicon® Ultra Centrifugal Filters.

[0345] An exemplary conjugate was fabricated according to the protocol described above, and the following ADC was constructed.

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

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

[0348] [Table 23]

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

[0350] The cytotoxicity data values ​​are the average IC values ​​for each test substance. 50 This may include data from different lots of the same construct. All data in this example are derived from a 3-day assay. A control ADC (control mAb(HC:S375C-MMAE)) consisting of a non-anti-TROP2 antibody conjugated to MP-AA-PABC-MMAE with cysteine ​​at position 375 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 tumor cells (BxPC3, Calu-3, HCC1806, HCC78, JIMT-1, and NCI-N87) and primary cells derived from ATCC (PCS-301 (primary small airway) and PCS-200 (primary keratinocyte)) 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, 10× 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). The culture medium (without cells) was used as Max_E, and cells were used as Min_E. Then, 5 μL of 10×ADC from the intermediate plate was added to the assay plate using a Bravo liquid handler at a very slow rate, without 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, Wisconsin, catalog no. G9242) was added to 50 μL of cell-containing medium using a Standard Cassette Combi. The contents were mixed in 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 measured using an integration time of 0.25-1 second per well and guidelines. 50 The values ​​were calculated. The activity data was normalized as an effect percentage according to the formula %E=((response-min_E) / (max_E-min_E)*100). The cytotoxic results are shown in Table 22.

[0352] [Table 24]

[0353] [Table 25] TIFF0007848412000072.tif159154

[0354] [Table 26] TIFF0007848412000074.tif173153

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

[0356] Cytotoxicity data are shown in Table 23. The reported values ​​are the average IC of each test substance. 50 Therefore, it may include data from different lots of the same construct. All data in this table are derived from a 4-day assay using various TROP2+ cells with different TROP2 surface densities. Specific ADCs were tested with other cells. [Table 27] [Table 28]

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

[0358] Experimental protocol: Female BALB / c nude mice aged 7-8 weeks were purchased from Gempharmatech Co., Ltd. (La Jolla, California). The mice were anesthetized with isoflurane inhalation and given 10x10 cubic centimeters of PBS:Matrigel (50:50) in 0.1 mL volume. 6 A single-cell suspension of BxPC3 pancreatic cancer cells (survival rate ≥ 95%) was subcutaneously inoculated into the right lower flank of the mouse. The body weight and tumor size of the mice were measured twice a week, and the formula V = 0.5[a * b 2 The tumor volume was calculated using the formula [ ]. In the formula, a and b are the long and short diameters of the tumor, respectively. To minimize the variation in tumor volume between groups, a computer-generated randomization procedure was used to assign 110 mice to 11 groups, each consisting of 10 mice. The tumor was 200 mm 3 Treatment was initiated when the average size was reached. PBS and MMAE conjugates were administered intravenously (IV) at a dose of 2 mg / kg according to Table 25.

[0359] [Table 29]

[0360] Figure 13 shows that a binding-modified sacituzumab variant conjugated to MP-AA-PABC-MMAE was effective in reducing tumor volume after therapeutic administration.

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

[0362] Experimental protocol: Female BALB / c nude mice aged 7-8 weeks were purchased from Gempharmatech Co., Ltd. The mice were anesthetized with isoflurane inhalation and given 10x10 in 0.1 mL of PBS:Matrigel (50:50).6 A single-cell suspension of BxPC3 pancreatic cancer cells (survival rate ≥ 95%) was subcutaneously inoculated into the right lower flank of the mouse. The body weight and tumor size of the mice were measured twice a week, and the formula V = 0.5[a * b 2 The tumor volume was calculated using the formula [ ]. In the formula, a and b are the long and short diameters of the tumor, respectively. To minimize the variation in tumor volume between groups, a computer-generated randomization procedure was used to assign 70 mice to 7 groups of 10 mice each. The tumor was 200 mm 3 Treatment was initiated when the average size was reached. PBS, a control mAb, and a bioconjugate were administered 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 therapeutic administration.

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

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

[0367] Figure 15 plots the plasma tAb, cAb, and released payload concentrations of αTROP2 (HC:BSM-YTE-S375C-MMAE) (LC:BSM-Y53D) over time. For the ADC, the measured tAb and cAb PK curves overlapped, indicating no detectable unconjugated MMAE in the plasma and demonstrating the linker's stability in vivo. The final half-life was 9.9 days.

[0368] Table 27 provides the sequences referred to in this disclosure. [Table 31] TIFF0007848412000080.tif234153TIFF0007848412000081.tif235154TIFF0007848412000082.tif234154TIFF0007848412000083.tif233154TIFF0007848412000084.tif233154TIFF0007848412000085.tif234155TIFF0007848412000086.tif234154TIFF0007848412000087.tif235154TIFF0007848412000088.tif230154TIFF0007848412000089.tif234154TIFF0007848412000090.tif232154TIFF0007848412000091.tif234153TIFF0007848412000092.tif233154TIFF0007848412000093.tif236153TIFF0007848412000094.tif233153TIFF0007848412000095.tif234154TIFF0007848412000096.tif235155TIFF0007848412000097.tif235153TIFF0007848412000098.tif232153TIFF0007848412000099.tif233153TIFF0007848412000100.tif232153TIFF0007848412000101.tif233154TIFF0007848412000102.tif234154TIFF0007848412000103.tif232154TIFF0007848412000104.tif237153TIFF0007848412000105.tif235154TIFF0007848412000106.tif234154TIFF0007848412000107.tif233155TIFF0007848412000108.tif236152TIFF0007848412000109.tif233154TIFF0007848412000110.tif236154TIFF0007848412000111.tif236153

[0369] The present invention is described herein with reference to the illustrated embodiments, but it should be understood that the invention is not limited thereto. Those skilled in the art and those with access to the teachings herein will recognize additional modifications and embodiments within that scope. In addition to those described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the claims. Accordingly, the present invention is limited only by the language of the claims herein and the claims appended herein.

[0370] Patents, patent applications, publications, product descriptions, and protocols are referenced throughout this application, and their disclosures are incorporated herein by reference in their entirety for any purpose.

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody that specifically binds to human TROP2, conjugated to a linker-monomethyllauristatin E (linker-MMAE) payload, Here, the antibody is It comprises two heavy chains (HC), each heavy chain comprising a variable domain (VH) and a constant domain (CH), wherein the VH comprises complementarity-determining regions (CDR) H1, CDRH2, and CDRH3, and It comprises two light chains (LCs), each light chain comprising a variable domain (VL) and a constant domain (CL), the VL comprising CDRL1, CDRL2 and CDRL3, Herein, the antibody-drug conjugate (ADC) comprises 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. The ADC according to claim 1, wherein the antibody 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 according to claim 1, wherein the LC comprises amino acid substitutions S20T, D60S, V85T and A100P, where the positions are relative to the amino acid sequence shown in SEQ ID NO: 13, and the HC comprises amino acid substitutions Q5L, K38R, A69S, T78Q, D89E, F95Y, S115T, R218K, E360D and M362L, where the positions are relative to the amino acid sequence shown in SEQ ID NO:

11.

4. The ADC according to claim 1, wherein the CH is IgG1 containing the M252Y, S254T and T256E amino acid substitutions, where its position follows the Eu numbering rules.

5. The ADC according to claim 1, wherein VH comprises the amino acid sequence of SEQ ID NO: 14, and VL comprises the amino acid sequence of SEQ ID NO:

16.

6. The ADC according to claim 1, wherein the antibody contains a cysteine ​​amino acid at position 375 of the CH.

7. The ADC according to claim 6, wherein HC comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 90, and LC comprises the amino acid sequence of SEQ ID NO:

22.

8. The ADC according to claim 6, wherein the linker-MMAE payload is conjugated to the cysteine.

9. The antibody has a thiol (SH) group of formula: MP-AA-PABC-MMAE: 【Chemistry 1】 The ADC according to claim 1, comprising a cysteine ​​residue conjugated to a linker-MMAE payload containing the following.

10. The aforementioned ADC is given by: 【Chemistry 2】 (In the formula, Ab is an anti-TROP2 antibody, and p is an integer from 1 to 8, where S is derived from the side chain of the cysteine ​​residue of the antibody.) The ADC according to claim 1, including the following:

11. The ADC, formula: 【Transformation 3】 (In the formula, Ab is an anti-TROP2 antibody containing a heavy-chain modified cysteine ​​residue or a light-chain modified cysteine ​​residue.) Here, the anti-TROP2 antibody containing the manipulated cysteine ​​residue is (gg) α-TROP2 (HC: BSM-YTE-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; And, Here, S is derived from the side chain of the manipulated cysteine ​​residue, and here, p is an integer selected from 1 or 2. The ADC according to claim 1, including the following:

12. The ADC, formula: 【Chemistry 4】 (In the formula, Ab is an anti-TROP2 antibody containing a heavy-chain modified cysteine ​​residue or a light-chain modified cysteine ​​residue.) Here, the anti-TROP2 antibody containing the manipulated cysteine ​​residue is (ff) α-TROP2 (HC: BSM-YTE-S375C) (LC: BSM-Y53D), comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 and two light chains having the amino acid sequence shown in SEQ ID NO: 22; (where S is derived from the side chain of the manipulated cysteine ​​residue, and where p is an integer selected from 1 or 2) The ADC according to claim 1, including the following:

13. An antibody-drug conjugate (ADC) comprising: 【Transformation 5】 (In the formula, Ab is an anti-Trop2 antibody comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 22, where p is 1 or 2, and where S is derived from the side chain of the manipulated cysteine ​​residue at position 375 of the constant domain of the heavy chain, the position being defined according to Eu numbering.) ADC, including.

14. An antibody-drug conjugate (ADC) comprising: 【Transformation 6】 (In the formula, Ab is an anti-TROP2 antibody comprising two heavy chains having the amino acid sequence shown in SEQ ID NO: 20 or SEQ ID NO: 90 and two light chains having the amino acid sequence shown in SEQ ID NO: 22, where p is 1 or 2, and where S is derived from the side chain of the manipulated cysteine ​​residue at position 375 of the constant domain of the heavy chain, the position being defined according to Eu numbering.) ADC, including.

15. A composition comprising the ADC according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. The composition according to claim 15, wherein the dominant 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 pyroglutamate.

17. A pharmaceutical composition comprising the ADC according to any one of claims 1 to 14 for treating cancer that overexpresses TROP2.

18. The pharmaceutical composition according to claim 17, 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 lymphoma, chronic lymphocytic lymphoma (CLL), Razi Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, gastric cancer, kidney cancer, salivary gland ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

19. Use of the ADC according to any one of claims 1 to 14 for manufacturing a pharmaceutical product for treating cancers that overexpress TROP2.

20. The use according to claim 19, 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 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, kidney cancer, salivary ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

21. An ADC according to any one of claims 1 to 14, for the treatment of cancers that overexpress TROP2.

22. The ADC according to claim 21, 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 lymphoma, chronic lymphocytic lymphoma (CLL), Razi Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, gastric cancer, kidney cancer, salivary gland ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

23. A pharmaceutical composition for the treatment of cancer, comprising an ADC according to any one of claims 1 to 14, for use in combination with a therapeutic agent, wherein the cancer is a cancer that overexpresses TROP2.

24. The pharmaceutical composition according to claim 23, wherein the therapeutic agent is a chemotherapeutic agent or a therapeutic antibody.

25. The pharmaceutical composition according to claim 24, wherein the therapeutic antibody is a checkpoint inhibitor.

26. The pharmaceutical composition according to claim 24, wherein the therapeutic antibody is an anti-PD1 antibody or an anti-PD-L1 antibody.

27. The pharmaceutical composition according to claim 26, 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 lymphoma, chronic lymphocytic lymphoma (CLL), Razi Burkitt lymphoma, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, thyroid cancer, bladder cancer, glioma, oral cancer, gastric cancer, kidney cancer, salivary gland ductal carcinoma, anaplastic thyroid carcinoma, neuroendocrine non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), colon cancer, sarcoma, esophageal cancer, cervical cancer, and uterine cancer.

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