Biomolecules bound to TROP2, pharmaceutical compositions, and methods

Abstract

Disclosed herein are antibody-drug conjugates (ADCs) in which a drug moiety is conjugated to an antibody or antigen-binding fragment that binds to TROP2, and methods of use thereof. Methods of use include the treatment and diagnosis of cancer. The ADCs of this disclosure may bind to the surface of specific cancer cells. Exemplary targets of the ADCs disclosed herein may include cancers such as lung cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 492,343, filed Mar. 27, 2023, and U.S. Provisional Patent Application No. 63 / 549,781, filed Feb. 5, 2024. The entire disclosures of the foregoing applications are incorporated herein by reference.

[0002] Sequence Listing This application is filed electronically in.xml format and includes a sequence listing that is incorporated herein by reference in its entirety. The.xml copy created on Mar. 26, 2024, is named "G3004 - 01900PCT_20240327_SeqListing.xml" and is 11 kilobytes in size. '

[0003] Field The present invention relates to antibody - drug conjugate (ADC) compositions and methods of using them for treating cancer. Also described herein are methods of using antibody - drug conjugate compounds for treating mammalian cells associated with a pathological condition. The present invention relates to antibodies and binding fragments thereof against TROP2, and includes pharmaceutical compositions comprising said antibodies and / or binding fragments. Further provided is a method of administering an ADC to a subject in an amount effective to inhibit cancer cells.

Background Art

[0004] Background of the Invention TROP2 (tumor-associated calcium signaling transducer 2, TACSTD2) is a common tumor surface antigen that mediates cell proliferation, adhesion, and migration. While TROP2 plays a crucial role in cancer development, its localization can lead to different functions within tumor cells. Patients with membrane-bound TROP2 expression have been shown to have poor clinical outcomes, while intracellular TROP2 is associated with higher survival rates and fewer recurrences in breast cancer patients (Ambrogi et al., (2014) PLOS ONE 9(5): e110606). TROP2 has been studied for its upregulation of PI3K / Akt-mediated epithelial-mesenchymal transition (EMT) in cervical cancer (Liu et al., (2013) PLOS ONE 8(9): e75864), gallbladder cancer (Chen et al., (2014) Tumor Biology 35(111): 11565-11569), and gastric cancer (Zhao et al., (2019) Cancer Med. 8(3): 1135-1147). The RAS-Raf-MEK-ERK pathway has been shown to be under the control of TROP2 to stimulate cell proliferation. TROP2 has been found to modulate cellular function through interactions with other membrane proteins, including IGF-1 (Sin et al., (2019) Gynecol. Oncol. 152(1): 185-193), MDK, and NRG1 (Zhang et al., (2014) Oncotarget. 5(19): 9281-9294). Identifying TROP2s that accompany and / or predict cancer, and developing antibody-drug conjugates (ADCs) against these markers for use in the diagnosis and treatment of a wide range of cancers, is of great interest. TROP2 can be designed as an ADC by combining its specific antibody and drug moiety via different linkers.

[0005] The use of antibody-drug conjugates (ADCs) for local delivery of cytotoxic or cell proliferation inhibitors, such as those used in cancer treatment to kill or inhibit tumor cells (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug. Del. Rev. 26:151-172; U.S. Patent No. 4975278), theoretically allows for targeted delivery of the drug portion to the tumor and its intracellular accumulation there. However, systemic administration of these unbound drugs can result in unacceptable levels of toxicity not only to the tumor cells being targeted but also to normal cells (Baldwin et al., 1986, Lancet pp. (March 15, 1986):603-05; Thorpe, 1985, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84): Biological and Clinical Applications, A. Pinchera et al., pp. 475-506). This seeks to achieve maximum efficacy with minimal toxicity. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., 1986, Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, supra). Some cytotoxic drugs tend to become inactive or lose activity when bound to large antibodies or protein receptor ligands.

[0006] Exatecan [IUPAC name: (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione; CAS number: 171335-80-1; Molecular formula: C 24 H 22 Exatecan (FN3O4) is a topoisomerase 1 inhibitor that is a structural analog of camptothecin with antineoplastic activity. In the past, exatecan was clinically evaluated as a standalone chemotherapy agent (exatecan mesylate, DX-8951f). Preclinical studies showed it to be more potent than SN-38-based irinotecan (CPT-11) in various tumor xenograft models, including CPT-11-resistant tumors. Although development of exatecan mesylate as a free drug has now been discontinued, exatecan remains a candidate for the cytotoxic drug portion of antibody-drug conjugates with excellent antitumor effects. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 4975278 [Non-patent literature]

[0008] [Non-Patent Document 1] Ambrogi et al., (2014) PLOS ONE 9(5): e110606 [Non-Patent Document 2] Liu et al., (2013) PLOS ONE 8(9): e75864 [Non-Patent Document 3] Chen et al., (2014) Tumor Biology 35(111): 11565-11569 [Non-Patent Document 4] Zhao et al., (2019) Cancer Med. 8(3): 1135-1147

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

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Non-Patent Document 11

Summary of the Invention

[0009] Therefore, the present invention is based on the discovery that TROP2 is abnormally expressed in a wide range of cancers. Cancers that express TROP2 include, but are not limited to, lung cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0010] In one embodiment, the present invention is characterized by an antibody or a conjugated fragment specific to TROP2.

[0011] In one embodiment, the antibody is an anti-TROP2 antibody.

[0012] In one embodiment, the anti-TROP2 antibody is R4702. An exemplary R4702 antibody is described in a PCT patent application (International Publication WO2022 / 222992), the contents of which are incorporated herein by reference in their entirety.

[0013] In one embodiment, the chemotherapeutic agent is a topoisomerase inhibitor, which includes topoisomerase I inhibitors and topoisomerase II inhibitors.

[0014] In one embodiment, the chemotherapeutic agent is a topoisomerase I inhibitor, which is either camptothecin (CPT) or non-camptothecin, and is selected from irinotecan, topotecan, camptothecin, rubitecan, MLN576, exatecan, berotecan, seconeoritosin, SN-38, Genz-644282, betulinic acid, β-rapacone, calenitecan, gimatecan, namithecan, edotecarin, SW044248, LMP744, T-2513, podocarpus flavone A, indimitecan, lulutotecan, TP3011, or 10-hydroxycamptothecin.

[0015] In one embodiment, the present invention provides antibody-drug conjugates (ADCs) in which an antibody is conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a toxin or fragment thereof having enzymatic activity derived from bacteria, fungi, plants, or animals), or a radioisotope (i.e., a radioconjugate). In one embodiment, the present invention is characterized by an antibody-drug conjugate (ADC) specific to TROP2.

[0016] In one embodiment, the drug is exatecan.

[0017] In one embodiment, the present invention provides a method for inhibiting the proliferation of cancer cells, the method comprising administering an effective amount of exemplary ADC (OBI-992) to a subject in need thereof, wherein the proliferation of cancer cells is inhibited.

[0018] In one embodiment, the present invention provides a method for treating cancer in a subject, the method comprising administering an effective dose of an exemplary ADC (OBI-992) described herein to a subject in need thereof.

[0019] In the disclosed compositions, both the ADC and other related components are present in immunogenic effective amounts. For each specific ADC, the optimal immunogenic effective amount should be determined experimentally (taking into account characteristics specific to the particular patient and / or type of treatment). Generally, this amount ranges from 0.01 μg to 250 mg per kilogram of body weight of the antibody that specifically targets TROP2. In some embodiments, the therapeutic effective amount (i.e., effective dose) of the therapeutic composition is about 0.001 μg / kg to about 250 mg / kg, 0.01 μg / kg to 100 mg / kg, or 0.1 μg / kg to 50 mg / kg, or about or at least: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0. 009;0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09;0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 ,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84 , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, or 250 grams or micrograms per patient's body weight in kilograms, or any range between any of the numbers specified herein, or any other range that would be apparent and understandable to a person skilled in the art without excessive experimentation. A person skilled in the art would understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatment, the subject's overall health or age, and other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject.

[0020] In one embodiment, the cancer is selected from the group consisting of lung cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0021] Details of one or more embodiments of the present invention are described below. Other features or advantages of the present invention will become apparent from the following drawings and detailed descriptions of some embodiments, as well as from the appended claims.

[0022] Brief explanation of the drawing A patent or application file must include at least one drawing created in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Office upon request and payment of the required fees.

[0023] A more complete understanding of the present invention may be obtained by considering the accompanying drawings in conjunction with the subsequent detailed description. The embodiments shown in the drawings are intended to illustrate the present invention only and should not be construed as limiting the present invention to the illustrated embodiments. [Brief explanation of the drawing]

[0024] [Figure 1A] Figure 1 shows the TROP2 immunohistochemical (IHC) expression in human normal tissue and tumor tissue. Figure 1A shows the TROP2 expression in tumor tissue of the human breast, lung, cervix, ovary, uterus, prostate, colon, esophagus, pancreas, larynx, stomach, and bladder. [Figure 1B] Figure 1B lists the TROP2 IHC expression H-scores calculated individually for each organ. [Figure 2A] Figure 2 shows the cytotoxicity assay of OBI-992 (R4702-exatecan ADC) in human cancer cell lines. Figure 2A shows that quantitative FACS values ​​were used to represent TROP2 expression. [Figure 2B]Figure 2B shows a positive correlation between OBI-992 cytotoxicity and TROP2 expression in cancer cells. [Figure 3] Figure 3 shows the weight changes of xenografts derived from BxPC-3 pancreatic cancer cells. Vehicle, R4702-E:R4702-exatecan, R4702-M:R4702-MMAE, R4702-S:R4702-SN38, IMMU-132, and DS-1062 were recorded three times a week until day 22. Data are shown as mean ± SEM (each group N=5, excluding the R4702-E-DAR8 10 mpk (N=4) and IMMU-132 (N=4) treatment groups, as outliers, which were tumors that proliferated subcutaneously rather than intramuscularly, were excluded). [Figure 4A] Figure 4 shows the tumor growth curves of xenografts derived from BxPC-3 pancreatic cancer cells (Figure 4A: Vehicle and test substance 10 mpk; Figure 4B: Vehicle and test substance 3 mpk). The vehicle, R4702-E: R4702-exatecan, R4702-M: R4702-MMAE, R4702-S: R4702-SN38, IMMU-132, and DS-1062 were recorded three times a week until day 22. The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%. Here, Ti and Ci represent the mean tumor volume in the treatment group and vehicle group at the end of the study (day 22). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment and vehicle groups at the start of test substance administration (day 1). Data are shown as mean ± SEM [Figure 4A: Excluding the R4702-E-DAR8 (N=4) and IMMU-132 (N=4) treatment groups, each group has N=5, and [Figure 4B: Excluding the IMMU-132 (N=4) treatment group, each group has N=5, due to the exclusion of outliers]. Statistical analysis was performed by Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). [Figure 4B] Same as above [Figure 5]Figure 5 shows the weight changes of xenografts derived from DU-145 prostate cancer cells. Weight was recorded twice a week for the vehicle, R4702-E:R4702-exatecan, R4702-M:R4702-MMAE, and DS-1062 until day 25. The latency period was the period after cancer cell transplantation but before the start of treatment. Data are shown as mean ± SEM (N=5 for each group except GP1 at 18 days and GP9 at 25 days). [Figure 6A] Figure 6 shows the tumor growth curves of xenografts derived from DU-145 prostate cancer cells (Figure 6A: Vehicle and test substance 10 mpk; Figure 6B: Vehicle and test substance 3 mpk; Figure 6C: Vehicle and test substance 1 mpk). Tumor volume for vehicle, R4702-E:R4702-exatecan, R4702-M:R4702-MMAE, and DS-1062 was recorded twice weekly until day 25. The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%. Here, Ti and Ci represent the mean tumor volume in the treatment and vehicle groups at the end of the study (day 25). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment and vehicle groups at the start of test substance administration (day 1). The incubation period was the period after cancer cell transplantation but before the start of treatment. Data are shown as mean ± SEM [Figure 6A: (N=5 in each group, excluding the control group (N=4; one animal was sacrificed on day 18 because its tumor volume exceeded 1500 mm3))], [Figure 6B: N=5 in each group, excluding the control group (N=4; one animal was sacrificed on day 18 because its tumor volume exceeded 1500 mm3) and the DS-1062 group (N=4; one animal was sacrificed on day 22 due to ulcerative tumor)], and [Figure 6C: (N=5 in each group, excluding the control group (N=4; one animal was sacrificed on day 18 because its tumor volume exceeded 1500 mm3))]. Statistical analysis was performed by Student's t-test (*p < 0.05). [Figure 6B] Same as above [Figure 6C] Same as above [Figure 7]Figure 7 shows the weight changes of xenografts derived from NCI-N87 gastric cancer cells. Weight was recorded twice a week for the vehicle, R4702-E:R4702-exatecan, and DS-1062 groups until day 23. Data are shown as mean ± SEM (N=5 for each group). [Figure 8A] Figure 8 shows the tumor growth curves of xenografts derived from NCI-N87 gastric cancer cells (Figure 8A: Vehicle and test substance 10 mpk; Figure 8B: Vehicle and test substance 3 mpk; Figure 8C: Vehicle and test substance 1 mpk). Tumor volume for vehicle, R4702-E:R4702-exatecan, and DS-1062 was recorded twice weekly until day 23. The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%. Here, Ti and Ci represent the mean tumor volume in the treatment and vehicle groups at the end of the study (day 23). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment and vehicle groups at the start of test substance administration (day 1). Data are presented as mean ± SEM [Figure 8A: (N=5 for G1-G4), [Figure 8B: (N=5 for each group)], [Figure 8C: (N=5 for each group)]. Statistical analysis was performed using Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001). [Figure 8B] Same as above [Figure 8C] Same as above [Figure 9] Figure 9 shows the weight changes of xenografts derived from NCI-H1975-C797S lung cancer cells. Weight was recorded twice a week for the vehicle, R4702-E:R4702-exatecan, DS-1062, osimertinib, and IMMU-132 until day 16. The latency period was the period after cancer cell transplantation but before the start of treatment. Data are shown as mean ± SEM (N=6 for each group, except for the control group (N=5) at 10 days). [Figure 10A]Figure 10 shows the tumor growth curves of xenografts derived from NCI-H1975-C797S lung cancer cells (Figure 10A: Vehicle and test substance 10 mpk; Figure 10B: Vehicle and test substance 3 mpk). Tumor volume for the vehicle, R4702-E:R4702-exatecan, DS-1062, osimertinib, and IMMU-132 was recorded twice weekly until day 16. The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%. Here, Ti and Ci represent the mean tumor volume in the treatment and vehicle groups at the end of the study (day 16). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment and vehicle groups at the start of test substance administration (day 1). The latency period was the period after cancer cell transplantation but before the start of treatment. Data are shown as mean ± SEM [Figure 10A: N=6 for each group, excluding the control group (N=5) at 10 days], and [Figure 10B: N=6 for each group, excluding the control group (N=5) at 10 days]. Statistical analysis was performed by Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). [Figure 10B] Same as above [Figure 11A] Figure 11 shows the nonspecific binding and off-target effects of OBI-992. Figure 11A: Whole blood samples from two donors (I and II) were incubated with fluorescein-labeled ADC for 4 hours to evaluate peripheral blood leukocyte (PBL) binding. The strength of cell binding was expressed by measuring the MFI of each leukocyte population. Figure 11B: THP-1 cells were incubated with ADC and subsequently detected using FITC-binding antibody. The strength of cell binding was expressed by measuring the MFI. Figure 11C: Cell viability of THP-1 cells treated with ADC for 4 days. The percentage of cell viability was calculated. Figure 11D: Differentiated neutrophils were treated with ADC for 6 days. Surviving neutrophils (CD66b+ / PI-) were identified by FACS. The relative percentage of CD66b+ / PI- cells is shown as mean and SD. [Figure 11B] Same as above [Figure 11C] Same as above [Figure 11D] Same as above [Figure 12A] Figure 12 shows that OBI-992 exhibits in vitro synthetic lethality with PARPi. Cell viability of MDA-MB-231 (Figure 12A), NCI-N87 (Figure 12B), and Capan-1 (Figure 12C) cells treated with PARP inhibitors and OBI-992 for 6 days. Percentage of cell viability was calculated, and results are shown as mean and SD (n=3). IC50 values ​​for each group were calculated using Prism. The multiplier change in IC50 values ​​of talazoparib treated with or without OBI-992 is labeled. [Figure 12B] Same as above [Figure 12C] Same as above [Figure 13A] Figure 13 shows that OBI-992 exhibits in vivo synthetic lethality with PARPi and anti-mPD-1. Figure 13A: Tumor-bearing mice were treated with OBI-992 or Talazoparib alone or in combination when their mean tumor volume reached 150-200 mm3. Tumor growth was monitored twice weekly until the end of the study (day 22). Figure 13B: Tumor-bearing mice were treated with OBI-992 or Olaparib alone or in combination when their mean tumor volume reached 150-200 mm3. Tumor growth was monitored twice weekly until the end of the study (day 22). Figure 13C: Tumor-bearing mice were treated with OBI-992 or anti-mPD-1 alone or in combination when their mean tumor volume reached 200-250 mm3. Tumor growth was monitored twice weekly until the end of the study (day 11). [Figure 13B] Same as above [Figure 13C] Same as above [Modes for carrying out the invention]

[0025] Detailed description of the invention Accordingly, methods and compositions of antibody-drug conjugates (ADCs) directed towards markers for use in the diagnosis and treatment of a wide range of cancers are provided. Antibody-drug conjugates (ADCs) in which a drug is conjugated to an antibody or antigen-binding fragment that binds to TROP2 have been developed and are disclosed herein. Uses include, but are not limited to, cancer treatment and diagnosis. The ADCs described herein can bind to cancer cells expressing a wide range of TROP2, thereby facilitating the diagnosis and treatment of cancer.

[0026] general definition The implementation of this invention will, unless otherwise specified, utilize conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art of those skilled in the art. Such techniques are well described in the literature. For example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, B. Perbal, A Practical Guide To Molecular Cloning (1984); Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer See also: and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986).

[0027] As used herein, the term “antigen” is defined as any substance that can trigger an immune response.

[0028] As used herein, the term “immunogenicity” refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0029] As used herein, the term “epitope” is defined as the portion of an antigen molecule that comes into contact with an antigen-binding site on an antibody or T cell receptor.

[0030] As used herein, the term “vaccine” refers to a preparation containing an antigen consisting of a disease-causing organism (either dead or attenuated) or components of such an organism, such as a protein, peptide, or polysaccharide, used to confer immunity to the disease caused by that organism. Vaccine preparations can be obtained naturally, synthetically, or by recombinant DNA technology.

[0031] As used herein, the term “antigen-specific” refers to the property of a cell population such that a supply of a particular antigen or fragment of an antigen results in specific cell proliferation.

[0032] As used herein, the term “specifically binds” refers to the interaction between a binding pair (e.g., an antibody and an antigen). In various examples, specific binding occurs at approximately 10 -6 moles / liter, approximately 10 -7 moles / liter, or about 10 -8 This can be embodied by affinity constants of moles / liter or less.

[0033] As used herein, the phrases “substantially similar,” “substantially identical,” “equivalent,” or “substantially equivalent” indicate a sufficiently high degree of similarity between two numerical values ​​(e.g., one relating to a nutrient and the other to a reference / comparison nutrient) to which a person skilled in the art would consider the difference between the two values ​​to be little or no biological and / or statistically significant in the context of the biological properties measured by the values ​​(e.g., Kd value, antiviral effect, etc.). The difference between the two values ​​is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10% as a function of the value of the reference / comparison nutrient.

[0034] As used herein, the phrases “substantially reduced” or “substantially different” indicate a sufficiently large difference between two numerical values ​​(generally one relating to the numerator and the other to a reference / comparison numerator) and a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological property measured by the value (e.g., the Kd value). The difference between the two values ​​is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value of the reference / comparison numerator.

[0035] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind quickly to antigens and remain bound for longer periods. Various methods for measuring binding affinity are known in the art and any of them can be used for the purposes of the present invention. Specific exemplary embodiments are described below.

[0036] Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. Antibodies exhibit binding specificity to specific antigens, while immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. The latter type of polypeptide is produced at low levels by the lymphatic system, for example, and at increased levels by myeloma.

[0037] The terms “antibody” and “immunoglobulin” are used interchangeably in their broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies insofar as they exhibit the desired biological activity), and may also include specific antibody fragments (described in more detail herein). Antibodies can be chimeric, humanized, and / or affinity-matured.

[0038] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. These domains are generally the most variable parts of the antibody and include the antigen-binding site.

[0039] The term "variable" refers to the fact that specific regions of a variable domain exhibit widely different sequences across antibodies, which are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework (FR). The natural heavy and light chain variable domains each contain four FR regions, primarily in a β-sheet structure, connected by three CDRs, forming loops and sometimes part of the β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in the binding of antibodies to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cell-mediated cytotoxicity.

[0040] Papain digestion of the antibody produces two identical antigen-binding fragments (called "Fab" fragments), each with a single antigen-binding site, and the remaining "Fc" fragment, named to reflect its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites that can still crosslink antigens.

[0041] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. In double-stranded Fv species, this region is formed by the association of dimers of variable domains from one heavy chain and one light chain via a strong non-covalent bond. In single-stranded Fv species, the variable domains from one heavy chain and one light chain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimer" structure similar to that of double-stranded Fv species. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0042] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' fragments in which the cysteine ​​residues of the constant domain have a free thiol group. The F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments having a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0043] The "light chains" of antibodies (immunoglobulins) derived from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0044] Depending on the amino acid sequence of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit and three-dimensional structures of different classes of immunoglobulins are well known and are commonly described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). Antibodies can be part of a larger fusion molecule formed by covalent or noncovalent bonding of an antibody to one or more other proteins or peptides.

[0045] The terms “full-length antibody,” “intact antibody,” and “complete antibody” are used interchangeably herein and refer to an antibody in substantially intact form, rather than an antibody fragment as defined below. This term particularly refers to an antibody having a heavy chain containing an Fc region.

[0046] An "antibody fragment" comprises only a portion of an intact antibody, the portion retaining at least one, and almost all, of the functions typically associated with that portion when present in an intact antibody. In one embodiment, the antibody fragment includes the antigen-binding site of an intact antibody and therefore retains the ability to bind to an antigen. In another embodiment, an antibody fragment containing, for example, an Fc region retains at least one of the biological functions typically associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may include an antigen-binding arm linked to an Fc sequence that can confer in vivo stability to the fragment.

[0047] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for any naturally occurring mutations that may be present in small amounts. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is not a discontinuous mixture of antibodies. Such a monoclonal antibody typically comprises an antibody containing a target-binding polypeptide sequence, where the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from multiple polypeptide sequences. For example, the selection process may be the selection of a unique clone from multiple clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. The selected target-binding sequence may be further modified, for example, to improve affinity for a target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, or to create a multispecific antibody, and it should be understood that an antibody containing a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations have the advantage of being typically free from contamination by other immunoglobulins. The modifier "monoclonal" indicates a characteristic of the antibody that it is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in this invention are produced by hybridoma (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.)., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567), phage display technology (e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (e.g., WO98 / 24893; WO96 / 34096; WO96 / 33735; WO91 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol.It can be produced by various techniques, including those described in 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0048] The monoclonal antibodies described herein include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and specifically include fragments of such antibodies to the extent that they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0049] The antibodies of the present invention also include chimeric or humanized monoclonal antibodies produced from the antibodies of the present invention.

[0050] Antibodies may be full-length or may include, but are not limited to, multispecific antibodies formed from antibody fragments, including, but are not limited to, Fab, F(ab')2, Fab', F(ab)', Fv, single-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragments (e.g., Ward et al, Nature, 341:544-546 (1989)), CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and antibody fragments, as well as one or more antibody fragments having antigen-binding moieties. Single-chain antibodies produced by conjugating antibody fragments using recombinant methods or synthetic linkers are also included in this invention. Bird et al. Science, 1988, 242:423-426. Huston et al, Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883. The antibody or its antigen-binding moiety according to the present invention may be monospecific, bispecific, or multispecific.

[0051] All antibody isotypes, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE (all classes and subclasses are included in this invention), are included in this invention. The antibody or its antigen-binding moiety may be a mammalian (e.g., mouse, human) antibody or its antigen-binding moiety. The antibody light chain may be kappa or lambda type.

[0052] Therefore, the anti-cancer antibody of the present invention may include a constant region, framework region, or any portion thereof of a non-mouse-derived, preferably human-derived, heavy chain or light chain in combination with a variable region of the heavy chain or light chain, and these can be incorporated into the antibody of the present invention.

[0053] An antibody having variable heavy chain regions and variable light chain regions that are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous to the variable heavy chain regions and variable light chain regions of an antibody produced by a reference antibody, and that can also bind to TROP2. The homology may be at either the amino acid or nucleotide sequence level.

[0054] The antibody or antigen-binding moiety may be a peptide. Such peptides may include variants, analogs, orthologues, homologs, and derivatives of the peptide that exhibit biological activity, such as binding to carbohydrate antigens. Peptides may include analogs of one or more amino acids (e.g., unnatural amino acids, amino acids naturally occurring only in unrelated biological systems, modified amino acids from mammalian systems, etc.), peptides with substitutional bonds, and other modifications known in the art.

[0055] Furthermore, within the scope of the present invention are antibodies or their antigen-binding moieties in which specific amino acids are substituted, deleted, or added. In exemplary embodiments, these modifications do not substantially affect the biological properties of the peptide, such as binding affinity. In another exemplary embodiment, an antibody may have amino acid substitutions in its framework region, for example, to improve the antibody's binding affinity to an antigen. In yet another exemplary embodiment, a select few acceptor framework residues may be replaced with corresponding donor amino acids. The donor framework may be a mature or germline human antibody framework sequence or consensus sequence. Guidance on methods for phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247: 1306-1310 (1990). Cunningham et al, Science, 244: 1081-1085 (1989). Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994). T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989). Pearson, Methods Mol. Biol. 243:307-31 (1994). Gonnet et al., Science 256: 1443-45 (1992).

[0056] Antibodies, or their antigen-binding moieties, can be derivatized or linked to other functional molecules. For example, an antibody can be functionally linked (by chemical coupling, gene fusion, non-covalent interactions, etc.) to one or more other molecular entities, such as another antibody, a detectable drug, a cytotoxic agent, a pharmaceutical, a protein or peptide capable of mediating association with another molecule (e.g., streptavidin core region or polyhistidine tag), an amino acid linker, a signal sequence, an immunogenicity carrier, or a ligand useful for protein purification, such as glutathione-S-transferase, a histidine tag, and Staphylococcus protein A. A single derivatized protein is produced by crosslinking two or more proteins (of the same or different types). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide) or homobifunctional (e.g., disuccinimidylsperiphosphate) linkers having two different reactive groups separated by a suitable spacer. Such linkers are available from Pierce Chemical Company, Rockford, 111. Useful detectable agents that can derivatize (or label) proteins include fluorescent compounds, various enzymes, prosthetic groups, luminescent substances, bioluminescent substances, and radioactive substances. Exemplary fluorescent detectable agents, not limited to these, include fluorescein, fluorescein isothiocyanate, rhodamine, and phycoerythrin. Proteins or antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, and glucose oxidase. Proteins can also be derivatized with prosthetic groups (e.g., streptavidin / biotin and avidin / biotin).

[0057] Nucleic acids encoding functionally active variants of the antibodies or their antigen-binding moieties are also included in the present invention. These nucleic acid molecules can hybridize with any of the antibodies or their antigen-binding moieties under medium-stringency, high-stringency, or very high-stringency conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY 6.3.1–6.3.6, 1989 (incorporated herein by reference). The specific hybridization conditions referred to herein are as follows: 1) moderate stringency hybridization conditions: one or more washes with 6 X SSC at approximately 45°C, followed by 0.2 X SSC at 60°C in 0.1% SDS; 2) high stringency hybridization conditions: one or more washes with 6 X SSC at approximately 45°C, followed by 0.2 X SSC at 65°C in 0.1% SDS; and 3) very high stringency hybridization conditions: one or more washes with 0.5 M sodium phosphate at 65°C in 7% SDS, followed by 0.2 X SSC at 65°C in 1% SDS.

[0058] The nucleic acid encoding the antibody or its antigen-binding portion of the present invention can be introduced into an expression vector that can be expressed in a suitable expression system, and the expressed antibody or its antigen-binding portion can subsequently be isolated or purified. Optionally, the nucleic acid encoding the antibody or its antigen-binding fragment of the present invention can be translated in a cell-free translation system. U.S. Patent No. 4,816,567. Queen et al, Proc Natl Acad Sci USA, 86: 10029-10033 (1989).

[0059] The antibody or its antigen-binding portion of the present invention can be produced by host cells transformed with DNA encoding the light and heavy chains (or a portion thereof) of the desired antibody. The antibody can be isolated and purified from these culture supernatants and / or cells using standard techniques. For example, host cells can be transformed with DNA encoding the light chain, heavy chain, or both of the antibody. Recombinant DNA techniques can also be used to remove a portion or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding, such as the constant region.

[0060] The nucleic acids of the present invention can be expressed in a variety of suitable cells, including prokaryotic and eukaryotic cells, such as bacterial cells (e.g., Escherichia coli), yeast cells, plant cells, insect cells, and mammalian cells. Many mammalian cell lines are known in the art and include immortalized cell lines available from the American Cell Culture and Cell Lineage Preservation Center (ATCC). Non-limiting examples of cells include, but are not limited to, parental cells, monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Derby bovine kidney (MDBK), myeloma and lymphoma cell derivatives and / or engineered variants, and all cell lines of mammalian origin or mammalian-like characteristics. Engineered variants include, for example, glycan profile modifications and / or site-specific integration site derivatives.

[0061] The present invention also provides cells comprising nucleic acids as described herein. The cells may be hybridomas or transfectants.

[0062] Alternatively, the antibody or its antigen-binding moiety of the present invention can be synthesized by solid-phase methods well known in the art. (References: Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton and RC Sheppard, published by IRL at Oxford University Press (1989). Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. MW Pennington and BM Dunn), chapter 7. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984). G. Barany and RB Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 1 and Vol. 2, Academic Press, New York, (1980), pp. 3-254. M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984).)

[0063] A “humanized” version of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient’s hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further improve the performance of the antibody. Generally, the humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. The humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically that of the human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also see the following reviews and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0064] As used herein, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains whose sequences are hypervariable and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Numerous descriptions of hypervariable regions are used and are incorporated herein. The Kabat complementarity-determining region (CDR), based on sequence variability, is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0065] A “framework” or “FW” residue is a variable domain residue other than a hypervariable region residue as defined herein.

[0066] "Kabat variable domain residue numbering" or "Kabat amino acid position numbering," and its variations, refer to the numbering system used for heavy chain or light chain variable domains in antibody compilations in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions of FR or HVR in the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a by Kabat) and a residue inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c by Kabat). The Kabat numbering of residues in a particular antibody can be determined by alignment of the antibody sequence with a homology region of a "standard" Kabat-numbered sequence.

[0067] "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0068] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, where the heavy chain variable domain (VH) is linked to the light chain variable domain (VL) within the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described more fully, for example, in EP 404,097; WO93 / 1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0069] A “human antibody” is one that has an amino acid sequence corresponding to that of an antibody produced by a human, and / or is prepared using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0070] Affinity-matured antibodies have one or more modifications to one or more of their HVRs, resulting in improved antibody affinity to an antigen compared to parent antibodies without these modifications. In one embodiment, affinity-matured antibodies have nanomolar or picomolar affinity to a target antigen. Affinity-matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0071] A "blocking" antibody or "antagonist" antibody inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0072] As used herein, "agonist antibody" is an antibody that mimics at least one of the functional activities of the target polypeptide.

[0073] "Disorder" is any condition that would benefit from treatment with the antibodies of the present invention. This includes chronic and acute disorders or diseases, including pathological conditions that make mammals susceptible to the disorder in question. Non-limiting examples of disorders treated herein include cancer.

[0074] The terms "proliferative disorder" and "proliferative disorder" refer to disorders related to a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0075] As used herein, “tumor” refers to the proliferation and growth of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive when used herein.

[0076] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by dysregulated cell proliferation / growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include lung cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0077] As used herein, “treatment” refers to a clinical intervention in an attempt to alter the natural course of an individual or cell being treated, which may be carried out for prevention or during the course of a clinicopathological condition. Desired effects of treatment include prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention or reduction of inflammation and / or tissue / organ damage, slowing of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of a disease or disorder.

[0078] The “individual” or “subject” is a vertebrate. In some embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock (such as cattle), sporting animals, pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, the vertebrate is a human.

[0079] For therapeutic purposes, “mammals” refers to all animals classified as mammals, including humans, livestock and farm animals, as well as zoo animals, sports animals, or pet animals (such as dogs, horses, cats, and cows). In some embodiments, the mammal is a human.

[0080] "Effective dose" refers to the amount that is effective in the required dosage and duration to achieve the desired therapeutic or prophylactic outcome.

[0081] The “therapeutic effective dose” of the substance / molecule of the present invention may vary depending on factors such as the disease state, age, sex, and body weight of the individual, as well as the ability of the substance / molecule to induce a desired response in the individual. The therapeutic effective dose is also the amount in which any toxic or harmful effects of the substance / molecule are outweighed by the therapeutically beneficial effects. The “prophylactic effective dose” refers to the amount that is effective in the required dosage and duration to achieve the desired prophylactic outcome. Typically, but not always, the prophylactic effective dose will be less than the therapeutic effective dose, as prophylactic doses are administered to subjects before or at an earlier stage of the disease.

[0082] "Combination" refers to combination therapy, which would be an amount of antibody-drug conjugate and / or other biological or chemical drugs that, when administered together (as co-administration and / or co-formulation) sequentially or simultaneously on the same or different days during a treatment cycle, is therapeutically effective and has a synergistic effect that is therapeutically additive or greater.

[0083] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by dysregulated cell proliferation. “Tumor” includes one or more cancer cells. Examples of cancer include, but are not limited to, lung cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0084] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with the function of cells and / or causes cell destruction. This term is intended to include toxins such as low molecular weight toxins or toxins with enzymatic activity derived from bacteria, fungi, plants or animals, including radioactive isotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 C, and radioisotopes of lutetium-177, strontium-89 and samarium ( 153 Sm), chemotherapeutic agents, and toxins such as synthetic analogs and their derivatives, including low molecular weight toxins or toxins with enzymatic activity derived from bacteria, fungi, plants or animals.

[0085] The term "photodynamic therapy (PDT)", also called photochemotherapy, is a form of phototherapy that involves light and a photosensitizing chemical substance and is used in combination with molecular oxygen to induce cell death (phototoxicity). It is clinically used to treat a wide range of conditions including wet age-related macular degeneration, psoriasis, and atherosclerosis, and has also shown some effectiveness in antiviral treatments including herpes. It also treats malignant cancers including head and neck, lung, bladder, skin and prostate cancers (Wang, SS et al. Cancer Journal. 8 (2): 154-63. 2002). "Photodynamic therapy agents" are selected from Photofrin, Laserphyrin, aminolevulinic acid (ALA), silicon phthalocyanine Pc 4, m-tetrahydroxyphenylchlorin (mTHPC), chlorin e6 (Ce6), Allumera, Levulan, Foscan, Metvix, Hexvix, Photochlor, Photosens, Photrex, Lumacan, Visonac, Amphinex, verteporfin, Purlytin, ATMPn, zinc phthalocyanine (ZnPc), protoporphyrin IX (PpIX), pyro pheophorbide a (PPa) or pheophorbide a (PhA).

[0086] "Chemotherapy agents" are compounds useful in the treatment of cancer. Examples of chemotherapy agents include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), meltansine (also called DM1), anthracyclines, pyrrolobenzodiazepines, α-amanitin, tubricin, benzodiazepines, erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), and PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), ronafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Alkylating agents such as Sugen, thiotepa, and CYTOXAN® cyclophosphamide; alkyl sulfonic acid esters such as busulfan, improsulfan, and biposulfan; aziridines such as benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylamelamines including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistostatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin;Duocalmycin (including synthetic analogues, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongstatin; chlorambucil, chlornafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine hydrochloride, melphalan, nobembiquin, fenestrine, prednimastine, trophosphamide, nitrogen mustards such as uracil mustard; nitrosoureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimustine; engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1) (Angew Chem. Intl. Ed. Engl. (1994)) 33:183-186); Antibiotics such as dynemycin including dynemycin A; bisphosphonates such as clodronate; esperamycin; and neocardinostatin chromophores and related chromoprotein enediin antibiotic chromophores), acrasinomycin, actinomycin, outramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pylori) Mitomycins such as nodoxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxlysine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; and bestrabusil ;Bisanthren;Edatrexate;Defofamine;Demecolsin;Diazicon;Elhornitine;Erptinium acetate;Epotilon;Etoglucid;Gallium nitrate;Hydroxyurea;Lentinan;Ronidinin;Mytansinoids such as Mytansin and Ansamitosin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitraerine;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK (Registered Trademark) Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Veraculine A, Loridine A and Angidin); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremofoll-Free, Albumin-Modified Nanoparticle Formulation of Paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate;Platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; XELODA; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®, Roche); and any of the above pharmaceutically acceptable salts, acids, or derivatives.

[0087] Furthermore, the definition of "chemotherapeutic agents" also includes: (i) anti-hormone agents that modulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), e.g., tamoxifen (including NOLVADEX® tamoxifen), raloxifen, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and FARESTON toremifene; (ii) adrenal glands Aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestan, fadrozol, RIVISOR® borozole, FEMARA® letrozole, and ARIMIIDEX® anastrozole; (iii) flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc. (iv) Antiandrogens; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); (v) Aromatase inhibitors; (v) Protein kinase inhibitors; (vi) Lipid kinase inhibitors; (vii) Antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in adherent cell proliferation, e.g., PKC-α, Ralf, and H-Ras; (viii) Ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; ( ix) Vaccines such as gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and (xi) salts, acids, or derivatives of any of the above that are acceptable as pharmaceuticals.

[0088] Protein kinase inhibitors include tyrosine kinase inhibitors that inhibit the tyrosine kinase activity of tyrosine kinases such as the ErbB receptor to some extent. Examples of tyrosine kinase inhibitors include EGFR-targeting drugs such as: (i) antibodies that bind to EGFR, MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (US Patent No. 4,943,533, see Mendelsohn et al.) and their variants, such as chimeric 225 (C225 or cetuximab; ERBITUX®, Imclone) and remodeled human 225 (H225) (WO 96 / 40210, Imclone Systems Inc.); antibodies that bind to type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind to EGFR (US Patent No. 5,891,996); and ABX-EGF (WO (ii) Human antibodies that bind to EGFR, such as 98 / 50433); (ii) Anti-EGFR antibodies conjugated with cytotoxic agents (EP 659439A2); and quinazolines such as ZD1839 or gefitinib (IRESSA®; Astra Zeneca), erlotinib HCl (CP-358774, TARCEVA®; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen), PD 153035, 4-(3-chloroanilino)quinazoline, pyridopyrimidines, pyridopyrimidines, pyrrolopyrimidines, e.g., CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide), tyrophostine containing the nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to nucleic acids encoding ErbB); quinoxaline (US Patent No. 5,804,396); triphostine (US Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG);Pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis / Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semacsanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); or U.S. Patent No. 5,804,396; WO 99 / 09016 (American Cyanamid); WO 98 / 43960 (American Cyanamid); WO 97 / 38983 (Warner Lambert); WO 99 / 06378 (Warner Lambert); WO Small molecules that bind to EGFR, such as those described in 99 / 06396 (Warner Lambert); WO 96 / 30347 (Pfizer, Inc); WO 96 / 33978 (Zeneca); WO 96 / 3397 (Zeneca); and WO 96 / 33980 (Zeneca).

[0089] "Anti-angiogenic agents" refer to compounds that block or, to some extent, inhibit the development of blood vessels. Anti-angiogenic factors may be small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis. An example of an anti-angiogenic agent is an antibody that binds to vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®, Genentech).

[0090] The term "cytokine" is a general term for proteins released by a population of cells that act on other cells as intercellular mediators. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and-β; Müllerian duct inhibitors; mouse gonadotropin-related peptides; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; and TGF-α and TGF-β. These include transforming growth factors (TGF); insulin-like growth factors-I and-II; erythropoietin (EPO); bone induction factors; interferons such as interferon-α, -β, and -γ; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligands (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell cultures, and bioactive equivalents of naturally occurring sequence cytokines.

[0091] As used in this application, the term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that exhibits lower cytotoxicity to tumor cells compared to the parent drug, and can be enzymatically activated or converted to a more active parent form. See, for example, Wilman, “Prodrugs in Cancer Chemotherapy,” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs that can be converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

[0092] Liposomes are vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for delivering drugs to mammals (such as anti-ErbB2 antibodies disclosed herein and, optionally, chemotherapeutic agents). The components of liposomes are typically arranged in a bilayer, similar to the lipid arrangement of biological membranes.

[0093] As used herein, the phrase “medically acceptable salt” refers to a medically acceptable organic or inorganic salt of ADC. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, saccharates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have multiple charged atoms in its structure. If multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0094] A "pharmaceutically acceptable solvate" refers to the association of one or more solvent molecules with an ADC. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0095] As used in this application, the term "imaging agent" refers to fluorophores, dyes, MRI contrast agents, or radionuclides. These agents are used in diagnostic assays for the therapeutic administration of ADC compounds.

[0096] A method for selecting patients for cancer treatment by imaging, the method being: (a) administering an effective dose of ADC to the patient; and (b) To detect the reporting signal of imaging agents in patients; Includes, Here, the imaging agent is a fluorophore, dye, MRI contrast agent, or radionuclide; and Here, the reporting signal is detected visually or instrumentally.

[0097] General characteristics of exemplary antibody-drug conjugates The compounds of the present invention include those useful for anticancer activity. In particular, the compounds contain an antibody covalently bound by a linker to a drug moiety that has cytotoxic or cell proliferation inhibitory effects when not bound to an antibody. The biological activity of the drug moiety is therefore regulated by binding to the antibody. The antibody-drug conjugates (ADCs) of the present invention can selectively deliver an effective amount of a cytotoxic agent to tumor tissue, thereby achieving higher selectivity, i.e., a lower effective dose.

[0098] Antibody-drug conjugates (ADCs) are formulated with formula I: Ab-(LD) n (I) Or it may be represented by a pharmaceutically acceptable salt or solvate thereof, where: Ab is an antibody that binds to TROP2, or an antibody that binds to one or more tumor-associated antigens or cell surface receptors; n is the drug-antibody ratio (DAR), ranging from 1 to 8.

[0099] An antibody-drug conjugate (ADC) contains an antibody covalently bound to one or more MMAE moieties by a linker. ADCs are represented by formula I: Ab-(LD) n (I) It can be represented as follows, where one or more exatecan drug moieties (D) are covalently bound to an antibody (Ab) by L. Ab is an antibody that targets TROP2, or an antibody that binds to one or more tumor-associated antigens or cell surface receptors. The linker L may be extracellular, i.e., stable outside the cell.

[0100] In another embodiment, the linker comprises maleimide or a derivative thereof bonded to the thio group of the antibody. In particular, the linker is 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (MCCa).

[0101] In one embodiment, a substantial amount of the drug portion remains uncleaved from the antibody until the antibody-drug conjugate enters a cell that has a cell surface receptor specific to the antibody of the antibody-drug conjugate, and the drug portion is cleaved from the antibody when the antibody-drug conjugate enters the cell.

[0102] In another embodiment, the ADC specifically binds to TROP2. The ADC may inhibit the proliferation of tumor cells that express TROP2.

[0103] In another embodiment, the antibody (Ab) of formula I is a human, chimeric, or humanized antibody.

[0104] In another embodiment, the anti-TROP2 antibody is selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamab, or sacituzumab.

[0105] Another aspect of the present invention is a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0106] Another embodiment provides a pharmaceutical combination agent comprising a compound of formula I and a second compound having anticancer properties or other therapeutic effects.

[0107] Another aspect includes the diagnostic and therapeutic use of the compounds and compositions disclosed herein.

[0108] Another embodiment is a method for killing tumor cells or cancer cells or inhibiting their growth, comprising treating cells with an amount effective in killing tumor cells or cancer cells or inhibiting their growth, an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof.

[0109] Another embodiment is a method for treating cancer, comprising administering a formulation of the compound of formula I to a patient. One method is for the treatment of cancer characterized by TROP2 expression in mammals. Mammals are, optionally, unresponsive or poorly responsive to treatment with unbound anti-TROP2 antibodies. The method comprises administering a therapeutically effective amount of antibody-drug conjugate compound to a mammal.

[0110] Another embodiment is a method for inhibiting the proliferation of tumor cells expressing TROP2, comprising administering to a patient an antibody-drug conjugate compound that specifically binds to the growth factor receptor and a chemotherapeutic agent, wherein the antibody-drug conjugate and the chemotherapeutic agent are each administered in an amount effective in inhibiting the proliferation of tumor cells in the patient.

[0111] Another embodiment is a method for treating a human patient who is susceptible to or diagnosed with a disorder characterized by TROP2 expression, comprising administering a combination of an antibody-drug conjugate compound of formula I and a chemotherapeutic agent.

[0112] Another embodiment is an assay method for detecting cancer cells, comprising exposing cells to an antibody-drug conjugate compound and determining the degree to which the antibody-drug conjugate compound binds to the cells.

[0113] Another aspect relates to a method for screening ADC drug candidates for the treatment of a disease or disorder characterized by TROP2 expression.

[0114] Another embodiment includes a manufactured product, i.e., a kit, comprising an antibody-drug conjugate, a container, and a package insert or label indicating the treatment.

[0115] Another embodiment includes a method for treating a disease or disorder characterized by overexpression of TROP2 in a patient using an antibody-drug conjugate compound.

[0116] Another embodiment includes methods for preparing, synthesizing, conjugating, and purifying antibody-drug conjugate compounds and intermediates for the preparation, synthesis, and conjugation of antibody-drug conjugate compounds.

[0117] ADC: Antibody The antibody unit (Ab-) of formula I includes, within its range, any unit of antibody that binds to, reactively associates with, or forms a complex with a receptor, antigen, or other receptor site associated with a particular target cell population. The antibody may be any protein or protein-like molecule that binds to, forms a complex with, or reacts with a portion of the cell population to be therapeutically or otherwise biologically modified. In one embodiment, the antibody unit acts to deliver a mytansinoid drug portion to the particular target cell population to which the antibody unit reacts. Such antibodies include, but are not limited to, high molecular weight proteins such as full-length antibodies and antibody fragments.

[0118] The antibodies constituting the antibody-drug conjugate of the present invention preferably retain the antigen-binding ability of their native wild-type counterparts. Therefore, the antibodies of the present invention can bind to antigens, preferably specifically.

[0119] In this specification, the term “antibody” is used in its broadest sense, specifically encompassing monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may originate from mouse, human, humanized, chimeric, or other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). Target antigens generally have numerous binding sites, also called epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have multiple corresponding antibodies. Antibodies include full-length immunoglobulin molecules or molecules containing an immunologically active portion of a full-length immunoglobulin molecule, i.e., an antigen-binding site that immune-specifically binds to an antigen or part of an antigen of a target of interest, such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass of an immunoglobulin molecule. Immunoglobulins may originate from any species. However, in one embodiment, the immunoglobulins are derived from humans, mice, or rabbits.

[0120] For example, antibodies may be full-length or include, but are not limited to, fragments of antibodies having antigen-binding moieties, such as Fab, F(ab')2, Fab', F(ab)', Fv, single-chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, and dAb fragments (e.g., Ward et al., Nature, 341:544-546 (1989)), isolated CDRs, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single-chain antibodies produced by linking antibody fragments using recombinant methods or synthetic linkers are also included in the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

[0121] All antibody isotypes, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, or IgE (all classes and subclasses are included in this invention), are included in this invention. The antibody or its antigen-binding moiety may be a mammalian (e.g., mouse, human) antibody or its antigen-binding moiety. The antibody light chain may be kappa or lambda type.

[0122] The variable region of the antibody or its antigen-binding fragment of the present invention may be non-human or human-derived. The framework of the antibody or its antigen-binding portion of the present invention may be human, humanized, non-human (e.g., a mouse framework modified to reduce antigenicity in humans), or synthetic (e.g., a consensus sequence).

[0123] In one embodiment, the antibody of the present invention, or its antigen-binding moiety, includes at least one heavy chain variable region and / or at least one light chain variable region.

[0124] The antibody or antigen-binding fragment of the present invention is attached to TROP2 for about 10-7 Less than M, approximately 10 -8 Less than M, approximately 10 -9 Less than M, approximately 10 -10 Less than M, approximately 10 -11 Less than M, or about 10 -12 It specifically binds with a dissociation constant (KD) of less than M. In one embodiment, the antibody or its antibody-binding moiety is 1 to 10 x 10 -9 The following dissociation constant (KD) is observed. In another embodiment, Kd is determined by surface plasmon resonance.

[0125] The antibodies constituting the antibody-drug conjugate of the present invention preferably retain the antigen-binding ability of their native wild-type counterparts. Therefore, the antibodies of the present invention can bind to antigens, preferably specifically. Such antigens include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell growth regulators, molecules involved in (e.g., functionally known or suspected) tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis, and molecules involved in (e.g., functionally known or suspected) angiogenesis. Tumor-associated antigens may be cluster differentiation factors (i.e., CD proteins). Antigens to which the antibodies of the present invention can bind may be members of one subset of the above categories, where other subsets of the said categories include other molecules / antigens having different characteristics (with respect to the antigen of interest).

[0126] In one embodiment, the antibody of the antibody-drug conjugate (ADC) specifically binds to TROP2.

[0127] In some embodiments, the antibody or its antigen-binding moiety includes, for example, a variable heavy chain and / or variable light chain of an anti-TROP2 antibody (R4702), as shown in Table 1.

[0128] In relevant embodiments, the exemplary antibody or its antigen-binding moiety includes, for example, the variable heavy chain CDR and / or variable light chain CDR of an anti-TROP2 antibody (R4702). Exemplary variable heavy chain and variable light chain CDRs and frameworks from these hybridoma clones are shown in Table 1.

[0129] [Table 1]

[0130] Antibodies having variable heavy chain regions and variable light chain regions that are at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homology to the variable heavy chain region and variable light chain region of an antibody bind to TROP2. Homology may exist at either the amino acid or nucleotide sequence level.

[0131] ADC targeting TROP2 One aspect of the present invention features a novel ADC (OBI-992) specific to TROP2.

[0132] Any of the exemplary antibodies described herein may be full-length antibodies or their antigen-binding fragments. In some examples, the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment. In some examples, the antibody is a human antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody.

[0133] Any of the exemplary antibodies described herein has one or more of the following characteristics: (a) being a recombinant antibody, monoclonal antibody, chimeric antibody, humanized antibody, human antibody, antibody fragment, bispecific antibody, monospecific antibody, monovalent antibody, IgG1 antibody, IgG2 antibody, or antibody derivative; (b) being a human, mouse, humanized, or chimeric antibody, antigen-binding fragment, or antibody derivative; (c) being a single-chain antibody fragment, multimer, Fab fragment, and / or immunoglobulin of IgG, IgM, IgA, IgE, IgD isotype and / or subclass thereof; (d) having one or more of the following characteristics: (i) being a cancer cell (ii) mediates ADCC and / or CDC; (iii) induces and / or promotes apoptosis in cancer cells; (iv) induces and / or promotes phagocytosis in cancer cells; and / or (v) induces and / or promotes the release of cytotoxic agents; (e) specifically binds to tumor-associated carbohydrate antigens, which are tumor-specific carbohydrate antigens; (f) does not bind to antigens expressed on non-cancer cells, non-tumor cells, benign cancer cells and / or benign tumor cells; and / or (g) specifically binds to tumor-associated carbohydrate antigens expressed on cancer stem cells and normal cancer cells.

[0134] Preferably, the binding of the antibody to each antigen is specific. The term “specific” is generally used to mean a situation in which one member of a binding pair does not show significant binding to any molecule other than its specific binding partner, and has, for example, less than about 30%, preferably less than 20%, 10%, or 1%, cross-reactivity with any other molecule other than those specified herein.

[0135] Antibody production Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to cloned cell lines that produce a single type of antibody, uses cells from various species, including mice (murine), hamsters, rats, and humans. Other methods for preparing MAbs, including chimeric and humanized antibodies, use genetic engineering, i.e., recombinant DNA technology.

[0136] Polyclonal antibodies can be produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for any naturally occurring mutations that may be present in small amounts.

[0137] Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, (1984) J. Immunol., 133:3001, and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The culture medium in which hybridoma cells are proliferating is assayed for the production of monoclonal antibodies directed against antigens. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by scachard analysis as described in Munson et al (1980) Anal. Biochem. 107:220.

[0138] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA is placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that otherwise do not produce antibody proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells (US 2005 / 0048572; US 2004 / 0229310). Reviews on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al (1993) Curr. Opinion in Immunol. 5:256-262 and Pluckthun (1992) Immunol. Revs. 130:151-188.

[0139] In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the technique described in McCafferty et al (1990) Nature 348:552-554; Clackson et al (1991) Nature 352:624-628; and Marks et al (1991) J. Mol. Biol., 222:581-597, respectively, describe the isolation of mouse and human antibodies using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al (1992) Bio / Technology 10:779-783), as well as combination infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al (1993) Nuc. Acids. Res. 21:2265-2266). Therefore, these techniques represent a viable alternative to conventional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0140] DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (U.S. Patent No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81:6851), or by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

[0141] Typically, such non-immunoglobulin polypeptides are used in place of the constant domain of an antibody, or they are used in place of the variable domain of one antigen-binding site of an antibody to create a chimeric bivalent antibody that includes one antigen-binding site having specificity for a certain antigen and another antigen-binding site having specificity for a different antigen.

[0142] ADC: Linker Exemplary ADC linker A suitable exemplary linker for ADCs is described, for example, in U.S. Patent No. 7,595,292 (WO2005 / 007197). The full description of linkers is incorporated herein by reference. Linker L conjugates the antibody to the drug moiety via a disulfide-free covalent bond. The linker is a bifunctional or polyfunctional moiety that can be used to link one or more drug moieties (D) to an antibody unit (Ab) to form an antibody-drug conjugate (ADC) of formula I. Antibody-drug conjugates (ADCs) can be easily prepared using linkers having reactive functional groups for binding to the drug and antibody. The cysteine ​​thiol or amine, or amino acid side chain such as lysine, of the antibody (Ab) can form bonds with the functional groups of the linker reagent, drug moiety, or drug-linker reagent.

[0143] The linker is preferably stable extracellularly. Prior to transport or delivery to cells, the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e., the antibody remains bound to the drug moiety. The linker is stable outside the target cell and can be cleaved intracellularly at some effective rate. An effective linker (i) maintains the specific binding properties of the antibody; (ii) enables intracellular delivery of the conjugate or drug moiety; (iii) remains stable and intact, i.e., uncleaved, until the conjugate is delivered or transported to its target site; and (iv) maintains the cytotoxic, cell-killing, or cell-proliferating inhibitory effects of the mytansinoid drug moiety. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC, and separation / analysis techniques such as LC / MS.

[0144] The covalent bonding of antibody and drug moieties requires the linker to have two reactive functional groups, i.e., be divalent in the sense of reactivity. Divalent linker reagents useful for conjugating two or more functionally or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are known, and methods for preparing the resulting complexes have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0145] An exemplary ADC linker may contain a biologically active compound of general formula II, where one of X and X' represents a polymer (particularly a toxin) and the other represents a hydrogen atom; each Q independently represents a linking group; W represents an electron-withdrawing moiety or a moiety that can be prepared by reduction of an electron-withdrawing moiety; or, if X' represents a polymer, XQW- together may represent an electron-withdrawing group; furthermore, if X represents a polymer, X' and the electron-withdrawing group W may form a ring with an intervening atom; Z 1 and Z 2 Each of these independently represents a group derived from a biological molecule, each of which is linked to A and B via a nucleophilic moiety; or Z 1 and Z 2Together, they represent groups derived from a single biological molecule linked to A and B via two nucleophilic moieties; A is C 1-5 It is an alkylene or alkenylene chain; B is a bond or C 1-4 The alkylene or alkenylene chain is formed, preferably by linking a suitable polymer to a suitable biologically active molecule via a disulfide crosslink or via a nucleophilic group in the molecule.

[0146] [ka]

[0147] In one embodiment, the disclosure provides a protein-polymer complex of formula III.

[0148] [ka] Here, X is a homo- or copolymer (especially toxins) selected from the group consisting of polyalkylene glycol, polyvinylpyrrolidone, polyacrylate, polymethacrylate, polyoxazoline, polyvinyl alcohol, polyacrylamide, polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(orthoester), polycarbonate, poly(iminocarbonate), polyamide, divinyl ether-maleic anhydride and styrene-maleic anhydride copolymers, polysaccharides, and polyglutamic acid; Q is a homo- or copolymer (especially toxins) selected from the group consisting of directly bonded, alkylene, optionally substituted aryl, and optionally substituted heteroaryl. A selected linking group, where alkylene, aryl, or heteroaryl may be terminated or interrupted by one or more oxygen atoms, sulfur atoms, keto groups, -O-CO- groups, -CO-O- groups, or -NR groups where R is an alkyl or aryl group; W is selected from the group consisting of keto groups, ester groups, sulfone groups, reduced keto groups, reduced ester groups, and reduced sulfone groups; X'-Q are hydrogen; A is a C1-5 alkylene or alkenylene chain; B is a link or a C1-4 alkylene or alkenylene chain; Z is a single protein linked to A and B via two thiol groups produced by the reduction of disulfide crosslinks in the protein.

[0149] Activity assay demonstrating the efficacy of exemplary ADCs The ADC(OBI-992) of the present invention can be characterized for their physical / chemical properties and biological functions by various assays known in the art.

[0150] The antibodies of the present invention, or their antigen-binding fragments, variants, or derivatives, may also be described or specified in relation to their binding affinity to antigens. The affinity of an antibody to a carbohydrate antigen can be experimentally determined using any suitable method (see, for example, Berzofsky et al, “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein). The measured affinity of a particular antibody-carbohydrate antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, the measurement of affinity and other antigen-binding parameters (e.g., KD, Ka, Ki) is preferably performed using standardized solutions of the antibody and antigen, and standardized buffers.

[0151] The antibodies or their antigen-binding moieties of the present invention have therapeutic, prophylactic, and / or diagnostic utility in vitro and in vivo. For example, these antibodies can be administered to cultured cells, for example, in vitro or ex vivo, or to subjects, for example, in vivo, to treat, inhibit, prevent recurrence of, and / or diagnose cancer.

[0152] Purified antibodies can be further characterized by a range of assays, including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high-performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0153] If necessary, the antibodies are analyzed for their biological activity. In some embodiments, the antibodies of the present invention are tested for their antigen-binding activity. Antigen-binding assays known in the art and available herein include, but are not limited to, any direct or competitive binding assays using techniques such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, fluorescence immunoassay, chemiluminescence immunoassay, nanoparticle immunoassay, aptamer immunoassay, and protein A immunoassay.

[0154] Humanized antibodies This invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “import” residues and are typically taken from an “import” variable domain. Humanization can be carried out by substituting a hypervariable region sequence in place of the corresponding sequence of a human antibody, essentially following the methods of Winter and collaborators (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536). Thus, such a “humanized” antibody is a chimeric antibody (U.S. Patent No. 4,816,567) in which substantially less of the intact human variable domain is replaced with the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which several hypervariable region residues and possibly several FR residues are replaced with residues from similar sites in rodent antibodies.

[0155] The selection of both light and heavy chain human variable domains used in the production of humanized antibodies can be crucial for reducing antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequences most closely resembling those of rodents are then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another method uses a specific framework derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0156] It is more generally desirable for antibodies to be humanized while retaining high affinity for antigens and other desirable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parent sequence and the humanized sequence using three-dimensional models of the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional structures of selected candidate immunoglobulin sequences. By examining these representations, it becomes possible to analyze the likely roles of residues in the function of the candidate immunoglobulin sequence, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that desired antibody properties, such as increased affinity for the target antigen(s), are achieved. Generally, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0157] Purpose The ADC(OBI-992) of the present invention can be used, for example, in vitro, ex vivo, and in vivo therapeutic methods. The ADC(OBI-992) of the present invention can be used as an antagonist to partially or completely block specific antigen activity in vitro, ex vivo, and / or in vivo. Thus, the ADC(OBI-992) of the present invention can be used, for example, in cell cultures containing an antigen, in human subjects, or in other mammalian subjects (e.g., chimpanzees, baboons, marmosets, cynomolgus monkeys and rhesus monkeys, pigs, or mice) that have antigens to which the ADC(OBI-992) of the present invention cross-reacts. In one embodiment, the ADC(OBI-992) of the present invention can be used to inhibit antigen activity by contacting the ADC(OBI-992) with the antigen so that the antigen activity is inhibited. In one embodiment, the antigen is a human protein molecule.

[0158] In one embodiment, the ADC(OBI-992) of the present invention may be used in a method to inhibit an antigen in a subject suffering from a disorder in which antigen activity is harmful, the method comprising administering the ADC(OBI-992) of the present invention to the subject so as to inhibit antigen activity in the subject. In one embodiment, the antigen is a human protein molecule, and the subject is a human subject. Alternatively, the subject may be a mammal expressing an antigen to which the ADC(OBI-992) of the present invention binds. Furthermore, the subject may be a mammal to which the antigen has been introduced (e.g., by administration of the antigen or by expression of an antigen transgene). The ADC(OBI-992) of the present invention may be administered to a human subject for therapeutic purposes. Furthermore, the ADC(OBI-992) of the present invention may be administered to a non-human mammal expressing an antigen (e.g., primates, pigs, or mice) to which the ADC(OBI-992) cross-reacts, for veterinary purposes or as an animal model of a human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic effects of the ADC (OBI-992) of the present invention (e.g., dose and time course studies).

[0159] The ADC (OBI-992) of the present invention may be used alone or in combination with other compositions in treatment. For example, the ADC (OBI-992) of the present invention may be administered co-administered with another antibody and / or adjuvant / therapeutic agent (e.g., steroid). For example, the ADC (OBI-992) of the present invention may be used in combination with anti-inflammatory agents and / or disinfectants in a treatment plan when treating any of the diseases described herein, including cancer, muscle disorders, ubiquitin pathway-related genetic disorders, immune / inflammatory diseases, neurological disorders, and other ubiquitin pathway-related disorders. Such combination therapies include co-administration (where two or more drugs are contained in the same or separate formulations) and separate administration, in which case the ADC (OBI-992) of the present invention may be administered before and / or after the administration of adjuvant therapy or multiple therapies.

[0160] The ADC (OBI-992) of the present invention may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and, if local treatment is desired, intrafocal administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Furthermore, the ADC (OBI-992) is appropriately administered by pulse infusion, particularly while gradually decreasing the dose of the ADC (OBI-992). Dosage may be by any suitable route, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic.

[0161] Therapeutic applications This specification describes a treatment method comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising one or more ADCs (OBI-992) as described herein.

[0162] In some embodiments, the subject requiring treatment (e.g., a human patient) is diagnosed with cancer, suspected of having cancer, or at risk of having cancer. Examples of cancer include, but are not limited to, lung cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

[0163] In preferred embodiments, ADC(OBI-992) can target cancer cells expressing TROP2. In some embodiments, ADC(OBI-992) can target TROP2 on cancer cells. In some embodiments, ADC(OBI-992) can target TROP2 in cancer.

[0164] Treatment results in a reduction in tumor size, elimination of malignant cells, prevention of metastasis, prevention of recurrence, reduction or elimination of disseminated cancer cells, extension of survival time, and / or extension of time to tumor progression.

[0165] In some embodiments, the treatment further includes administering an additional therapy to the subject before, during, or after the administration of ADC (OBI-992). In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiotherapy.

[0166] The method of the present invention is particularly advantageous for treating and preventing early-stage tumors, thereby preventing progression to more advanced stages, resulting in a reduction in morbidity and mortality associated with advanced cancer. The method of the present invention is also advantageous for preventing tumor recurrence or regrowth, such as dormant tumors that persist after removal of the primary tumor, or for reducing or preventing tumor development.

[0167] Subjects treated by the methods described herein may be mammals, more preferably humans. Mammals include, but are not limited to, livestock, sports animals, pets, primates, horses, dogs, cats, mice, and rats. Human subjects requiring treatment may be human patients who have, are at risk of, or are suspected of having cancer, including, but are not limited to, lung cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer. Subjects with cancer may be identified by routine health examinations.

[0168] As used herein, “effective dose” refers to the amount of each activator required to produce a therapeutic effect in a subject, either alone or in combination with one or more other activators. The effective dose varies, as will be recognized by those skilled in the art, depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, the duration of treatment, the nature of any combination therapy, the specific route of administration, and similar factors within the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed without going beyond routine experiments. Generally, it is preferable to use the maximum dose of the individual component or combination thereof, i.e., the maximum safe dose as determined by sound medical judgment. However, it will be understood by those skilled in the art that a patient may insist on a lower dose or a tolerable dose for medical, psychological, or substantially any other reason.

[0169] As used herein, the term “treatment” means the application or administration of a composition comprising one or more activators to a subject having cancer, cancer symptoms, or a predisposition to cancer, for the purpose of curing, restoring, alleviating, reducing, modifying, treating, improving, enhancing, or influencing cancer, cancer symptoms, or a predisposition to cancer.

[0170] The “onset” or “progression” of cancer means the initial signs and / or subsequent progression of cancer. Cancer onset can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to undetectable progression. For the purposes of this disclosure, onset or progression refers to the biological course of the condition. “Onset” includes appearance, recurrence, and emergence. As used herein, “emergence” or “appearance” of cancer includes initial emergence and / or recurrence.

[0171] Depending on the type of disease being treated or the site of the disease, conventional methods known to those skilled in the art of medicine may be used to administer the pharmaceutical composition to a subject. The composition may also be administered via other conventional routes, such as orally, parenterally, inhaled spray, topically, rectally, nasally, cheek, vaginally, or implanted reservoir. As used herein, the term “parenterally” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-sacral, intra-sternal, intrathecal, intrafocal, and intracranial injection or infusion techniques. Furthermore, it may be administered to a subject via an injectable depot administration route, such as using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods.

[0172] The injectable compositions may include various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol). For intravenous injection, water-soluble ADC (OBI-992) can be administered by infusion, injecting a pharmaceutical formulation containing ADC (OBI-992) and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients.

[0173] Administration of antibody-drug conjugate pharmaceuticals Therapeutic antibody-drug conjugates (ADCs) can be administered via any route appropriate to the condition being treated. ADCs are typically administered parenterally, i.e., by infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, bolus, intratumoral injection, or epidural injection (Shire et al (2004) J. Pharm. Sciences 93(6):1390-1402). Therapeutic antibody-drug conjugates (ADCs) are typically prepared for parenteral administration in unit-dose injectable form using a pharmaceutically acceptable parenteral vehicle. Antibody-drug conjugates (ADCs) of the desired purity may be mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

[0174] Acceptable parenteral vehicles, diluents, carriers, excipients, and stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin The materials include proteins such as methyl ester, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). For example, lyophilized anti-ErbB2 antibody preparations are described in WO97 / 04801, which are expressly incorporated herein by reference. Exemplary formulations of ADCs such as trastuzumab-SMCC-DM1 contain approximately 100 mg / ml of trehalose (2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5-triol; C 12 H 22 O 11 (CAS No. 99-20-7) and approximately 0.1% TWEEN® 20 (Polysorbate 20; 2-[2-[3,4-bis(2-hydroxyethoxy)tetrahydrofuran-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl dodecanoate; C 26 H 50 O 10 It contains (CAS number 9005-64-5) at approximately pH 6.

[0175] Therapeutic antibody-drug conjugate (ADC) pharmaceutical formulations may contain certain amounts of unreacted drug portion (D), antibody-linker intermediate (Ab-L), and / or drug-linker intermediate (DL) as a result of excess reagents, impurities, and incomplete purification and separation of by-products during the manufacturing process of the ADC, or hydrolysis or decomposition due to time / temperature during storage of bulk ADC or formulated ADC compositions.

[0176] Active pharmaceutical ingredients can also be encapsulated, for example, by coacervation technology or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0177] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing ADCs, the matrices in the form of films or molded articles such as microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0178] Preparations used for in vivo administration must be sterile, which can be easily achieved by filtration using a sterile filtration membrane.

[0179] The formulations include those suitable for the aforementioned routes of administration. The formulations may be presented in convenient unit dosage forms and may be prepared by any method well known in the field of pharmaceutical technology. The techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methods involve associating the active ingredient with a carrier constituting one or more auxiliary components. Generally, formulations are prepared by homogeneously and closely associating the active ingredient with a liquid carrier, a fine powder solid carrier, or both, and then, if necessary, shaping the product.

[0180] Aqueous suspensions contain the active substance in a mixture with excipients suitable for the preparation of aqueous suspensions. Such excipients include suspending agents, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum, as well as dispersing or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxides and long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), and condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[0181] The pharmaceutical composition of ADC may be in the form of a sterile injectable formulation, such as a sterile injectable aqueous or oily suspension. This suspension may be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents as mentioned above. The sterile injectable formulation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol, or may be prepared as a lyophilized powder. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic saline. Furthermore, sterile fixative oils may be conventionally used as solvents or suspension media. For this purpose, any non-irritating fixative oil, including synthetic monoglycerides or diglycerides, may be used. Furthermore, fatty acids such as oleic acid may also be used in the preparation of injectable formulations.

[0182] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. For example, an aqueous solution intended for intravenous infusion may contain approximately 3 to 500 μg of active ingredient per milliliter of solution to ensure that an appropriate volume is infused at a rate of approximately 30 mL / hour. Subcutaneous (bolus) administration may be performed with a total volume of approximately 1.5 ml or less and a concentration of approximately 100 mg ADC / ml. For ADCs requiring frequent and chronic administration, subcutaneous routes may be employed, such as using pre-filled syringes or auto-injector device technology.

[0183] As a general suggestion, the effective initial dose of ADC administered per dose ranges from approximately 0.01 to 100 mg / kg, i.e., approximately 0.1 to 20 mg / kg of patient body weight per day, with a typical initial range of 0.3 to 15 mg / kg / day for the compound used. For example, a human patient may initially receive approximately 1.5 mg of ADC per kg of patient body weight. The dose may be escalated up to the maximum tolerated dose (MTD). The administration schedule may be approximately every three weeks, but may be more frequent or less frequent depending on the diagnosed condition or response. The dose may be further adjusted during the course of treatment to remain below the MTD, which can be safely administered over several cycles of approximately four or more.

[0184] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the intended recipient's blood; as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

[0185] Oral administration of protein-based therapeutics is generally undesirable due to limited absorption and low bioavailability resulting from hydrolysis or denaturation in the intestines. However, formulations of ADCs suitable for oral administration can be prepared as individual units such as capsules, cachets, or tablets, each containing a predetermined amount of ADC.

[0186] The formulations may be packaged in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized state requiring only the addition of a sterile liquid carrier, such as sterile water for injection, immediately before use. Injectable solutions and suspensions prepared at the time of use are prepared from the sterile powders, granules, and tablets of the types described above. Exemplary unit dosage forms contain the active ingredient in a daily dose, a unit sub-daily dose, or an appropriate fraction thereof.

[0187] The present invention further provides veterinary compositions comprising at least one active ingredient as defined above, together with a veterinary carrier. The veterinary carrier is a material useful for administering the composition and may be a solid, liquid, or gaseous material, which is inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally, or by any other desired route.

[0188] For the prevention or treatment of disease, the appropriate dose of ADC depends on the type of disease being treated, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to antibodies, and the discretion of the attending physician. The molecule is administered to the patient in a single dose or appropriately over a series of treatments. Depending on the type and severity of the disease, approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 to 20 mg / kg) of the molecule is an initial candidate dose for administration to the patient, for example, by one or more separate doses or by continuous infusion. A typical daily dose may be approximately 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. An exemplary dose of ADC administered to a patient is in the range of approximately 0.1 to approximately 10 mg / kg of the patient's body weight.

[0189] For repeated administrations over several days or longer, treatment is continued, depending on the patient's condition, until the desired suppression of disease symptoms occurs. An exemplary dosing regimen includes administering an initial loading dose of approximately 4 mg / kg, followed by a weekly maintenance dose of approximately 2 mg / kg of anti-ErbB2 antibody. Other dosing regimens may also be useful. The progression of this treatment is readily monitored by conventional techniques and assays.

[0190] Combination therapy Antibody-drug conjugates (ADCs) can be combined with a second compound having anticancer properties in a combination drug formulation or a combination therapy regimen. Preferably, the second compound in the combination drug formulation or regimen has complementary activity to the ADC of the combination drug so as not to adversely affect each other.

[0191] The second compound may be a chemotherapeutic agent, cytotoxic agent, cytokine, proliferation inhibitor, antihormone agent, aromatase inhibitor, protein kinase inhibitor, lipid kinase inhibitor, antiandrogen, antisense oligonucleotide, ribozyme, gene therapy vaccine, anti-angiogenic agent, and / or cardioprotective agent. Such molecules are appropriately present in combination in amounts effective for the intended purpose. Pharmaceutical compositions containing ADC may also have therapeutically effective amounts of chemotherapeutic agents such as tubulin formation inhibitors, topoisomerase inhibitors, or DNA binding agents.

[0192] Metabolites are radiolabeled (for example, 14 C or 3 H) ADCs can be prepared and parenterally administered to animals such as rats, mice, guinea pigs, monkeys, or humans at a detectable dose (e.g., greater than approximately 0.5 mg / kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours), and then identified by isolating the conversion products from urine, blood, or other biological samples. These products are readily isolated because they are labeled (others are isolated by the use of antibodies that can bind to epitopes remaining in the metabolites). The structure of the metabolites is determined by conventional methods, e.g., MS, LC / MS, or NMR analysis. Generally, the analysis of metabolites is carried out in the same manner as conventional drug metabolism studies well known to those skilled in the art. The conversion products are useful in diagnostic assays for therapeutic dosing of ADC compounds unless otherwise found in vivo.

[0193] Metabolites include the products of in vivo cleavage of ADCs, where cleavage occurs in any of the bindings linking the drug moiety to the antibody. Therefore, metabolic cleavage can result in a naked antibody or an antibody fragment. Antibody metabolites can be linked to some or all of the linker. Metabolites can also result in the production of the drug moiety or a portion of it. Metabolites of the drug moiety can be linked to some or all of the linker.

[0194] manufactured goods In another embodiment, a product or “kit” is provided containing an ADC and materials useful for treating the disorders described above. The product includes a container and a label or accompanying information on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, or blister packs. Containers may be formed from a variety of materials, such as glass or plastic. The container holds an antibody-drug conjugate (ADC) composition effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). At least one activator in the composition is an ADC. The label or accompanying information indicates that the composition is used to treat a selected condition, such as cancer.

[0195] Without further detailed explanation, those skilled in the art will likely be able to make the most of the present invention based on the above description. Accordingly, some embodiments described below should be construed as merely illustrative and not limit the remainder of this disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referred herein. [Examples]

[0196] Example 1: TROP2 immunohistochemistry (IHC) expression in normal human tissue and tumor tissue 1.1 Materials and equipment 1.1.1 Chemicals / Reagents (a) EnVision® FLEX, High pH, ​​(Link) Kit: (Dako, Cat No. K8002) (b) EnVision FLEX High pH TRS (c) EnVision FLEX Peroxidase-Blocking Reagent (d) EnVision FLEX / HRP (e) Envision FLEX Wash Buffer 20X (f) DAB Substrate Buffer & Chromogen (g) EnVision® FLEX Hematoxylin (Dako, Cat No. K8008) (h) Rabbit isotype control antibody (Abcam, Cat No. ab172730) (i) Primary antibody: Rabbit monoclonal anti-TROP2 antibody, clone SP294 (Abcam, Cat No. ab227690) (j) Antibody diluent containing background reduction component (Dako, Cat No. S3022) (k) Xylene (Leica, Cat No. 3803665) (l) Micromount Mounting Medium (Leica, Cat No. 3801731) 1.1.2 Equipment / Supplies (a) Positively charged slides (Fisherbrand Superfrost Plus Slides or equivalent) (b) Drying oven (c) Antigen retrieval chamber, Dako PT Link (d) Autostainer Link 48 (Dako) 1.1.3 Tissue / Antibody Controls (a) Positive control: SKBR3 cell line block or human TNBC tissue (b) Negative control: Human normal colon tissue (c) Antibody isotype control: Rabbit isotype antibody

[0197] 1.2 Procedure 1.2.1 Reagent Preparation (a) EnVision FLEX High pH TRS: Dilute 30 mL of 50x TRS solution with 1470 mL of deionized water to a final concentration of 1x by a 1:50 dilution. Note: Discard after two uses or after 5 days have passed since dilution. (b) Envision FLEX washing buffer: Dilute 100 mL of 20x washing buffer concentrate with 1900 mL of deionized water to a ratio of 1:20. Note: Diluted solutions are usable at room temperature for one week, or can be stored at 2-8°C for one month. Discard the diluted solution if it is cloudy. (c) Envision FLEX DAB+: Add 1 drop of Liquid DAB+ Chromogen to 1 mL of DAB+ substrate buffer and mix well. Note: The prepared substrate colorant (DAB) is stable for 5 days when stored at 2-8°C. (d) TROP2 antibody: The concentrated antibody is supplied at 0.11 mg / mL. The concentrated antibody is diluted 1:150 with Dako background reduction antibody diluent to a final concentration of 0.73 μg / mL. Note: The working solution will expire one day after preparation. (e) Isotype control antibody: The concentrated antibody is supplied at 1.675 mg / mL. The concentrated antibody is diluted 1:2284 with Dako background reduction antibody diluent to a final concentration of 0.73 μg / mL. Note: The working solution will expire one day after preparation.

[0198] 1.2.2 Slide pretreatment and activation (a) Section the FFPE tissue block to 4-5 μm, mount it on a positively charged slide, and air dry it. Store the slide at room temperature before use. (b) Heat the slides in a dry oven at 60±2℃ for 60 minutes. (c) Create labels for the slides to be stained with TROP2 panels: Under the "New Slides" tab, enter the case number in the "Case Number" field. Under "Slides," select the "TROP2" panel under "Protocol." Click "Add Slide." The default staining is 150 μL per drop zone, applied to the two drop zones closest to the label; this position can be adjusted at the user's discretion. Click "Case Complete" to print the label. Attach the label to the frosted edge of the slide. (d) Fill the PT Link tanks with 1.5 L of 1x working solution EnVision FLEX High pH TRS (Agilent) per tank. Configure the PT Link module for TROP2 as follows: Set the activation temperature to 97°C and the time to 30 minutes. Set both the "Preheat" and "Cool End" temperatures to 65°C. (e) Press "Run" on the PT Link module to preheat the TRS to 65°C (this takes about 20 minutes), then place the slide rack into the PT Link module. (f) Press "Run" on the PT Link module to heat the TRS to the activation temperature of 97°C (this takes about 20 minutes). Once the desired temperature is reached, the slide will be activated at 97°C for 30 minutes. (g) After activation, the slides are cooled to 65°C in the module (this takes about 30 minutes), then removed from the PT Link and placed in a PT rinse station tank filled with 1.5 L of 1x FLEX washing buffer for 5 minutes to equilibrate.

[0199] 1.2.3 Reagent bottle preparation and slide loading (a) Using the reagents from the EnVision® FLEX, High pH Kit, perform the following steps on the Dako Link 48 IHC platform: Note: All reagents / antibodies must be equilibrated to room temperature before immunostaining. All staining steps are performed at room temperature on an Autostainer. (b) In the Workflow tab, highlight the slide to be stained and click the "Reagents" button to check the "Reagents Required" window. For antibody and DAB+ reagent vial preparation: Select User Fill Reagent; select "Fill Bottle" and enter the bottle serial number; select the appropriate vial code (5 mL, 12 mL, or 25 mL); set the fill volume based on the amount of reagent prepared; set the expiration date and enter the lot number; select Save and print the label. (c) Place all necessary reagents in the reagent rack and put it into the Link48 Autostainer. Ensure that the buffer and deionized water bottles are full and the waste container is empty. (d) Lift the slide rack from the PT Link rinse tank, rinse the paraffin off the slides using a squeeze bottle filled with 1x wash buffer, and then load the slide rack into the Dako Autostainer Link48. (e) Select the Instrument tab and click "Prime buffer and water" to prime the buffer first, then the water. Click "Start" and Steiner will scan the barcodes on the slides and reagents. After confirming that all reagents and slides have been scanned correctly, click "Start Run".

[0200] 1.2.4 TROP2 Automated IHC Staining Procedure Note: All staining steps are performed at room temperature on the Autostainer. (a) Rinse the slide with wash buffer, incubate with EnVision FLEX Peroxidase Block in a 300 μL (150 μL x 2 zones) aliquot for 5 minutes, then rinse with FLEX wash buffer. (b) Incubate the slides with primary antibody (isotype control or TROP2 antibody, final concentration 0.73 μg / mL in Dako background reduction antibody dilution) in 300 μL (150 μL x 2 zones) for 60 minutes, then rinse with wash buffer. (c) Incubate the slides with EnVision FLEX HRP in 300 μL (150 μL x 2 zones) for 30 minutes, then rinse with FLEX wash buffer, followed by incubation in FLEX wash buffer for 5 minutes. (d) Visualization was achieved by incubating EnVision FLEX DAB+ with a 300 μL (150 μL x 2 zones) aliquot for 10 minutes, followed by rinsing with wash buffer. (e) Counterstain the slide with EnVision FLEX Hematoxylin in a volume of 300 μL (150 μL x 2 zones) for 5 minutes, then rinse with deionized water. (f) Rinse the slides with washing buffer, incubate for 5 minutes, and then rinse again with deionized water.

[0201] 1.2.5 Run End and Slide Mounting (a) Unload the slides from the Autostainer. Dehydration and clearing are performed manually at stepwise alcohol and xylene stations. (b) Next, manually cover slip the slide with mounting medium.

[0202] 1.2.6 Data Analysis: Semi-Quantitative Evaluation Criteria for Pathologists (a) The percentage and intensity of TROP2 IHC expression are assessed in tumor cells and read by a certified pathologist. TROP2 IHC staining primarily reveals membrane and cytoplasmic expression patterns. The percentage of tumor cells with membrane and / or cytoplasmic TROP2 expression is recorded at each staining intensity (0, 1+, 2+, and 3+). (b) The H-score is used to indicate the TROP2 expression level on tumor tissue. The H-score ranges from 0 to 300 and is calculated by the following formula: H-score = (percentage of low intensity x 1) + (percentage of moderate intensity x 2) + (percentage of high intensity x 3).

[0203] 1.3 Results Figure 1 shows TROP2 IHC expression in normal human tissue and tumor tissue. Figure 1A shows TROP2 expression in tumor tissue of the human breast, lung, cervix, ovary, uterus, prostate, colon, esophagus, pancreas, larynx, stomach, and bladder. Figure 1B further lists the calculated TROP2 H-scores for each organ individually. Furthermore, recent references have also reported that the Trop2 antigen was overexpressed in (a) 58% of oral squamous cell carcinomas; (b) 64% of lung adenocarcinomas, 75% of lung squamous cell carcinomas, and 18% of high-grade lung neuroendocrine neoplasms; (c) 80% of urothelial carcinomas and 71% of prostate cancers; (d) 58.6% of epithelial ovarian cancers and 71.8% of endometrioid carcinomas; (e) 82.5% of papillary thyroid carcinomas; (f) 80% of breast cancers and 88% of TNBCs; (g) 55% of pancreatic cancers; (h) 56% of gastric cancers; and (i) 68.4% of colon cancers (Shutan Liao et al., (2021) Drug Dev Res, 82(8): 1096-1110).

[0204] Example 2: Selection of candidate TROP2 antibodies and topoisomerase inhibitors To determine the binding kinetics of TROP2 antibody to human TROP2, surface plasmon resonance (SPR) experiments were performed using a Biacore 8K instrument (GE Healthcare). TROP2 antibody (10 nM) was covalently immobilized on the surface of a protein A sensor chip using HBS-EP+ buffer, pH 7.4. In essence, the protein A chip binds to the antibody (primarily human) in an orientation-specific manner via its Fc region. The reference flow cell was processed without ligand. The kinetics experiments were performed by 2-fold serial dilutions to obtain a range of TROP2 Ab concentrations in HBS-EP+ buffer, pH 7.4 (0.7, 1.41, 2.81, 5.63, 11.25, 22.5, 45, and 90 nM). For analysis of association and dissociation rates, the same experimental conditions (flow rate 30 μL / min, contact time 150 sec and dissociation time 600 sec, re-contact time 30 sec) were used in each measurement cycle. Chip regeneration was performed by washing with glycine HCl, pH 1.5. The level of interaction on the sensor chip is represented as a change in response units (RU). Data analysis was performed using BIA evaluation software with a 1:1 coupled Langmuir fit model.

[0205] To evaluate the in vitro binding affinity of the TROP2 antibody to the TROP2 protein, surface plasmon resonance (SPR) analysis using Biacore was employed. The results showed that R4702 had a relatively faster association rate (7.45 x 10⁻¹) to the TROP2 protein than datopotamab and sacituzumab. 5 ( / Ms) and slow dissociation rate (1.50x10) -4 It was revealed that the values ​​shown are ( / s). Furthermore, the equilibrium dissociation constant (KD) values ​​for R4702, datopotamab, and sacituzumab are 2.01 x 10⁻¹⁰, respectively. -10 M, 6.43x10 -8 M, and 1.67x10 -9It was M (Table 2). These results showed that R4702 had a binding affinity for TROP2 protein that was 320-fold and 8-fold superior to datopotamab and sacituzumab, respectively. The much higher affinity compared to datopotamab of R4702 was due to R4702 showing a faster on-rate and a slower off-rate in binding to the TROP2 protein.

[0206]

Table 2

[0207] To investigate the potential of topoisomerase 1 (TOP1) inhibitors as effective drug moieties of ADCs, the cytotoxicities of exatecan, deruxtecan (DXd), and SN-38 across different cancer cell types were evaluated. Exatecan showed relatively high toxicity in triple-negative breast cancer (MDA-MB-231), gastric cancer (NCI-N87), and pancreatic cancer (Capan-1) cells compared to DXd and SN-38. Generally, exatecan showed 2- to 5-fold superior efficacy compared to the others against three different cell lines, and the IC 50 is summarized in Table 3.

[0208]

Table 3

[0209] Example 3: Binding process of exemplary OBI-992 (TROP2 antibody-drug conjugate) 3.1 Linker binding of 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (MCCa) of R4702 R4702 is a TROP2 monoclonal antibody disclosed in WO2022 / 222992; exatecan is a commercially available anti-cancer agent; the 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (MCCa) linker is modified from commercially available succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). The complete chemical structure of OBI-992 (average DAR=4) is shown below:

[0210] [ka]

[0211] 3.2 Synthesis of MCCa linker with drug portion exatecan Product N-PM-0017 was produced from a mixture of N-DT-0013 and N-PM-0018. The manufacturing scheme and process are listed below:

[0212] [ka]

[0213] (a) Step 1: To a suspension of exatecan mesylate in DMF, N-PM-0015 and DIPEA were added at room temperature. The suspension became a clear brown solution within 5 minutes. This mixture was stirred at room temperature for 20 hours. After the reaction was complete, the reaction mixture was added to TBME while stirring over 30 minutes to obtain a precipitate. After stirring for 30 minutes, the solid was collected by filtration and subsequently dried under high vacuum to obtain crude N-PM-0016. This crude product was used in the next step without further purification. (b) Step 2: A stirred suspension of N-PM-0016 in DCM was cooled to -20°C. Pre-cooled TFA liquid to -10°C was added to the N-PM-0016 solution over 60 minutes. This mixture was stirred at -20°C for 10 hours. After the reaction was complete, the reaction mixture was added to a stirred TBME over 30 minutes to obtain a precipitate. After stirring for 30 minutes, the solid was collected by filtration and subsequently dried under high vacuum to obtain crude N-PM-0018. This crude product was used in the next step without further purification. (c) Step 3: HATU and NMM were added to a solution of N-DT-0013 in DMF. This mixture was stirred at room temperature for 2 hours. A solution of N-PM-0018 and NMM in DMF was added to the N-DT-0013 solution over 30 minutes at room temperature. This mixture was stirred for a further 2 hours at room temperature. After the reaction was complete, the reaction mixture was added to a stirred TBME over 30 minutes to obtain a precipitate. After stirring for 30 minutes, the solid was collected by filtration and purified by reverse-phase chromatography (eluent: ACN / water). The pure fractions were combined and extracted with 10% MeOH / DCM to obtain N-PM-0017.

[0214] 3.3 OBI-992 coupling process The overall coupling process for OBI-992 (R4702-N-PM-0017) is listed as follows:

[0215] [ka]

[0216] Anti-TROP2 antibody R4702 (10 mg / mL, total 50 mL) in reaction buffer (50 mM histidine, 20 mM EDTA, pH 7.0) was cooled to 12-16°C. R4702 was treated with TCEP.HCl (2.29 mg; 0.00799 mmol) in reaction buffer (0.46 mL) at 12-16°C for 2-6 hours. To reduce the antibody solution, payload-linker N-PM-0017 (39.94 mg; 0.0183 mmol) in DMSO was added and conjugated at 12-16°C for 1 hour. After binding was complete, the OBI-992 buffer was replaced with a storage buffer (20 mM sodium acetate, pH 5.0, containing 0.1% (w / w) polysorbate 80) via a UF / DF dialysis membrane to achieve the final concentration of OBI-992 (10.14 mg / mL, total 41.9 mL). The drug-antibody ratio (DAR) of the final ADC was determined by hydrophilic interaction chromatography (HIC) and was 4.15.

[0217] Example 4: Cytotoxicity assay of OBI-992 in human cancer cell lines 4.1 Cell lines Human prostate cancer DU145 cells were cultured in Eagle's Minimum Essential Medium (Corning) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). Human pancreatic cancer Capan-1 cells were cultured in Iskov-modified Dulbecco's medium (IMDM; Gibco) containing 20% ​​FBS and 1% P / S. Human ovarian cancer ES2 cells were cultured in McCoy's 5A (Gibco) containing 5% FBS and 1% P / S. Human breast cancer MCF7 cells were cultured in Minimum Essential Medium (Gibco) containing 10% FBS, 1% P / S, and 0.01 mg / mL human recombinant insulin. Human lung cancer H1975, H460, and NCI-N87, human pancreatic cancer BxPC3, and human acute monocytic leukemia THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) containing 10% FBS and 1% P / S. All of the above cell lines were cultured at 37°C in a humidified incubator with 5% CO2. Human breast cancer MDA-MB-231 cells were cultured at 37°C in a humidified incubator without CO2 in Leibovitz's L-15 medium (Gibco) containing 10% FBS and 1% P / S. All cell lines except MCF-7 were purchased from the American Cell Culture and Cell Lineage Preservation Corporation (ATCC).

[0218] 4.2 Quantitative flow cytometry and cell viability (a) Cells were stained with AF488-conjugated mouse anti-human TROP2 antibody or isotype control antibody and then analyzed by BD Canto II flow cytometry. The number of binding sites of anti-human TROP2 antibody on cells was calculated using the Quantum® Simply Cellular® (QSC) anti-mouse IgG kit (Bangs Laboratories, Inc.) according to the manufacturer's instructions. Briefly, QSC calibration microspheres coated with increasing amounts of capture antibody were labeled until saturated with the same antibody used to label the cells. The mean fluorescence index (MFI) of the labeled calibration microspheres and cells was recorded and analyzed using the QuickCal® analysis template (Bangs Laboratories, Inc.) to determine the antibody binding capacity value for each cell. (b) Cells grown on 96-well white plates were treated with the indicated compounds for 144 hours. Viable cells were detected using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Briefly, cells treated with the compound were lysed for 10 minutes using a 1:1 mixture of CellTiter-Glo® Reagent and culture medium. After incubation, luminescence was recorded using a SpectraMax M2 reader. The half maximal inhibitory concentration (IC 50 ) was calculated using GraphPad Prism 6.

[0219] 4.3 Results OBI-992 is a conjugate of R4702 and exatecan via a hydrophilic linker. To evaluate the correlation between the cytotoxicity of OBI-992 and TROP2 expression levels in cancer cells, various cancer cell lines were used for the evaluation. TROP2 expression levels were determined by flow cytometry (Figure 2A). The results, as shown by Spearman correlation (r = -0.8, p = 0.0138), indicate that the IC 50We revealed a positive correlation between this and TROP2 expression levels (Figure 2B). This demonstrated the potent cancer cell-killing effect of OBI-992 against various cancer cell lines. Example 5: Demonstration of efficacy: Measurement of the antitumor activity of exemplary TROP2 ADC (OBI-992) in BxPC-3 human pancreatic cancer cell-derived xenografts in BLAB / c nude mice.

[0220] 5.1 Test substance and dosing pattern (a) R4702-Exatecan-DAR4 (5.50 mg / mL) (b) R4702-Exatecan-DAR8 (5.11 mg / mL) (c) R4702-MMAE-DAR4 (4.96 mg / mL) (d) R4702-SN38-DAR8 (4.0 mg / mL) (e) IMMU-132 (5.0 mg / mL) (f) DS-1062 (4.95 mg / mL)

[0221] [Table 4]

[0222] 5.2 Cell line: BxPC-3 cells (high TROP2 expression; human pancreatic cancer)

[0223] 5.3 Animals (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1 nu / CrlBltw (BALB / c nude) (c) Supplier: BioLasco Taiwan (d) Gender: Female (e) Age at the start of the study: 7 weeks (f) Starting body weight range: 15-25 g (g) Animal grouping: The mice were divided into 13 groups, each containing 5 mice. A total of 65 mice were involved in the experiment.

[0224] 5.4 Apparatus and Materials (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolation positive / negative pressure verified cage rearing system (TECNIPLAST / Blue Line) (d) Caliper (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0225] 5.5 Method (a) Establishment of xenograft mouse models Subcutaneous inoculation of tumor cells: 5x10 6 BxPC-3 cells were mixed with an equal amount of Matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). The subcutaneous injection volume was 100 μL / mouse. (b) Route and administration of the test product: Average tumor volume is 150-200 mm 3 The first day on which the target was reached was designated as day 1. All test substances (test substances 1-6) or reference substance (sodium citrate solution) were administered intravenously to G2-G13 mice on days 1, 8, and 15. Injections were performed using an insulin syringe at doses of 10 mg / kg or 3 mg / kg, with an injection volume of 5 mL / kg. (c) Weight measurement Measurements began the day after tumor inoculation. The animals' weight was measured and recorded three times a week. (d) Calculation of tumor growth inhibition rate The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%, where Ti and Ci represent the mean tumor volume in the treatment group and vehicle group at the end of the study (day 22). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment group and vehicle group at the start of test substance administration (day 1). (e) Blood sampling: Blood samples were collected using mandibular blood collection on days 0, 7, and 14. The collected blood samples were centrifuged at 4°C and 1500g for 15 minutes to separate the serum from the pellet. The supernatant serum was collected and stored at -80°C. The experiment was completed on day 22. (f) Tumor excision At the end of the experiment, the mice were euthanized with CO2. The connective tissue surrounding the tumor was excised, and the tumor sample was immediately frozen in liquid nitrogen and stored at -80°C. (g) Statistical analysis The results are presented as the mean and the standard error of the mean (mean ± SEM). All data collected for each treatment group were compared with the vehicle group using Student's t-test. A p < 0.05 value was considered statistically significant.

[0226] 5.6 Results A summary of group body weights is shown in Figure 3 and Table 5. No significant difference in mean body weight was observed between the treatment group and the vehicle control group during the study period.

[0227] [Table 5] TIFF2026520085000012.tif228169TIFF2026520085000013.tif204169

[0228] A summary of group tumor volumes is shown in Figure 4 and Table 6. Tumor volume was measured three times per week on days 1, 3, 6, 8, 10, 13, 15, 17, 20, and 22. Animals were sacrificed on day 22, and tumors were collected for further pharmacokinetic analysis.

[0229] [Table 6] TIFF2026520085000015.tif235169TIFF2026520085000016.tif210169

[0230] On day 22, compared to the vehicle group, Figure 4A shows that the inhibitory effects in the 10 mg / kg group, from highest to lowest, were R4702-MMAE (TGI: 132%, p < 0.001), R4702-exatecan-DAR4 (TGI: 114%, p < 0.01), R4702-exatecan-DAR8 (TGI: 111%, p < 0.01), DS-1062 (TGI: 105%, p < 0.01), IMMU-132 (TGI: 66%, p > 0.05), and R4702-SN38 (TGI: 61%, p < 0.05). Furthermore, Figure 4B shows that at 3 mg / kg, R4702-MMAE and R4702-exatecan remained potent and significant. R4702-MMAE showed the highest efficacy (TGI: 131%, p<0.001), followed by R4702-exatecan-DAR8 (TGI: 120%, p<0.001), R4702-exatecan-DAR4 (TGI: 101%, p<0.01), and DS-1062 (TGI: 85%, p<0.01). However, IMMU-132 (TGI: 54%, p>0.05) and R4702-SN-38 (TGI: 36%, p>0.05) showed significantly less and negligible antitumor effects compared to the other treatment groups. In this study, all anti-TROP2 ADCs showed significant tumor inhibitory effects. R4702-MMAE showed the best antitumor effect, followed by R4702-exatecan, DS-1062, IMMU-132, and R4702-SN38.

[0231] Example 6: Demonstration of efficacy: Measurement of the antitumor activity of exemplary TROP2 ADC (OBI-992) in xenografts derived from DU-145 human prostate cancer cells. 6.1 Test substance and dosing pattern (a) R4702-Exatecan-DAR4 (5.57 mg / mL) (b) R4702-Exatecan-DAR8 (5.11 mg / mL) (c) DS-1062 (4.80 mg / mL) (d) R4702-MMAE-DAR4 (4.86 mg / mL)

[0232] [Table 7]

[0233] 6.2 Cell line: DU-145 cells (moderate TROP2 expression; human prostate cancer)

[0234] 6.3 Animals (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1 nu / CrlBltw (BALB / c nude) (c) Supplier: BioLasco Taiwan (d) Gender: Female (e) Age at the start of the study: 7 weeks (f) Starting body weight range: 15-25 g (g) Animal grouping: The mice were divided into 13 groups, each containing 5 mice. A total of 65 mice were involved in the experiment.

[0235] 6.4 Apparatus and Materials (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolation positive / negative pressure verified cage rearing system (TECNIPLAST / Blue Line) (d) Caliper (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0236] 6.5 Method (a) Establishment of xenograft mouse models Subcutaneous inoculation of tumor cells: 5x10 6 One DU-145 cell was mixed with an equal volume of Matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). The subcutaneous injection volume was 100 mL / mouse. (b) Route and administration of the test product: Average tumor volume is 100-150 mm 3 The first day on which the target was reached was designated as day 1. All test substances (test substance 1 to test substance 4) were administered intravenously once on day 1 to mice in groups G2 to G13. The injection was performed using an insulin syringe at doses of 10 mg / kg, 3 mg / kg, or 1 mg / kg, with an injection volume of 5 mL / kg. The reference substance (sodium citrate solution) was administered to G1 mice. (c) Weight measurement The animals' weight was measured and recorded twice a week starting the day after tumor inoculation. (d) Calculation of tumor growth inhibition rate The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%, where Ti and Ci represent the mean tumor volume in the treatment group and vehicle group at the end of the study (day 22). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment group and vehicle group at the start of test substance administration (day 1). (e) Blood sampling: Blood samples were collected using mandibular blood collection on days 0 and 14. A blood sample was also collected using cardiac puncture at the time of animal slaughter on day 23. The collected blood samples were centrifuged at 4°C and 1500g for 15 minutes to separate the serum from the pellet. The supernatant serum was collected and stored at -80°C. The experiment was completed on day 25. (f) Tumor excision At the end of the experiment, the mice were euthanized with CO2. The connective tissue surrounding the tumor was excised, and the tumor sample was immediately frozen in liquid nitrogen and stored at -80°C. (g) Statistical analysis The results are presented as the mean and the standard error of the mean (mean ± SEM). All data collected for each treatment group were compared with the vehicle group using Student's t-test. A p < 0.05 value was considered statistically significant.

[0237] 6.6 Results A summary of group weights is shown in Figure 5 and Table 8. No significant differences in mean weight were observed among the G1-G13 groups during the study period.

[0238] [Table 8] TIFF2026520085000019.tif235168TIFF2026520085000020.tif234169TIFF2026520085000021.tif51169

[0239] A summary of group tumor volumes is shown in Figure 6 and Table 9. Due to their large tumor size, two animals were euthanized before scheduled human euthanasia. One mouse in the vehicle group had a tumor volume of 1500 mm³. 3 The animals were euthanized on day 18 due to exceeding the limit, and one animal in the DS-1062 (3 mg / kg) group was euthanized on day 22 due to an ulcerative tumor. Mean tumor volume was recorded on days 1, 4, 8, 11, 15, 18, 22, and 25. The experiment was completed, and the animals were euthanized on day 25.

[0240] [Table 9] TIFF2026520085000023.tif213169TIFF2026520085000024.tif214169TIFF2026520085000025.tif46169

[0241] All TROP2-targeted ADCs showed potent and statistically significant antitumor effects at 10 mg / kg on day 25 (Figure 6A). Compared to vehicle controls, R4702-MMAE showed the best tumor growth inhibition (TGI: 103%, p<0.001), followed by R4702-exatecan-DAR8 (TGI: 99%, p<0.001), R4702-exatecan-DAR4 (TGI: 91%, p<0.01), and DS-1062 (TGI: 81%, p<0.01). The degree of tumor growth inhibition was dose-dependent for the ADCs tested, ranging from 1 to 10 mg / kg. At 3 mg / kg (Figure 6B), the antitumor effect was potent and equivalent across all ADCs, with no significant differences among R4702-MMAE (TGI: 88%), R4702-exatecan-DAR8 (TGI: 88%), R4702-exatecan-DAR4 (TGI: 92%), and DS-1062 (TGI: 83%). At 1 mg / kg (Figure 6C), the antitumor effect of R4702-MMAE decreased to a TGI of 57%, but TGI was maintained relatively high in R4702-exatecan-DAR8 (TGI: 81%), R4702-exatecan-DAR4 (TGI: 78%), and DS-1062 (TGI: 85%). The results showed that R4702-exatecan ADC was superior to or equally effective than the benchmark DS-1062 in a TROP2 intermediate-level expressing DU-145 xenograft model. Example 7: Demonstration of efficacy: Measurement of the antitumor activity of exemplary TROP2 ADC (OBI-992) in xenografts derived from NCI-N87 human gastric cancer cells.

[0242] 7.1 Test substance and administration pattern (a) R4702-Exatecan-DAR4 (5.57 mg / mL) (b) R4702-Exatecan-DAR8 (5.11 mg / mL) (c) DS-1062 (4.80 mg / mL)

[0243] [Table 10]

[0244] 7.2 Cell line: NCI-N87 cells (high TROP2 expression; human gastric cancer)

[0245] 7.3 Animals (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1 nu / CrlBltw (BALB / c nude) (c) Supplier: BioLasco Taiwan (d) Gender: Female (e) Age at the start of the study: 7 weeks (f) Starting body weight range: 15-25 g (g) Animal grouping: The mice were divided into 13 groups, each containing 5 mice. A total of 65 mice were involved in the experiment.

[0246] 7.4 Apparatus and Materials (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolation positive / negative pressure verified cage rearing system (TECNIPLAST / Blue Line) (d) Caliper (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0247] 7.5 Method (a) Establishment of xenograft mouse models Subcutaneous inoculation of tumor cells: 2.5 x 10 6 NCI-N87 cells were mixed with an equal amount of Matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). The subcutaneous injection volume was 100 μL / mouse. (b) Route and administration of the test product: Average tumor volume is 100-150 mm 3The first day on which the target was reached was designated as day 1. All test substances (test substance 1 to test substance 3) were administered intravenously once on day 1 to mice in groups G2 to G13. The injections were given using an insulin syringe at doses of 10 mg / kg, 3 mg / kg, 1 mg / kg, or 0.3 mg / kg, with an injection volume of 5 mL / kg. The reference substance (sodium citrate solution) was administered to G1 mice. (c) Weight measurement The animals' weight was measured and recorded twice a week starting the day after tumor inoculation. (d) Calculation of tumor growth inhibition rate The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%, where Ti and Ci represent the mean tumor volume in the treatment group and vehicle group at the end of the study (day 22). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment group and vehicle group at the start of test substance administration (day 1). (e) Blood sampling: Blood samples were collected using mandibular blood collection on days 0 and 14. A blood sample was also collected using cardiac puncture at the time of animal slaughter on day 23. The collected blood samples were centrifuged at 4°C and 1500g for 15 minutes to separate the serum from the pellet. The supernatant serum was collected and stored at -80°C. The experiment was completed on day 23. (f) Tumor excision At the end of the experiment, the mice were euthanized with CO2. The connective tissue surrounding the tumor was excised, and the tumor sample was immediately frozen in liquid nitrogen and stored at -80°C. (g) Statistical analysis The results are presented as the mean and the standard error of the mean (mean ± SEM). All data collected for each treatment group were compared with the vehicle group using Student's t-test. A p < 0.05 value was considered statistically significant.

[0248] 7.6 Results A summary of group body weights is shown in Figure 7 and Table 11. No significant differences in mean body weight were observed among the G1-G13 groups during the study period.

[0249] [Table 11] TIFF2026520085000028.tif235168TIFF2026520085000029.tif49169

[0250] A summary of group tumor volumes is shown in Figure 8 and Table 12. Mean tumor volumes were recorded on days 1, 3, 6, 9, 13, 16, 20, and 23. The experiment was completed, and the animals were sacrificed on day 23.

[0251] [Table 12] TIFF2026520085000031.tif212169TIFF2026520085000032.tif49169

[0252] On day 23, compared to vehicle controls, a single dose of 10 mg / kg of R4702-exatecan-DAR8 showed the best inhibitory effect with complete tumor regression in all treated animals (Figure 8A), followed by R4702-exatecan-DAR4 (TGI: 118%, p<0.001) and DS-1062 (TGI: 111%, p<0.001). At 3 mg / kg (Figure 8B), the antitumor effect remained significant and statistically substantial for all three ADCs. R4702-exatecan-DAR8 was the most potent (TGI: 116%, p<0.001), followed by R4702-exatecan-DAR4 (TGI: 110%, p<0.001) and DS-1062 (TGI: 96%, p<0.01). At 1 mg / kg (Figure 8C), R4702-exatecan-DAR8 showed a significant antitumor effect (TGI: 86%, p<0.01), while the effects of R4702-exatecan-DAR4 (TGI: 49%, p>0.05) and DS-1062 (TGI: 53%, p>0.05) did not reach statistical significance. In this study, single doses of 3 or 10 mg / kg of R4702-exatecan-DAR8, R4702-exatecan-DAR4, or DS-1062 showed significant and pronounced inhibition of tumor growth in the NCI-N87 xenograft tumor model. R4702-exatecan-DAR8 was the most potent of the three ADCs tested, with complete tumor regression observed at 10 mg / kg.

[0253] Example 8: Demonstration of efficacy: Measurement of the antitumor activity of exemplary TROP2 ADC (OBI-992) in xenografts derived from NCI-H1975-C797S human lung cancer cells. 8.1 Test substance and dosing pattern (a) R4702-Exatecan-DAR4 (5.12 mg / mL) (b) R4702-Exatecan-DAR8 (5.11 mg / mL) (c) DS-1062 (4.84 mg / mL) (d) IMMU-132 (5.0 mg / mL) (e) Osimertinib (25.0 mg / mL)

[0254] [Table 13]

[0255] 8.2 Cell line: NCI-H1975-C797S cells (high TROP2 expression; human lung cancer)

[0256] 8.3 Animals (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1 nu / CrlBltw (BALB / c nude) (c) Supplier: BioLasco Taiwan (d) Gender: Female (e) Age at the start of the study: 7 weeks (f) Starting body weight range: 15-25 g (g) Animal grouping: The mice were divided into 9 groups, with each group containing 6 mice. A total of 54 mice were involved in the experiment.

[0257] 8.4 Apparatus and Materials (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolation positive / negative pressure verified cage rearing system (TECNIPLAST / Blue Line) (d) Caliper (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0258] 8.5 Method (a) Establishment of xenograft mouse models Subcutaneous inoculation of tumor cells: 1.0 x 10 7 NCI-H1975-C797S cells were mixed with an equal volume of Matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). The subcutaneous injection volume was 100 μL / mouse. (b) Route and administration of the test product: Average tumor volume is 100-150 mm 3 The first day on which the target was reached was designated as day 1. All test substances (test substance 1 to test substance 4) were administered intravenously to mice. Injections were performed using an insulin syringe at doses of 10 mg / kg and 3 mg / kg, with an injection volume of 5 mL / kg. The reference substance (sodium citrate solution) was administered intravenously to the vehicle group. Osimertinib (test substance 5) was administered orally via gastric tube. (c) Weight measurement The animals' weight was measured and recorded twice a week starting the day after tumor inoculation. (d) Calculation of tumor growth inhibition rate The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%, where Ti and Ci represent the mean tumor volume in the treatment group and vehicle group at the end of the study (day 16). Meanwhile, T1 and C1 represent the mean tumor volume in the treatment group and vehicle group at the start of test substance administration (day 1). (e) Blood sampling: On day 0, a blood sample was collected using mandibular blood collection. On day 16, a blood sample was collected using cardiac puncture. The collected blood samples were centrifuged at 4°C and 1500g for 15 minutes to separate the serum from the pellet. The supernatant serum was collected and stored at -80°C. The experiment was completed on day 16. (f) Tumor excision At the end of the experiment, the mice were euthanized with CO2. The connective tissue surrounding the tumor was excised, and the tumor sample was immediately frozen in liquid nitrogen and stored at -80°C. (g) Statistical analysis The results are presented as the mean and the standard error of the mean (mean ± SEM). All data collected for each treatment group were compared with the vehicle group using Student's t-test. A p < 0.05 value was considered statistically significant.

[0259] 8.6 Results A summary of group weights is shown in Figure 9 and Table 14. No significant differences in mean weight were observed among the G1-G9 groups during the study period.

[0260] [Table 14] TIFF2026520085000035.tif236169TIFF2026520085000036.tif123169

[0261] A summary of group tumor volumes is shown in Figure 10 and Table 15. Mean tumor volumes were recorded on days 1, 3, 7, 10, 14, and 16. The experiment was completed, and the animals were sacrificed on day 16.

[0262] [Table 15] TIFF2026520085000038.tif212169TIFF2026520085000039.tif134170

[0263] All TROP2-targeted ADCs except DS-1062 showed significant and statistically substantial tumor inhibitory effects. Compared to vehicle controls, a single dose of 10 mg / kg of R4702-exatecan-DAR8 showed the best tumor growth inhibition (TGI: 106%, p<0.001) (Figure 10A), followed by R4702-exatecan-DAR4 (TGI: 80%, p<0.001), and DS-1062 (TGI: 40%, p>0.05). At 3 mg / kg (Figure 10B), R4702-exatecan-DAR8 (TGI: 64%, p<0.01) and R4702-exatecan-DAR4 (TGI: 59%, p<0.05) maintained potent antitumor effects, while DS-1062 showed a negligible low response (TGI: 24%, p>0.05). The benchmark ADC IMMU-132 at 12.5 mg / kg in a different dosing regimen (2 weeks, twice weekly) showed a significant antitumor effect at a TGI of 44% (P<0.05), which was lower than the inhibitory response caused by R4702-exatecan-DAR8 and R4702-exatecan-DAR4 at 3 or 10 mg / kg. R4702-exatecan-DAR8 and R4702-exatecan-DAR4 demonstrated far superior antitumor efficacy compared to the benchmarks DS-1062 and IMMU-132 in the NCI-H1975-C797S human lung cancer xenograft model, which has three EGFR mutations and is resistant to TKI therapy.

[0264] Example 9: Off-target effects of OBI-992 9.1 Cellular association of ADCs with PBLs Off-target association of TROP2 ADCs with peripheral blood leukocytes (PBLs) was evaluated by ex vivo assay. Normal human blood was purchased from the Taipei Blood Center in Taiwan. Fluorescein-labeled ADCs were mixed with whole blood obtained from two healthy donors and incubated at 37°C for 4 hours. After incubation, whole blood samples were treated with BD FACS Lysing solution to remove red blood cells (RBCs), followed by FACS analysis. Based on cell size and granularity, three PBL populations—lymphocytes, monocytes, and granulocytes—were identified and distinguished by gating on an FSC / SSC plot. The mean fluorescence intensity (MFI) of each population was measured to indicate the level of cell association.

[0265] 9.2 In vitro evaluation of ADC toxicity to differentiated neutrophils Bone marrow-derived human CD34 + Stem cells were grown in SFEM II medium containing CC100 supplement, followed by three stages of neutrophil differentiation. The progression of neutrophil differentiation was monitored by measuring the granulocyte marker CD66b, and approximately 25%, 45%, and 85% of CD66b were observed after the first, second, and third stages of differentiation, respectively. + The cells were identified. For ADC toxicity, differentiated neutrophils were incubated with ADC for 6 days, followed by CD66b and propidium iodide (PI) staining. Surviving neutrophils were CD66b positive and PI negative (CD66b + / PI - They were distinguished by being ). The absolute cell count was determined by referring to CountBright Beads (Invitrogen, Cat. No. C36950) uniformly added to each sample immediately before FACS analysis. Viable CD66b in each sample was determined according to the manufacturer's instructions. + Cell counts were determined. The number of viable cells was normalized to the untreated control, and relative CD66b was calculated. + / PI - The results are shown as a percentage of cells. The concentration at which haploid growth is inhibited (IC) is the IC25 concentration. 50 The result was calculated using GraphPad Prism 6.

[0266] 9.3 Results Several reports suggest that nonspecific binding of mAbs to the Fcγ receptor (FcγR) may cause off-target toxicity. To evaluate the binding ability of ADCs to immune cells, whole blood was incubated with fluorescein-labeled OBI-992 or Dato-DXd for 4 hours. Leukocyte populations of lymphocytes, monocytes, and granulocytes were fractionated by flow cytometry. Limited binding signals were observed in the lymphocyte population with both OBI-992 and Dato-DXd treatment. Conversely, the monocyte population treated with Dato-DXd showed higher mean fluorescence intensity (MFI) ranging from 3 to 5 times higher compared to OBI-992 (Figure 11A), suggesting stronger association between Dato-DXd and monocytes. Furthermore, Dato-DXd showed dose-dependent binding to monocyte THP-1 cells, unlike OBI-992 (Figure 11B). Dato-DXd showed higher cytotoxicity against THP-1 cells and IC 50 It was 95 nM, and was 5 times more toxic than OBI-992 (546 nM) (Figure 11C). Neutropenia is frequently observed in cancer patients treated with ADCs. Therefore, neutrophils differentiated from hematopoietic stem cells (CD66b + The survival rate of ) was investigated by staining with propidium iodide (PI). The results showed that neutrophils were more sensitive to Dato-DXd than to OBI-992, and IC 50 The results showed that the levels were 2.9 nM versus 93.4 nM (Figure 11D). These findings suggest that OBI-992 has a lower potential for off-target effects on immune cells compared to Dato-DXd.

[0267] Example 10: Synthetic lethality in vitro and in vivo between OBI-992 and PARPi 10.1 In vitro synergistic effects of OBI-992 Talazoparib was further evaluated with OBI-992 for potential combination therapy. IC of talazoparib in the presence of OBI-992. 50 The values ​​decreased significantly, and a strong synergistic effect was observed in different cancer cell lines (MDA-MB-231, NCI-N87, Capan-1). In MDA-MB-231 cells, talazoparib IC50 The value improved from 368 nM to 11 nM, representing a 32-fold increase in efficacy (Figure 12A). Furthermore, the efficacy improvements in NCI-N87 and Capan-1 cells were 9-fold and 4-fold, respectively (Figures 12B and 12C). These results suggest that PARP inhibitors are promising candidates for combination therapy with OBI-992 in cancer treatment.

[0268] 10.2 In vivo synergistic effects of OBI-992 Xenotransplantation trials include CAnN.Cg-Foxn1 nu / CrlBltw(BALB / c nude) mice were used. C57BL / 6NCrl(B6) mice were used as a syngeneic mouse model. Mice were housed in individual ventilated cages (IVCs) with sterile bedding, in controlled environments of 22±3°C, 50±20% relative humidity, and a 12 / 12-hour light / dark cycle, in groups of up to 5 or 6, under specific pathogen-free (SPF) conditions. Food (LabDiet 5010, PMI, USA) and water (sterile RO water) were freely available throughout the entire study period. Mice were acclimatized for at least 3 days prior to the start of the study. After transplantation of cancer cells, tumor size was measured with calipers, and tumor volume was calculated using the ellipsoid equation ((major axis × minor axis × minor axis) × (π / 6)) according to the records. The tumor growth inhibition (TGI) rate was calculated using tumor volume according to the following formula: TGI (%) = [1 - (Ti - T1) / (Ci - C1)] × 100%. Ti and Ci represent the mean tumor volume in the treatment and vehicle groups at the end of the study, while T1 and C1 represent the mean tumor volume in the treatment and vehicle groups at the start of test substance administration (day 1). All animal protocols were reviewed and approved by the Institutional Animal Management and Use Committee of the National Center for Experimental Animals in Taiwan.

[0269] The synergistic effects of OBI-992 and PARP inhibitors (olaparib and talazoparib) were evaluated using xenograft tumors derived from Capan-1 human pancreatic cancer cells in BALB / c nude mice. 7Cells ( / mouse) were mixed with an equal amount of Matrigel (BD) and subcutaneously injected into the right flank of female BALB / c nude mice (BioLasco Taiwan, 100 mL / mouse). The average tumor volume was 100-150 mm². 3 Mice were divided into groups when they reached a certain age. OBI-992 was administered at a suboptimal dose of 0.1 or 0.3 mg / kg to demonstrate a synergistic effect and avoid potential excessive toxicity when combined with PARP inhibitors. Olaparib (Combi-Blocks, Inc.) was administered at 80 mg / kg, and talazoparib (AmBeed, Inc.) at 0.1 mg / kg. OBI-992 was administered intravenously as a single dose on day 1. Olaparib and talazoparib were administered by oral gastric tube in a 5-day consecutive, 2-day rest cycle. Tumor volume, body weight, mortality, and animal behavior were monitored twice weekly until the end of the study (day 22). Throughout the study period, all mice were in good condition, and no adverse effects were observed. Figure 13A showed that tumor growth inhibition (TGI) of OBI-992 alone and OBI-992 + talazoparib was 31-40% and 97-103%, respectively (P<0.001). Similarly, Figure 13B also showed that tumor growth inhibition (TGI) of OBI-992 alone and OBI-992 + olaparib was 31-40% and 83-98%, respectively (P<0.001). These results also suggest that PARP inhibitors are promising candidates for combination therapy with OBI-992 in cancer treatment.

[0270] To evaluate the synergistic effect of OBI-992 and PD-1 blockade, we used MC38 mouse colon cancer cells (MC38 / hTROP2) that ectopically express human TROP2. Surviving MC38 / hTROP2 cells (1 x 10⁶) 6 Cells ( / mouse) were mixed with an equal amount of Matrigel (BD) and subcutaneously injected into the right flank of female B6 mice (BioLasco Taiwan, 100 mL / mouse). The average tumor volume was 200-250 mm². 3Tumor-bearing mice were divided into groups when they reached a certain stage of development. OBI-992 was administered either alone at 3 mg / kg or in combination with 5 mg / kg of anti-mPD-1 (Leinco Technologies, Inc.). OBI-992 was administered as a single intravenous dose, and anti-mPD-1 was administered intravenously twice a week. Tumor volume, body weight, mortality, and animal behavior were monitored twice a week until the end of the study (day 11). All mice were in good condition throughout the study period, and no adverse effects were observed. Figure 13C shows that the tumor growth inhibition (TGI) of OBI-992 + anti-mPD-1 was 96% (P<0.05). This result also demonstrated the synergistic effect of anti-PD-1 and OBI-992 in cancer treatment.

[0271] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as those generally understood by those skilled in the art of the invention. Any composition, method, kit, and means of communication similar to or equivalent to those described herein may be used in carrying out the invention, but preferred compositions, methods, kits, and means of communication are described herein.

[0272] All references cited herein are incorporated herein by reference to the maximum extent permitted by law. Any discussion of those references is intended merely to summarize the claims made by their authors. No acknowledgment is made that any reference (or any part of any reference) constitutes relevant prior art. The applicant reserves the right to challenge the accuracy and validity of any cited references.

Claims

1. An antibody-drug conjugate (ADC) comprising a drug portion and an antibody or antigen-binding fragment thereof that binds to tumor-associated calcium signal transducer 2 (TROP2), wherein the ADC is an antibody-drug conjugate represented by formula (I). Ab-(L-D) ｎ (I) (In the formula, The drug portion (D) is covalently bound to the antibody or its antigen-binding fragment (Ab) via the linker (L); The antibody is an anti-TROP2 antibody; and, n is an integer between 1 and 8.

2. The ADC according to claim 1, wherein the antibody is a monoclonal antibody, an antigen-binding fragment, a chimeric antibody, or a humanized antibody.

3. The antigen-binding fragment is Fab, F(ab') ２ The ADC according to claim 2, wherein the ADC is Fv or scFv.

4. The ADC according to claim 1, wherein the anti-TROP2 antibody is hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamab, or sacituzumab.

5. The ADC according to claim 1, wherein the drug portion is exatecan, deruxtecan, SN-38, or MMAE.

6. The ADC according to claim 1, wherein the linker comprises maleimide or a derivative thereof bonded to the thio group of the antibody.

7. The ADC according to claim 6, wherein the linker is 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (MCCa).

8. The ADC according to claim 1, wherein the drug portion is a chemotherapeutic agent, a photodynamic therapy agent, or a biological preparation.

9. The ADC according to claim 8, wherein the photodynamic therapeutic agent is Photofrin, Lezaphyrin, aminolevulinic acid (ALA), silicon phthalocyanine Pc4, m-tetrahydroxyphenylchlorine (mTHPC), chlorin e6 (Ce6), Almera, Reblanc, Foscan, Metovix, Hexivix, Photochlor, Photosense, Phototrex, Lumacan, Bisonac, Amfinex, Verteporfin, Pruritin, ATMPn, zinc phthalocyanine (ZnPc), protoporphyrin IX (PpIX), pyropheoforbid a (PPa), or pheoforbid a (PhA).

10. The ADC according to claim 1, wherein the drug portion is an antiproliferative agent.

11. The aforementioned antiproliferative agents include exatecan, irinotecan, topotecan, camptothecan, rubitecan, MLN576, exatecan, berotecan, seconeoritosin, SN-38, Genz-644282, betulinic acid, β-rapacone, calenitesin, gimatecan, namithecan, edotecarin, SW044248, LMP744, T-2513, podocarpus flavone A, indimitecan, lulutotecan, TP3011 or 10-hydroxycamptothecin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), meltansine (DM1), anthracycline, pyrrolobenzodiazepine, α-amanitin, tubulicin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, leucovorin, rapamycin, lapatinib, ronafarnib (SARASAR, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agents; alkyl sulfonates; aziridine; ethyleneimine; methylamelamine; acetogenin; camptothecin; bryostatin; callistostatin; CC-1065; cryptophycin; dorastatin; duocalmycin; eryuterobin; pancratistatin; sarcodictiin; spongstatin; chlorambucil; chlornafadin; colophosphamide; estramustine; ifosfamide; mechloretamine; mechloretamine oxide hydrochloride; melphalan; nobembiquin; phenesterine; prednimastine; trophosphamide; uracil mustard ;carmustine;chlorozotocin;fotemustine;lomustine;nimustine;ranimustine;calicheamycin;dynemycin;clodronate;esperamycin;neocardinostatin chromophore;aclasinomycin;actinomycin;autoramycin;azaselin;bleomycin;kactinomycin;karabicin;kaminomycin;cardinophylline;chromomycin;dactinomycin;daunorubicin;detorubicin;6-diazo-5-oxo-L-norleucine;doxorubicin;epirubicin;esorubicin;idarubicin;marcelomycin;mitomycin;mycophenolic acid;nogaramycin;olibomycin;Peplomycin; potophyllomycin; puromycin; queramycin; rhodorubicin; streptonigrin; streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; methotrexate; 5-fluorouracil (5-FU); denopterin; pteropterin; trimethrexate; fludarabine; 6-mercaptopurine; thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; enocitabine; floxlysine; Carsterone; dromostanolone propionate; epithiostanol; mepithiostan; testolactone; aminoglutethimide; mitotane; trilostane; floric acid; acegraton; aldofhamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diazicon; elformitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; mytansin; ansamitosin ; Mitoguazone; Mitoxantrone; Mopidammol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Trichothecene; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside; Cyclophosphamide; Thiotepa; Ta The ADC according to claim 10, wherein the ADC is a xoid; paclitaxel; docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; cisplatin; carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoid or capecitabine.

12. A pharmaceutical composition comprising the ADC described in claim 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent, carrier, or excipient.

13. The pharmaceutical composition according to claim 12, further comprising an additional anticancer agent.

14. A method for treating cancer in a patient, comprising administering to a patient in need of treatment an effective amount of the ADC described in claim 1 and a pharmaceutically acceptable carrier, or the pharmaceutical composition described in claim 12.

15. The method according to claim 14, wherein the cancer is a TROP2-expressing cancer selected from the group consisting of lung cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

16. The method according to claim 14, further comprising administering an additional anticancer agent to the patient.

17. The method according to claim 16, wherein the combination of the ADC and the additional anticancer agent produces a synergistic or additive effect in cancer treatment, thereby improving the therapeutic effect.

18. A method for inducing or enhancing an immune response in patients requiring treatment, A method comprising administering an effective amount of the pharmaceutical composition according to claim 12 and performing one or more steps selected from the following: (a) Administering the pharmaceutical composition two or more times; (b) Adjusting the time interval and / or dosage regimen between two consecutive doses; (c) Adjusting the route of administration and / or changing the site of administration; or (d) Administer additional anticancer drugs.

19. The method according to claim 18, wherein the administration may be modified and / or complemented by the addition of an immune response enhancer.

20. The method according to claim 14 or 18, wherein the effective dose is 0.001 μg to 250 mg per kg of the patient's body weight.

21. The method according to claim 18, wherein the method comprises administering an additional anticancer agent, the combination of the pharmaceutical composition and the additional anticancer agent resulting in a synergistic or additive effect in inducing or enhancing an immune response.

22. Use of the ADC according to claim 1 in the manufacture of a pharmaceutical product for use in combination with an effective amount of an additional agent selected from the group consisting of anticancer agents, immunosuppressants, and anti-infective agents for the treatment of lung cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer.

23. A method of selecting patients for cancer treatment using diagnostic imaging, (a) administering an effective amount of the ADC described in claim 1 to the patient; and (b) To detect the reporting signal of the imaging agent in the patient. Includes, The imaging agent is a fluorophore, a dye, an MRI contrast agent, or a radionuclide; The aforementioned reporting signal is detected visually or instrumentally. method.

24. The method according to claim 23, wherein the patient has detectable cancer, and the method further detects cancer metastasis.

25. An antibody-drug conjugate (ADC) that binds to TROP2, (a) An antibody comprising a heavy chain variable domain and a light chain variable domain, The aforementioned heavy chain variable domain i. The first heavy chain complementarity determination region (HCDR1) having the amino acid sequence of SEQ ID NO: 1; ii. A second heavy chain complementarity determination region (HCDR2) having the amino acid sequence of SEQ ID NO: 2; and iii. A third heavy chain complementarity-determining region (HCDR3) having the amino acid sequence of SEQ ID NO: 3 Includes, The aforementioned light chain variable domain i. A first light chain complementarity determination region (LCDR1) having the amino acid sequence of SEQ ID NO: 4; ii. A second light chain complementarity determination region (LCDR2) having the amino acid sequence of SEQ ID NO: 5; and iii. A third light chain complementarity determination region (LCDR3) having the amino acid sequence of SEQ ID NO: 6 Antibodies, including (b) The drug portion; (c) Linker and; Antibody-drug conjugates containing such conjugates.

26. The aforementioned antibody (a) Heavy chain variable domains containing amino acid sequences that are 90% to 100% identical to the amino acid sequence of SEQ ID NO: 7; and (b) Light chain variable domains containing amino acid sequences that are 90% to 100% identical to the amino acid sequence of Sequence ID No. 8; The ADC according to claim 25, further comprising:

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