Trop2 conjugated biological molecules, pharmaceutical compositions and methods

Abstract

Antibody drug conjugate (ADC) comprising a drug moiety conjugated to antibody or antigen binding fragments thereof that bind to TROP2 disclosed herein, as well as methods of use thereof. Methods of use include, without limitation, cancer therapies and diagnostics. The ADC of the disclosure can bind to certain cancer cell surfaces. Exemplary targets of the ADC disclosed herein can include carcinomas, such as lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

Description

PATENT Attorney Docket No.: G3004-01900PCT TROP2 CONJUGATED BIOLOGICAL MOLECULES, PHARMACEUTICAL COMPOSITIONS AND METHODS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit to and priority of U.S. Provisional Patent Applications Nos. 63 / 492,343, filed on March 27, 2023, and 63 / 549,781, filed on February 5, 2024. The entirety of the aforementioned application is incorporated herein by reference. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml copy, created on March 26, 2024, is named “G3004-01900PCT_20240327_SeqListing.xml” and is 11 kilobytes in size. FIELD

[0003] The present disclosure is directed to antibody-drug conjugates (ADCs) compositions and methods of use thereof to treat cancer. Also described herein are methods of using antibody-drug conjugate compounds for treatment of mammalian cells associated with pathological conditions. The present disclosure relates to antibodies and binding fragments thereof to TROP2, including pharmaceutical compositions comprising said antibody and / or binding fragments. Further, methods are provided for administering ADCs to a subject in an amount effective to inhibit cancer cells. BACKGROUND OF THE INVENTION

[0004] TROP2 (Tumor-associated calcium signal transducer 2, TACSTD2) is a prevalent tumor surface antigen and mediates cell growth, adhesion, and migration. TROP2 plays an important role in cancer development, however its localization may be shown different function in tumor cell. Patients are shown worse clinical outcome with membranous TROP2 expression while intracellular TROP2 is with better survival rate and less relapse happened in breast cancer patients (Ambrogi et al., (2014) PLOS ONE 9(5): e110606). TROP2 has been studied in up-regulation of epithelial-mesenchymal transition (EMT) through PI3K / Akt 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). RAS-Raf-MEK-ERK pathway has been indicated under regulation of TROP2 to stimulate cell proliferation. TROP2 has been discovered in interaction with other membrane proteins to regulate cellularPATENT Attorney Docket No.: G3004-01900PCT function 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). It is of great interest to identify TROP2 associated with and / or predictive of cancers, and develop antibody-drug conjugates (ADCs) against the markers for use in diagnosing and treating a broad spectrum of cancers. TROP2 can be designed as an ADC by combining its specific antibodies with drug moiety through different linkers.

[0005] The use of antibody-drug conjugates (ADCs) for the local delivery of cytotoxic or cytostatic agents, e.g., drugs to kill or inhibit tumor cells in the treatment of cancer (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 targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, while systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., 1986, Lancet pp. (Mar.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). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been reported as 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 be inactive or less active when conjugated 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] indolizino[1,2-b] quinoline-10,13-dione; CAS No.: 171335-80-1; Molecular Formula: C24H22FN3O4) is a topoisomerase 1 inhibitor which is a structural analog of camptothecin with antineoplastic activity. In the past, exatecan has been clinically evaluated as a stand- alone chemotherapy drug (exatecan mesylate, DX-8951f), as preclinical studies indicated it was more potent than SN-38-based irinotecan (CPT-11) against various tumor xenograft models, including CPT-11 -resistant tumors. Currently the development of exatecan mesylate as free drug is discontinued but exatecan is still a candidate for cytotoxic drug moiety of the anti-body drug conjugate with excellent antitumor effect. SUMMARY OF THE INVENTION

[0007] Accordingly, the present disclosure is based on the discovery that TROP2 are aberrantly expressed in a broad spectrum of cancers. Cancers expressing TROP2 include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer,PATENT Attorney Docket No.: G3004-01900PCT colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0008] In one aspect, the present disclosure features an antibody or binding fragment thereof specific to TROP2.

[0009] In certain embodiments, the antibody is an Anti-TROP2 antibody.

[0010] In certain embodiments, the Anti-TROP2 antibody is R4702. Exemplary R4702 antibody is as described in PCT patent application (PCT publication number WO2022 / 222992), the contents of which are incorporated by reference in its entirety.

[0011] In certain embodiments, the chemotherapeutic agent is Topoisomerase inhibitor, which comprises Topoisomerase I inhibitor and Topoisomerase II inhibitor.

[0012] In certain embodiments, the chemotherapeutic agent is Topoisomerase I inhibitor, which is Camptothecin (CPT) or Non-camptothecins, selected from Irinotecan, Topotecan, Camptothecin, Rubitecan, MLN576, Exatecan, Belotecan, Seconeolitsine, SN-38, Genz-644282, Betulinic acid, β-Lapachone, Karenitecin, Gimatecan, Namitecan, Edotecarin, SW044248, LMP744, T-2513, Podocarpusflavone A, Indimitecan, Lurtotecan, TP3011 or 10-hydroxycamptothecin.

[0013] In one aspect, the invention provides antibody-drug conjugates (ADCs), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In certain embodiments, the present disclosure features an antibody-drug conjugate (ADC) thereof specific to TROP2.

[0014] In certain embodiments, the drug is Exatecan.

[0015] In one aspect, the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising the administering of an effective amount of an exemplary ADC (OBI-992) to a subject in need thereof, wherein the proliferation of cancer cells is inhibited.

[0016] In certain embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the exemplary ADC (OBI-992) described herein.

[0017] In the disclosed compositions, both the ADC or any other relevant components are present in immunogenically effective amounts. For each specific ADC, the optimal immunogenically effective amount should be determined experimentally (taking into consideration specific characteristics of a given patient and / or type of treatment). Generally, this amount is in the range of 0.01 μg-250 mg per kilogram bodyPATENT Attorney Docket No.: G3004-01900PCT weight of an antibody which was specifically targeting TROP2. In some embodiments, a therapeutically effective amount of a therapeutic composition (i.e., an effective dosage) may range from about 0.001 ^g / 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 kilogram of patient body weight, or any range between any of the numbers listed herein, or other ranges that would be apparent and understood by artisans without undue experimentation. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present.

[0018] In certain embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer thyroid cancer and oral cancer.

[0019] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims. BRIEF DESCRIPTION OF THE FIGURES

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] A more complete understanding of the invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.

[0022] Figure 1 showed TROP2 Immunohistochemistry (IHC) expression in human normal and tumor tissues. Figure 1A indicated TROP2 expressed in human breast, lung, cervix, ovary, uterus, prostate, colon,PATENT Attorney Docket No.: G3004-01900PCT esophagus, pancreas, larynx, stomach and bladder tumor tissues. Figure 1B listed the calculated TROP2 IHC expression H-score in every organ individually.

[0023] Figure 2 showed cytotoxicity assay of OBI-992 (R4702-exatecan ADC) in human cancer cell lines. Figure 2A indicated the quantitative FACS value was used to represent TROP2 expression. Figure 2B indicated the positive correlation of OBI-992 cytotoxicity with TROP2 expression in cancer cells.

[0024] Figure 3 showed BxPC-3 pancreatic carcinoma cell-derived xenograft body weight changes. Vehicle, R4702-E: R4702-exatecan, R4702-M: R4702-MMAE, R4702-S: R4702-SN38, IMMU-132, and DS-1062 were recorded three times weekly till Day 22. Data was shown as the mean ± SEM (N=5 for each group except to R4702-E-DAR810 mpk (N=4) and IMMU-132 (N=4) treatment groups due to exclusion of outliers, whose tumors were growing intramuscularly not subcutaneously).

[0025] Figure 4 showed BxPC-3 pancreatic carcinoma cell-derived xenograft tumor growth curves (Figure 4A: 10 mpk of vehicle and test substances; Figure 4B: 3 mpk of vehicle and test substances). Vehicle, R4702-E: R4702-exatecan, R4702-M: R4702-MMAE, R4702-S: R4702-SN38, IMMU-132, and DS-1062 was recorded three times weekly till Day 22. Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 22). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). Data was shown as the mean ± SEM [Figure 4A: N=5 for each group except to R4702-E-DAR8 (N=4) and IMMU-132 (N=4) and [Figure 4B: N=5 for each group except to IMMU-132 (N=4)] treatment groups due to exclusion of outliers, which tumor was growing intramuscularly not subcutaneously). Statistical analyses were performed by Student’s t-test. (*p < 0.05, **p < 0.01, ***p < 0.001).

[0026] Figure 5 showed DU-145 prostate carcinoma cell-derived xenograft body weight changes. The body weight of vehicle, R4702-E: R4702-exatecan, R4702-M: R4702-MMAE and DS-1062 were recorded twice weekly till Day 25. The latent day was the period after cancer cell implantation but before start treatment. Data was shown as the mean ± SEM (N=5 for each group except to GP1 after day 18 and GP9 on day 25).

[0027] Figure 6 showed DU-145 prostate carcinoma cell-derived xenograft tumor growth curves (Figure 6A: 10 mpk of vehicle and test substances; Figure 6B: 3 mpk of vehicle and test substances; Figure 6C: 1 mpk of vehicle and test substances). The tumor volume of vehicle, R4702-E: R4702-exatecan, R4702-M: R4702-MMAE and DS-1062 were recorded twice a week till Day 25. Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1)PATENT Attorney Docket No.: G3004-01900PCT / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 25). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). Latent day was the period after cancer cell implantation but before start treatment. Data was shown as the mean ± SEM [Figure 6A: (N=5 for each group except for control group (N=4; one sacrificed on Day 18 due to tumor volume exceeding 1500 mm3)], [Figure 6B: N=5 for each group except for control group (N=4; one sacrificed on Day 18 due to tumor volume exceeding 1500 mm3) and DS-1062 group (N=4; one sacrificed on Day 22 due to ulcerous tumor)], and [Figure 6C: (N=5 for each group except for control group (N=4; one sacrificed on Day 18 due to tumor volume exceeding 1500 mm3)]. Statistical analyses were performed by Student’s t-test. (*p < 0.05)

[0028] Figure 7 showed NCI-N87 gastric carcinoma cell-derived xenograft body weight changes. The body weight of vehicle, R4702-E: R4702-exatecan, and DS-1062 were recorded twice weekly till Day 23. Data was shown as the mean ± SEM (N=5 for each group).

[0029] Figure 8 showed NCI-N87 gastric carcinoma cell-derived xenograft tumor growth curves (Figure 8A: 10 mpk of vehicle and test substances; Figure 8B: 3 mpk of vehicle and test substances; Figure 8C: 1 mpk of vehicle and test substances). The tumor volume of vehicle, R4702-E: R4702-exatecan, and DS-1062 were recorded twice a week till Day 23. Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 23). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). Data was shown as the mean ± SEM [Figure 8A: (N=5 for G1-G4)], [Figure 8B: (N=5 for each group)], [Figure 8C: (N=5 for each group)]. Statistical analyses were performed by Student’s t-test. (*p < 0.05, **p<0.01, ***p<0.001)

[0030] Figure 9 showed NCI-H1975-C797S lung carcinoma cell-derived xenograft body weight changes. The body weight of vehicle, R4702-E: R4702-exatecan, DS-1062, Osimertinib and IMMU-132 were recorded twice weekly till Day 16. The latent day was the period after cancer cell implantation but before start treatment. Data was shown as the mean ± SEM (N=6 for each group except for control group (N=5) after day 10).

[0031] Figure 10 showed NCI-H1975-C797S lung carcinoma cell-derived xenograft tumor growth curves (Figure 10A: 10 mpk of vehicle and test substances; Figure 10B: 3 mpk of vehicle and test substances). The tumor volume of vehicle, R4702-E: R4702-exatecan, DS-1062, Osimertinib and IMMU-132 were recorded twice a week till Day 16. Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicatePATENT Attorney Docket No.: G3004-01900PCT the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 16). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). Latent day was the period after cancer cell implantation but before start treatment. Data was shown as the mean ± SEM [Figure 10A: N=6 for each group except for control group (N=5) after day 10], and [Figure 10B: N=6 for each group except for control group (N=5) after day 10]. Statistical analyses were performed by Student’s t-test. (*p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0032] Figure 11 showed non-specific binding and off-target effect of OBI-992. Figure 11A: Whole blood samples from two donors (I and II) were incubated with the fluorescein-labeled ADCs for four hours to assess the peripheral blood leukocyte (PBL) association. MFI of each leukocyte population was measured to represent the strength of cell association. Figure 11B: THP-1 cells were incubated with the ADCs, followed by detection using FITC-conjugated antibodies. MFI was measured to represent the strength of cell association. Figure 11C: Cell viability of THP-1 cells treated with ADCs for four days. Percentages of cell viability were calculated. Figure 11D: The differentiated neutrophils were treated with the ADCs for six days. Viable neutrophils (CD66b+ / PI-) were identified by FACS. Relative percentage of CD66b+ / PI- cells were shown with mean and SD.

[0033] Figure 12 showed OBI-992 demonstrates 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 inhibitor with OBI-992 for six days. Percentages of cell viability were calculated, and results were shown with mean and SD (n=3). The IC50 values of each group were calculated by Prism. Fold change of the IC50 value of talazoparib treated with or without OBI-992 was labeled.

[0034] Figure 13 showed OBI-992 demonstrates 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 as the average tumor volume reached 150-200 mm3. Tumor growth was monitored twice weekly till the end of study (Day 22). Figure 13B: Tumor bearing mice were treated with OBI-992 or Olaparib alone or in combination as the average tumor volume reached 150-200 mm3. Tumor growth was monitored twice weekly till the end of study (Day 22). Figure 13C: Tumor bearing mice were treated with OBI-992 or anti- mPD-1 alone or in combination as the average tumor volume reached 200-250 mm3. Tumor growth was monitored twice weekly till the end of study (Day 11). DETAILED DESCRIPTION OF THE INVENTION

[0035] Accordingly, antibody-drug conjugate (ADC) methods and compositions directed to the markers for use in diagnosing and treating a broad spectrum of cancers are provided. An antibody-drug conjugatePATENT Attorney Docket No.: G3004-01900PCT (ADC) comprising a drug conjugated to an antibody or an antigen-binding fragment that binds TROP2 was developed and disclosed herein. Methods of use include, without limitation, cancer therapies and diagnostics. The ADC described herein can bind to a broad spectrum of TROP2-expressing cancer cells, thereby facilitating cancer diagnosis and treatment. General Definitions

[0036] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, 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 (D. N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. 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 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 (D. M. Weir and C. C. Blackwell, eds., 1986).

[0037] As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

[0038] As used herein, the term “immunogenicity” refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

[0039] As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

[0040] As used herein, the term “vaccine” refers to a preparation that contains an antigen, consisting of whole disease-causing organisms (killed or weakened) or components of such organisms, such as proteins, peptides, or polysaccharides, that is used to confer immunity against the disease that the organisms cause. Vaccine preparations can be natural, synthetic or derived by recombinant DNA technology.

[0041] As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.PATENT Attorney Docket No.: G3004-01900PCT

[0042] As used herein, the term "specifically binding," refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10-6moles / liter, about 10-7moles / liter, or about 10-8moles / liter, or less.

[0043] The phrase “substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said 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 for the reference / comparator molecule.

[0044] The phrase “substantially reduced,” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said 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 for the reference / comparator molecule.

[0045] “Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

[0046] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.PATENT Attorney Docket No.: G3004-01900PCT

[0047] The terms “antibody” and “immunoglobulin” are used interchangeably in the 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 so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein). An antibody can be chimeric, human, humanized and / or affinity matured.

[0048] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of heavy or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0049] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0050] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0051] “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, evenPATENT Attorney Docket No.: G3004-01900PCT a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigens, although at a lower affinity than the entire binding site.

[0052] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0053] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0054] Depending on the amino acid sequences 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, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0055] The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably, to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

[0056] “Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen-binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to an intact antibody. For example, such anPATENT Attorney Docket No.: G3004-01900PCT antibody fragment may comprise an antigen-binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[0057] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (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, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567), phage display technologies (See, 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 technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, 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. Pat.PATENT Attorney Docket No.: G3004-01900PCT Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 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.14: 845-851 (1996); Neuberger, Nature Biotechnol.14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol.13: 65-93 (1995).

[0058] The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0059] Antibodies of the present invention also include chimerized or humanized monoclonal antibodies generated from antibodies of the present invention.

[0060] The antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but 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 fragment (e.g., Ward et al, Nature, 341 :544-546 (1989)), an CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al, Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

[0061] The antibodies or antigen-binding portions thereof of the present invention may be monospecific, bi-specific or multispecific.

[0062] All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD, or IgE (all classes and subclasses are encompassed by the present invention). The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof. The light chains of the antibody may be of kappa or lambda type.

[0063] Thus, anti-cancer antibodies of the present invention include in combination with a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, of non-murine origin, preferably of human origin, which can be incorporated into an antibody of the present invention.PATENT Attorney Docket No.: G3004-01900PCT

[0064] Antibodies with a variable heavy chain region and a variable light chain region 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%o, 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 region and variable light chain region of the antibody produced by the reference antibody, and can also bind to TROP2. Homology can be present at either the amino acid or nucleotide sequence level.

[0065] The antibodies or antigen-binding portions may be peptides. Such peptides can include variants, analogs, orthologs, homologs and derivatives of peptides, that exhibit a biological activity, e.g., binding of a carbohydrate antigen. The peptides may contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), peptides with substituted linkages, as well as other modifications known in the art.

[0066] Also within the scope of the invention are antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted or added. In an exemplary embodiment, these alternations do not have a substantial effect on the peptide's biological properties such as binding affinity. In another exemplary embodiment, antibodies may have amino acid substitutions in the framework region, such as to improve binding affinity of the antibody to the antigen. In yet another exemplary embodiment, a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids. The donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make 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, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989). Pearson, Methods Mol. Biol.243:307-31 (1994). Gonnet et al., Science 256: 1443-45 (1992).

[0067] The antibody, or antigen-binding portion thereof, can be derivatized or linked to another functional molecule. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag), amino acid linkers, signal sequences, immunogenic carriers, or ligands useful in protein purification, such as glutathione-S-transferase,PATENT Attorney Docket No.: G3004-01900PCT histidine tag, and staphylococcal protein A. One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types). Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m- maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111. Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting, exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase and the like. A protein can also be derivatized with a prosthetic group (e.g., streptavidin / biotin and avidin / biotin).

[0068] Nucleic acids encoding a functionally active variant of the present antibody or antigen-binding portion thereof are also encompassed by the present invention. These nucleic acid molecules may hybridize with a nucleic acid encoding any of the present antibody or antigen-binding portion thereof 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, N.Y.6.3.1-6.3.6, 1989, which is incorporated herein by reference. Specific hybridization conditions referred to herein are as follows: 1) medium stringency hybridization conditions: 6 X SSC at about 45 °C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 60 °C; 2) high stringency hybridization conditions: 6 X SSC at about 45 °C, followed by one or more washes in 0.2XSSC, 0.1% SDS at 65 °C; and 3) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65 °C, followed by one or more washes at 0.2XSSC, 1% SDS at 65 °C.

[0069] A nucleic acid encoding the present antibody or antigen-binding portion thereof may be introduced into an expression vector that can be expressed in a suitable expression system, followed by isolation or purification of the expressed antibody or antigen-binding portion thereof. Optionally, a nucleic acid encoding the present antibody or antigen-binding portion thereof 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).

[0070] The present antibodies or antigen-binding portions thereof can be produced by host cells transformed with DNA encoding light and heavy chains (or portions thereof) of a desired antibody. Antibodies can be isolated and purified from these culture supernatants and / or cells using standard techniques. For example, a host cell may be transformed with DNA encoding the light chain, the heavy chain, or both, of an antibody. Recombinant DNA technology may also be used to remove some or all of thePATENT Attorney Docket No.: G3004-01900PCT DNA encoding either or both of the light and heavy chains that is not necessary for binding, e.g., the constant region.

[0071] The present nucleic acids can be expressed in various suitable cells, including prokaryotic and eukaryotic cells, e.g., bacterial cells, (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting examples of the cells include all cell lines of mammalian origin or mammalian-like characteristics, including but not limited to, parental cells, derivatives and / or engineered variants of 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-Darby bovine kidney (MDBK), myeloma and lymphoma cells. The engineered variants include, e.g., glycan profile modified and / or site-specific integration site derivatives.

[0072] The present invention also provides for cells comprising the nucleic acids described herein. The cells may be a hybridoma or transfectant.

[0073] Alternatively, the present antibody or antigen-binding portion thereof can be synthesized by solid phase procedures well known in the art. Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989). Methods in Molecular Biology, Vol.35: Peptide Synthesis Protocols (ed. M. W. Pennington and B. M. Dunn), chapter 7. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984). G. Barany and R. B. 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).

[0074] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulinPATENT Attorney Docket No.: G3004-01900PCT sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a 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). See also the following review articles and 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).

[0075] The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are 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 refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0076] “Framework” or “FW” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0077] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0078] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables 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).PATENT Attorney Docket No.: G3004-01900PCT

[0079] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in 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 the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO93 / 1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0080] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0081] An “affinity matured” antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the 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 CDR and / or framework residues is 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).

[0082] A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0083] An “agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

[0084] A “disorder” is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.

[0085] The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.PATENT Attorney Docket No.: G3004-01900PCT

[0086] “Tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

[0087] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0088] As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and / or tissue / organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

[0089] An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

[0090] “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.

[0091] An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0092] A “therapeutically effective amount” of a substance / molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.PATENT Attorney Docket No.: G3004-01900PCT

[0093] A “combination” refers to combination therapy would be the amount of the antibody-drug conjugate and / or the amount of other biological or chemical drugs that when administered together (either as co-administration and / or co-formulation), either sequentially or simultaneously, on the same or different days during a treatment cycle, have a synergistic effect that is therapeutically effective and more than therapeutically additive.

[0094] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0095] The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g.,211At,131I,125I,90Y,186Re,188Re,153Sm,212Bi,32P,60C, and radioactive isotopes of lutetium-177, strontium- 89 and samarium (153Sm) ), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0096] The term “photodynamic therapy (PDT)’, sometimes called photochemotherapy, is a form of phototherapy involving light and a photosensitizing chemical substance, used in conjunction with molecular oxygen to elicit cell death (phototoxicity). It is used clinically to treat a wide range of medical conditions, including wet age-related macular degeneration, psoriasis, atherosclerosis and has shown some efficacy in anti-viral treatments, including herpes. It also treats malignant cancers including head and neck, lung, bladder, skin and prostate cancer (Wang, SS et al. Cancer Journal.8 (2): 154–63.2002). The “photodynamic therapeutic agent” is 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), Pyropheophorbidea (PPa) or Pheophorbide a (PhA).

[0097] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (also called DM1), anthracycline, pyrrolobenzodiazepine, ^-amanitin, tubulysin, benzodiazepine, erlotinib (TARCEVA®), Genentech / OSI Pharm.), bortezomib (VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sunitinib (SUTENT®, SU11248, Pfizer), letrozole (FEMARA®), Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®,PATENT Attorney Docket No.: G3004-01900PCT Sanofi), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafarnib (SARASAR®, SCH 66336), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®polysaccharidePATENT Attorney Docket No.: G3004-01900PCT complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE®doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil; 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 pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0098] Also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON. toremifene; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN®exemestane, formestanie, fadrozole, RIVISOR®vorozole, FEMARA®letrozole, and ARIMIDEX®anastrozole; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in adherent cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, 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) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0099] Protein kinase inhibitors include tyrosine kinase inhibitors which inhibit to some extent tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor. Examples of tyrosine kinase inhibitors include EGFR-targeted drugs such as: (i) antibodies which bind to EGFR, including MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see,PATENT Attorney Docket No.: G3004-01900PCT U.S. Pat. No.4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®, Imclone) and reshaped human 225 (H225) (WO 96 / 40210, Imclone Systems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR (U.S. Pat. No.5,891,996); and human antibodies that bind EGFR, such as ABX-EGF (WO 98 / 50433); (ii) anti-EGFR antibody conjugated with a cytotoxic agent (EP 659439A2); and small molecules that bind to EGFR including ZD1839 or Gefitinib (IRESSA™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA™; Genentech / OSI) and AG1478, AG1571 (SU 5271; Sugen), quinazolines such as PD 153035,4-(3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3- d]pyrimidines, curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to ErbB- encoding nucleic acid); quinoxalines (U.S. Pat. No.5,804,396); tryphostins (U.S. Pat. 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); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); or as described in: U.S. Pat. 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 99 / 06396 (Warner Lambert); WO 96 / 30347 (Pfizer, Inc); WO 96 / 33978 (Zeneca); WO 96 / 3397 (Zeneca); and WO 96 / 33980 (Zeneca).

[0100] An “anti-angiogenic agent” refers to a compound which blocks, or interferes with to some degree, the development of blood vessels. The anti-angiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. An exemplary anti-angiogenic agent is an antibody that binds to Vascular Endothelial Growth Factor (VEGF) such as bevacizumab (AVASTIN®, Genentech).

[0101] The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone 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); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transformingPATENT Attorney Docket No.: G3004-01900PCT growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) 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, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0102] The term “prodrug” as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., 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 this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate- containing prodrugs, peptide-containing prodrugs, D-amino acid-modified pro drugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.

[0103] A “liposome” is a small vesicle composed of various types of lipids, phospholipids and / or surfactant which is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

[0104] The phrase “pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an ADC. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1′-methylene- bis-(2-hydroxy-3-naphthoate)) 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 may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, aPATENT Attorney Docket No.: G3004-01900PCT pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion.

[0105] “Pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and 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.

[0106] The term “imaging agent” as used in this application refers to a fluorophore, a dye, an MRI contrast agent or a radionuclide. These agents are used in diagnostic assays for therapeutic dosing of the ADC compounds.

[0107] A method of selecting a patient for cancer therapy by imaging, wherein the method comprises: (a) Administering to the patient an effective amount of the ADC; and (b) Detecting a reporting signal of an imaging agent in the patient; wherein the imaging agent is a fluorophore, a dye, an MRI contrast agent or a radionuclide; and wherein the reporting signal is detected visually or instrumentally. General Features of Exemplary Antibody-Drug Conjugates

[0108] The compounds of the invention include those with utility for anticancer activity. In particular, the compounds include an antibody conjugated, i.e. covalently attached by a linker, to a drug moiety where the drug when not conjugated to an antibody has a cytotoxic or cytostatic effect. The biological activity of the drug moiety is thus modulated by conjugation to an antibody. The antibody-drug conjugates (ADCs) of the invention may selectively deliver an effective dose of a cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose may be achieved.

[0109] Antibody-drug conjugates (ADCs) may be represented by Formula I: Ab-(L-D)n (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is an antibody which binds TROP2, or which binds to one or more tumor-associated antigens or cell- surface receptors; n is the Drug-to-antibody ratio (DAR) and ranging from 1 to 8.

[0110] An antibody-drug conjugate (ADC) comprise an antibody covalently attached by a linker to one or more MMAE moieties. ADC may be represented by Formula I: Ab-(L-D)n(I)PATENT Attorney Docket No.: G3004-01900PCT wherein one or more Exatecan drug moieties (D) are covalently linked by L to an antibody (Ab). Ab is an antibody which targets TROP2 or which binds to one or more tumor-associated antigens or cell-surface receptors. The linker L may be stable outside a cell, i.e. extracellular.

[0111] In another embodiment, wherein the linker comprises a maleimide or a derivative thereof conjugated to a thio group of the antibody. Especially, the linker is a 4-(N-Maleimidomethyl)-cyclohexane- 1-carboxylate (MCCa).

[0112] In one embodiment, a substantial amount of the drug moiety is not cleaved from the antibody until the antibody-drug conjugate enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibody-drug conjugate does enter the cell.

[0113] In another embodiment, the ADC specifically binds to TROP2. The ADC may inhibit growth of tumor cells which expresses TROP2.

[0114] In another embodiment, the antibody (Ab) of Formula I is a human, chimeric or humanized antibody.

[0115] In another embodiment, the anti-TROP2 antibody is selected from hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamb, or sacituzumab.

[0116] Another aspect of the invention is a pharmaceutical composition including a Formula I compound, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0117] Another aspect provides a pharmaceutical combination comprising a Formula I compound and a second compound having anti-cancer properties or other therapeutic effects.

[0118] Another aspect includes diagnostic and therapeutic uses for the compounds and compositions disclosed herein.

[0119] Another aspect is a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating the cells with an amount of an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.

[0120] Another aspect are methods of treating cancer comprising administering to a patient a formulation of a Formula I compound. One method is for the treatment of cancer in a mammal, wherein the cancer is characterized by the expression of TROP2. The mammal optionally does not respond, or responds poorly, toPATENT Attorney Docket No.: G3004-01900PCT treatment with an unconjugated Anti-TROP2 antibody. The method comprises administering to the mammal a therapeutically effective amount of an antibody-drug conjugate compound.

[0121] Another aspect is a method of inhibiting the growth of tumor cells that expresses TROP2 comprising administering to a patient an antibody-drug conjugate compound which binds specifically to said growth factor receptor and a chemotherapeutic agent wherein said antibody-drug conjugate and said chemotherapeutic agent are each administered in amounts effective to inhibit growth of tumor cells in the patient.

[0122] Another aspect is a method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by expression of TROP2, comprising administering a combination of an antibody- drug conjugate compound of Formula I and a chemotherapeutic agent.

[0123] Another aspect is an assay method for detecting cancer cells comprising: exposing cells to an antibody-drug conjugate compound, and determining the extent of binding of the antibody-drug conjugate compound to the cells.

[0124] Another aspect concerns methods of screening ADC drug candidates for the treatment of a disease or disorder where the disease or disorder is characterized by the expression of TROP2.

[0125] Another aspect includes articles of manufacture, i.e. kits, comprising an antibody-drug conjugate, a container, and a package insert or label indicating a treatment.

[0126] Another aspect includes methods of treating a disease or disorder characterized by the overexpression of TROP2 in a patient with the antibody-drug conjugate compounds.

[0127] Another aspect includes methods of making, methods of preparing, methods of synthesis, methods of conjugation, and methods of purification of the antibody-drug conjugate compounds, and the intermediates for the preparation, synthesis, and conjugation of the antibody-drug conjugate compounds. ADCs: antibodies

[0128] The antibody unit (Ab-) of Formula I includes within its scope any unit of an antibody that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population. An antibody can be any protein or protein-like molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified. In one aspect, the antibody unit acts to deliver the maytansinoid drug moiety to the particular target cell population with which the antibody unit reacts. Such antibodies include, but are not limited to, large molecular weight proteins such as, full-length antibodies and antibody fragments.PATENT Attorney Docket No.: G3004-01900PCT

[0129] Antibodies comprising the antibody-drug conjugates of the invention preferably retain the antigen binding capability of their native, wild type counterparts. Thus, antibodies of the invention are capable of binding, preferably specifically, to antigens.

[0130] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full- length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.

[0131] For example, the antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but 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 fragment (e.g., Ward et al., Nature, 341:544-546 (1989)), an isolated CDR, diabodies, triabodies, tetrabodies, linear antibodies, single- chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker, are also encompassed by the present invention. Bird et al. Science, 1988, 242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85:5879-5883.

[0132] All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE (all classes and subclasses are encompassed by the present invention). The antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof. The light chains of the antibody may be of kappa or lambda type.PATENT Attorney Docket No.: G3004-01900PCT

[0133] The variable regions of the present antibodies or antigen-binding portions thereof can be from a non-human or human source. The framework of the present antibodies or antigen-binding portions thereof can be human, humanized, non-human (e.g., a murine framework modified to decrease antigenicity in humans), or a synthetic framework (e.g., a consensus sequence).

[0134] In one embodiment, the present antibodies, or antigen-binding portions thereof, comprise at least one heavy chain variable region and / or at least one light chain variable region.

[0135] The present antibodies or antigen-binding portions thereof specifically bind to TROP2 with a dissociation constant (KD) of less than about 10E-7 M, less than about 10E-8 M, less than about 10E-9 M, less than about 10E-10 M, less than about 10E-11 M, or less than about 10E-12 M. In one embodiment, the antibody or the antibody binding portion thereof has a dissociation constant (KD) of 1˜10×10E-9 or less. In another embodiment, the Kd is determined by surface plasmon resonance.

[0136] Antibodies comprising the antibody-drug conjugates of the invention preferably retain the antigen binding capability of their native, wild type counterparts. Thus, antibodies of the invention are capable of binding, preferably specifically, to antigens. Such antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules associated with (for e.g., known or suspected to contribute functionally to) tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis and molecules associated with (for e.g., known or suspected to contribute functionally to) angiogenesis. The tumor-associated antigen may be a cluster differentiation factor (i.e., a CD protein). An antigen to which an antibody of the invention is capable of binding may be a member of a subset of one of the above-mentioned categories, wherein the other subset(s) of said category comprise other molecules / antigens that have a distinct characteristic (with respect to the antigen of interest).

[0137] In one embodiment, the antibody of the antibody-drug conjugates (ADCs) specifically binds to TROP2

[0138] In some embodiments, the antibodies or antigen-binding portions thereof include, for example, the variable heavy chains and / or variable light chains of the Anti-TROP2 antibody (R4702), as shown in Table 1.

[0139] In related embodiments, the exemplary antibodies or antigen-binding portions thereof include, for example, the CDRs of the variable heavy chains and / or the CDRs of the variable light chains of Anti- TROP2 antibody (R4702). The exemplary CDRs and frameworks of the variable heavy chains and the variable light chains from these hybridoma clones are shown in Table 1.PATENT Attorney Docket No.: G3004-01900PCT Table 1. Anti-TROP2 antibody (R4702) amino acid sequence Variable Region Amino Acid Sequences SEQ ID NO. Heavy Chain CDR1 GYSFTTHWIH 1 [ eastabout 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 region and variable light chain region of the antibody bind to TROP2. Homology can be present at either the amino acid or nucleotide sequence level. ADC Targeting TROP2

[0141] One aspect of the present disclosure features the new ADC (OBI-992) specific to TROP2.

[0142] Any of the exemplary antibodies described herein can be a full length antibody or an antigen- binding fragment thereof. In some examples, the antigen binding fragment is a Fab fragment, a F(ab')2fragment, or a single-chain Fv fragment. In some examples, the antigen binding fragment is a Fab fragment,PATENT Attorney Docket No.: G3004-01900PCT a 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.

[0143] Any of the exemplary antibodies described herein has one or more characteristics of: (a) is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, an antibody fragment, a bispecific antibody, a monospecific antibody, a monovalent antibody, an IgG1 antibody, an IgG2 antibody, or derivative of an antibody; (b) is a human, murine, humanized, or chimeric antibody, antigen-binding fragment, or derivative of an antibody; (c) is a single-chain antibody fragment, a multibody, a Fab fragment, and / or an immunoglobulin of the IgG, IgM, IgA, IgE, IgD isotypes and / or subclasses thereof; (d) has one or more of the following characteristics: (i) mediates ADCC and / or CDC of cancer cells; (ii) induces and / or promotes apoptosis of cancer cells; (iii) inhibits proliferation of target cells of cancer cells; (iv) induces and / or promotes phagocytosis of cancer cells; and / or (v) induces and / or promotes the release of cytotoxic agents; (e) specifically binds the tumor-associated carbohydrate antigen, which is a tumor-specific carbohydrate antigen; (f) does not bind an antigen expressed on non- cancer cells, non-tumor cells, benign cancer cells and / or benign tumor cells; and / or (g) specifically binds a tumor-associated carbohydrate antigen expressed on cancer stem cells and on normal cancer cells.

[0144] Preferably the binding of the antibodies to their respective antigens is specific. The term "specific" is generally used to refer to the situation in which one member of a binding pair will not show any significant binding to molecules other than its specific binding partner (s) and e.g. has less than about 30%, preferably 20%, 10%, or 1 % cross-reactivity with any other molecule other than those specified herein. Production of Antibodies

[0145] Various methods have been employed to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans. Other methods to prepare MAbs, including chimeric and humanized antibodies, uses genetic engineering, i.e. recombinant DNA techniques.

[0146] Polyclonal antibodies may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0147] Human myeloma and mouse-human heteromyeloma cell lines also have 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,PATENT Attorney Docket No.: G3004-01900PCT 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Binding specificity of monoclonal antibodies produced by hybridoma cells may be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al (1980) Anal. Biochem.107:220.

[0148] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells (US 2005 / 0048572; US 2004 / 0229310). Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al (1993) Curr. Opinion in Immunol.5:256-262 and Plückthun (1992) Immunol. Revs.130:151-188.

[0149] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques 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 describe the isolation of murine and human antibodies, respectively, 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 combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al (1993) Nuc. Acids. Res.21:2265- 2266). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0150] The DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567); and Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81:6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0151] Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.PATENT Attorney Docket No.: G3004-01900PCT ADCs: the linkers Exemplary ADC linker

[0152] Suitable exemplary linkers for the ADC are described in, for example, US Patent No.7595292 (WO2005 / 007197). The entire content directed to linkers is hereby incorporated by reference herein. The linker, L, attaches the antibody to a drug moiety through covalent bonds, not comprising a disulfide group. The linker is a bifunctional or multifunctional moiety which can be used to link one or more drug moieties (D) and an antibody unit (Ab) to form antibody-drug conjugates (ADCs) of Formula I. Antibody-drug conjugates (ADCs) can be conveniently prepared using a linker having reactive functionality for binding to the Drug and to the Antibody. A cysteine thiol, or an amine, e.g. N-terminus or amino acid side chain such as lysine, of the antibody (Ab) can form a bond with a functional group of a linker reagent, drug moiety or drug-linker reagent.

[0153] The linkers are preferably stable extracellularly. Before transport or delivery into a cell, the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the antibody remains linked to the drug moiety. The linkers are stable outside the target cell and may be cleaved at some efficacious rate inside the cell. An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e. not cleaved, until the conjugate has been delivered or transported to its targeted site; and (iv) maintain a cytotoxic, cell-killing effect or a cytostatic effect of the maytansinoid drug moiety. Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation / analysis technique LC / MS.

[0154] Covalent attachment of the antibody and the drug moiety requires the linker to have two reactive functional groups, i.e. bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p234-242).

[0155] Exemplary ADC Linkers can include biologically active compounds of the general formula II in which one of X and X′ represents a polymer (especially 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 preparable by reduction of an electron-withdrawing moiety; or, if X′ represents a polymer, X-Q-W- together may represent an electron withdrawing group; and in addition, if X represents a polymer, X′ and electron withdrawing group W together with the interjacent atoms may form a ring; each of Z1and Z2independently represents a group derived from a biological molecule, each of which is linked to A and B via a nucleophilicPATENT Attorney Docket No.: G3004-01900PCT moiety; or Z1and Z2together represent a single group derived from a biological molecule which is linked to A and B via two nucleophilic moieties; A is a C1-5alkylene or alkenylene chain; and B is a bond or a C1-4alkylene or alkenylene chain; are formed by conjugating a suitable polymer to a suitable biologically active molecule via nucleophilic groups in said molecule, preferably via a disulphide bridge.provides a protein-polymer conjugate of formula IIIpolymer (especially a toxin) selected from the group consisting of polyalkylene glycols, polyvinylpyrrolidones, polyacrylates, polymethacrylates, polyoxazolines, polyvinylalcohols, polyacrylamides, polymethacrylamides, HPMA copolymers, polyesters, polyacetals, poly(ortho ester)s, polycarbonates, poly(imino carbonate)s, polyamides, copolymers of divinylether-maleic anhydride and styrene-maleic anhydride, polysaccharides, and polyglutamic acids; Q is a linking group selected from the group consisting of a direct bond, alkylenes, optionally-substituted aryls, and optionally-substituted heteroaryls, wherein the alkylene, aryl, or heteroaryl may be terminated or interrupted by one or more oxygen atoms, sulphur atoms, keto groups, —O—CO— groups, —CO—O— groups, or —NR groups in which R is an alkyl or aryl group; W is selected from the group consisting of a keto group, an ester group, a sulphone group, a reduced keto group, a reduced ester group, and a reduced sulphone group; X′-Q is hydrogen; A is a C1-5 alkylene or alkenylene chain; B is a bond or a C1-4 alkylene or alkenylene chain; and Z is a single protein linked to A and B via two thiol groups generated by reduction of a disulfide bridge in the protein. Activity Assays Demonstrating the efficacy of the exemplary ADCs

[0157] ADC of the invention (OBI-992) can be characterized for their physical / chemical properties and biological functions by various assays known in the art.

[0158] Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. The affinity of an antibody for a carbohydrate antigen can be determined experimentally using any suitable method (see, e.g., BerzofskyPATENT Attorney Docket No.: G3004-01900PCT et al, "Antibody- Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular antibody-carbohydrate antigen interaction can vary if measured under different conditions {e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Ka, Ka) are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

[0159] The present antibodies or antigen-binding portions thereof have in vitro and in vivo therapeutic, prophylactic, and / or diagnostic utilities. For example, these antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and / or diagnose cancer.

[0160] Purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

[0161] Where necessary, antibodies are analyzed for their biological activity. In some embodiments, antibodies of the invention are tested for their antigen binding activity. The antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, chemiluminescent immunoassays, nanoparticle immunoassays, aptamer immunoassays, and protein A immunoassays. Humanized Antibodies

[0162] The invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.PATENT Attorney Docket No.: G3004-01900PCT

[0163] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is 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 particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. 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.

[0164] It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. Uses

[0165] An ADC of the invention (OBI-992) may be used in, for example, in vitro, ex vivo and in vivo therapeutic methods. ADC of the invention (OBI-992) can be used as an antagonist to partially or fully block the specific antigen activity in vitro, ex vivo and / or in vivo. Accordingly, ADCs of the invention (OBI-992) can be used to inhibit a specific antigen activity, e.g., in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an ADC of the invention (OBI-992) cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse). In one embodiment, an ADC of the invention (OBI-992) can be used for inhibiting antigen activities by contacting the ADC (OBI-992) with the antigen such that antigen activity is inhibited. In one embodiment, the antigen is a human protein molecule.

[0166] In one embodiment, an ADC of the invention (OBI-992) can be used in a method for inhibiting an antigen in a subject suffering from a disorder in which the antigen activity is detrimental, comprisingPATENT Attorney Docket No.: G3004-01900PCT administering to the subject an ADC of the invention (OBI-992) such that the antigen activity in the subject is inhibited. In one embodiment, the antigen is a human protein molecule and the subject is a human subject. Alternatively, the subject can be a mammal expressing the antigen with which an ADC of the invention (OBI-992) binds. Still further the subject can be a mammal into which the antigen has been introduced (e.g., by administration of the antigen or by expression of an antigen transgene). An ADC of the invention (OBI- 992) can be administered to a human subject for therapeutic purposes. Moreover, an ADC of the invention (OBI-992) can be administered to a non-human mammal expressing an antigen with which the ADC (OBI- 992) cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of ADCs of the invention (OBI-992) (e.g., testing of dosages and time courses of administration).

[0167] ADCs of the invention (OBI-992) can be used either alone or in combination with other compositions in a therapy. For instance, an ADC of the invention (OBI-992) may be co-administered with another antibody, and / or adjuvant / therapeutic agents (e.g., steroids). For instance, an ADC of the invention (OBI-992) may be combined with an anti-inflammatory and / or antiseptic in a treatment scheme, e.g. in treating any of the diseases described herein, including cancer, muscular disorders, ubiquitin-pathway- related genetic disorders, immune / inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders. Such combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations), and separate administration, in which case, administration of the ADC of the invention (OBI-992) can occur prior to, and / or following, administration of the adjunct therapy or therapies.

[0168] An ADC of the invention (OBI-992) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the ADC (OBI-992) is suitably administered by pulse infusion, particularly with declining doses of the ADC (OBI-992). Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Therapeutic Applications

[0169] Described herein are therapeutic methods that include administering to a subject in need of such treatment a therapeutically effective amount of a composition that includes one or more ADCs (OBI-992) described herein.PATENT Attorney Docket No.: G3004-01900PCT

[0170] In some embodiments, the subject (e.g., a human patient) in need of the treatment is diagnosed with, suspected of having, or at risk for cancer. Examples of the cancer include, but are not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer.

[0171] In preferred embodiments, the ADC (OBI-992) is capable of targeting TROP2-expressing cancer cells. In some embodiments, the ADC (OBI-992) is capable of targeting TROP2 on cancer cells. In some embodiments, the ADC (OBI-992) is capable of targeting TROP2 in cancers.

[0172] The treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and / or prolongation of time to tumor cancer progression.

[0173] In some embodiments, the treatment further comprises administering an additional therapy to said subject prior to, during or subsequent to said administering of the ADCs (OBI-992). In some embodiments, the additional therapy is treatment with a chemotherapeutic agent. In some embodiments, the additional therapy is radiation therapy.

[0174] The methods of the invention are particularly advantageous in treating and preventing early stage tumors, thereby preventing progression to the more advanced stages resulting in a reduction in the morbidity and mortality associated with advanced cancer. The methods of the invention are also advantageous in preventing the recurrence of a tumor or the regrowth of a tumor, for example, a dormant tumor that persists after removal of the primary tumor, or in reducing or preventing the occurrence of a tumor.

[0175] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having cancer, which include, but not limited to, lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer and oral cancer. A subject having cancer can be identified by routine medical examination.

[0176] “An effective amount” as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specificPATENT Attorney Docket No.: G3004-01900PCT route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0177] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer.

[0178] "Development" or "progression" of cancer means initial manifestations and / or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein "onset" or “occurrence” of cancer includes initial onset and / or recurrence.

[0179] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0180] Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble ADCs (OBI-992) can be administered by the drip method, whereby a pharmaceutical formulation containing the ADC (OBI-992) and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.PATENT Attorney Docket No.: G3004-01900PCT Administration of Antibody-Drug Conjugate Pharmaceutical Formulations

[0181] Therapeutic antibody-drug conjugates (ADCs) may be administered by any route appropriate to the condition to be treated. The ADC will typically be administered parenterally, i.e. infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, bolus, intratumor injection or epidural (Shire et al (2004) J. Pharm. Sciences 93(6):1390-1402). Pharmaceutical formulations of therapeutic antibody-drug conjugates (ADCs) are typically prepared for parenteral administration with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form. An antibody-drug conjugate (ADC) having the desired degree of purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers, in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

[0182] Acceptable parenteral vehicles, diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). For example, lyophilized anti-ErbB2 antibody formulations are described in WO 97 / 04801, expressly incorporated herein by reference. An exemplary formulation of an ADC such as trastuzumab-SMCC-DM1 contains about 100 mg / ml of trehalose (2-(hydroxymethyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-tetrahydropyran-3,4,5- triol; C12H22O11; CAS Number 99-20-7) and about 0.1% TWEEN™ 20 (polysorbate 20; dodecanoic acid 2- [2-[3,4-bis(2-hydroxyethoxy)tetrahydrofuran-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl ester; C26H50O10; CAS Number 9005-64-5) at approximately pH 6.

[0183] Pharmaceutical formulations of a therapeutic antibody-drug conjugate (ADC) may contain certain amounts of unreacted drug moiety (D), antibody-linker intermediate (Ab-L), and / or drug-linker intermediate (D-L), as a consequence of incomplete purification and separation of excess reagents, impurities, and by- products, in the process of making the ADC; or time / temperature hydrolysis or degradation upon storage of the bulk ADC or formulated ADC composition.PATENT Attorney Docket No.: G3004-01900PCT

[0184] The active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0185] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the ADC, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma- ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

[0186] The formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0187] The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0188] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one orPATENT Attorney Docket No.: G3004-01900PCT more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

[0189] The pharmaceutical compositions of ADC may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation 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-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

[0190] The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL / hour can occur. Subcutaneous (bolus) administration may be effected with about 1.5 ml or less of total volume and a concentration of about 100 mg ADC per ml. For ADC that require frequent and chronic administration, the subcutaneous route may be employed, such as by pre-filled syringe or autoinjector device technology.

[0191] As a general proposition, the initial pharmaceutically effective amount of ADC administered per dose will be in the range of about 0.01-100 mg / kg, namely about 0.1 to 20 mg / kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg / kg / day. For example, human patients may be initially dosed at about 1.5 mg ADC per kg patient body weight. The dose may be escalated to the maximally tolerated dose (MTD). The dosing schedule may be about every 3 weeks, but according to diagnosed condition or response, the schedule may be more or less frequent. The dose may be further adjusted during the course of treatment to be at or below MTD which can be safely administered for multiple cycles, such as about 4 or more.

[0192] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.PATENT Attorney Docket No.: G3004-01900PCT

[0193] Although oral administration of protein therapeutics are generally disfavored due to poor bioavailability due to limited absorption, hydrolysis or denaturation in the gut, formulations of ADC suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the ADC.

[0194] The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Exemplary unit dosage formulations contain a daily dose or unit daily sub-dose, or an appropriate fraction thereof, of the active ingredient.

[0195] The invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

[0196] For the prevention or treatment of disease, the appropriate dosage of an ADC will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g.0.1-20 mg / kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg / kg of patient weight.

[0197] For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg / kg, followed by a weekly maintenance dose of about 2 mg / kg of the anti-ErbB2 antibody. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. Combination TherapyPATENT Attorney Docket No.: G3004-01900PCT

[0198] An antibody-drug conjugate (ADC) may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the ADC of the combination such that they do not adversely affect each other.

[0199] The second compound may be a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, aromatase inhibitor, protein kinase inhibitor, lipid kinase inhibitor, anti-androgen, antisense oligonucleotide, ribozyme, gene therapy vaccine, anti-angiogenic agent and / or cardio protectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. A pharmaceutical composition containing an ADC may also have a therapeutically effective amount of a chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.

[0200] Metabolite products may be identified by preparing a radiolabeled (e.g.14C or3H) ADC, administering it parenterally in a detectable dose (e.g. greater than about 0.5 mg / kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the metabolite). The metabolite structures are determined in conventional fashion, e.g. by MS, LC / MS or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well-known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the ADC compounds.

[0201] Metabolites include the products of in vivo cleavage of the ADC where cleavage of any bond occurs that links the drug moiety to the antibody. Metabolic cleavage may thus result in the naked antibody, or an antibody fragment. The antibody metabolite may be linked to a part, or all, of the linker. Metabolic cleavage may also result in the production a drug moiety or part thereof. The drug moiety metabolite may be linked to a part, or all, of the linker. Articles of Manufacture

[0202] In another embodiment, an article of manufacture, or “kit”, containing ADC and materials useful for the treatment of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, or blister pack. The containers may be formed from a variety of materialsPATENT Attorney Docket No.: G3004-01900PCT such as glass or plastic. The container holds an antibody-drug conjugate (ADC) composition which is 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 having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an ADC. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.

[0203] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES Example 1: TROP2 immunohistochemistry (IHC) expression in human normal and tumor tissue

[0204] 1.1 Material 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 with Background-Reducing components (Dako, Cat No. S3022) (k) Xylene (Leica, Cat No.3803665) (l) Micromount mounting medium (Leica, Cat No.3801731) 1.1.2 Equipment / supplies (a) Positive- charged slides (Fisherbrand Superfrost Plus Slides or equivalent) (b) Drying oven (c) Antigen Retrieval Chamber, Dako PT Link (d) Autostainer Link 48 (Dako)PATENT Attorney Docket No.: G3004-01900PCT 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

[0205] 1.2 Procedure 1.2.1 Reagent Preparation (a) EnVision FLEX High pH TRS: Dilute 30mL of 50x TRS solution with 1470 mL of deionized water, 1:50 dilution for a final concentration of 1x. Note: Discard after 2 uses and no longer than 5 days after dilution (b) Envision FLEX Wash Buffer: Dilute 1:20 by adding 100 mL of 20x Wash Buffer concentrate to every 1900 mL of deionized water. Note: Diluted solution may be used for one week at ambient temperature or stored at 2-8 °C for one month. Discard diluted solution if cloudy in appearance (c) Envision FLEX DAB+: Add a drop of Liquid DAB+ Chromogen per 1 mL of DAB+ Substrate Buffer and mix well. Note: Prepared Substrate Chromogen Solution (DAB) is stable for 5 days when stored at 2-8 °C (d) TROP2 antibody: Concentrated antibody is supplied as 0.11 mg / mL. Dilute 1:150 of concentrated antibody to Dako Background-Reducing Antibody diluent, for a final concentration of 0.73 µg / mL. Note: Working solution expires after 1 day from preparation (e) Isotype control antibody: Concentrated antibody is supplied as 1.675 mg / mL. Dilute 1:2284 of concentrated antibody to Dako Background-Reducing Antibody diluent, for a final concentration of 0.73 µg / mL. Note: Working solution expires after 1 day from preparation. 1.2.2 Slide Pretreatment and Retrieval (a) FFPE tissue blocks are sectioned at 4-5 µm, mounted on positively charged slides, and allowed to air- dry. Slides are stored at room temperature before use. (b) Slides are heated in a 60±2 °C drying oven for 60 minutes. (c) Make labels for slides to be stained with TROP2 panel: Under the “New Slides” tab, enter the case number in the ”Case Number” field. Under “Slides”, choosePATENT Attorney Docket No.: G3004-01900PCT “TROP2” panel in “Protocol”. Click “Add Slide”. Default staining is 2 drop zones nearest the label, 150 ^L per drop zone, which position can be adjusted per user discretion. Click “Case Complete” to print labels. Place label on the frosted edge of the slide. (d) Fill PT Link tank with 1.5 L per tank of the 1x working solution of EnVision FLEX High pH TRS (Agilent). Set the PT Link module as follows for TROP2: retrieval temperature should be set at 97 °C, time set at 30 minutes. “Preheat” and “Cool End” temperature both be set at 65 °C. (e) Press “Run” on the PT Link module to preheat the TRS to 65 °C (this takes approximately 20 minutes) and then place rack of slides into the PT Link module. (f) Press “Run” on the PT Link module to warm up TRS to the retrieval temperature of 97 °C (this takes approximately 20 minutes). Slides will be retrieved for 30 minutes at 97 °C once desired temperature reached. (g) Slides are allowed to cool to 65 °C in the module following retrieval (this takes approximately 30 minutes), then removed from the PT Link and placed in the PT Rinse Station Tank filled with 1.5 L 1x FLEX wash buffer to equilibrate for 5 minutes. 3 Reagent bottle preparation and slide loading (a) Reagents from the EnVision™ FLEX, High pH Kit are used to perform the following steps on Dako Link 48 IHC platform. Note: All reagents / antibodies should be equilibrated to ambient temperature prior to immunostaining. All staining steps are performed on Autostainer at ambient temperature (b) In Workflow tab, highlight the slides to be stained and click the “Reagents” button to check the window of “Reagents Required”. For antibodies and DAB+ reagent vials preparation: Select a user-filled reagent; select “Fill Bottle” and input the bottle serial number; Select the appropriate vial code (5 mL, 12 mL or 25 mL); Set the filled quantity based on how much reagent was prepared; Set expiration date and input the lot number; select save then print the label. (c) Place all the required reagents into the reagent rack and place them into the Link48 Autostainer. Ensure buffer and deionized water bottles are filled, and waste containers are empty. (d) Lift racks of slides from PT Link Rinse tank and rinse paraffin off of slides using squeeze bottle filled with 1x Wash Buffer, then load rack(s) of slides onto Dako Autostainer Link48. (e) Select the Instrument tab and click “Prime buffer and water” to prime buffer then water. Click “Start”, the Stainer will scan the slides and reagent barcodes. Click “Start Run” after confirming all reagents and slides are properly scanned.PATENT Attorney Docket No.: G3004-01900PCT 1.2.4 TROP2 Automated IHC Staining Procedures Note: All staining steps are performed on Autostainer at ambient temperature (a) Slides are rinsed with wash buffer and incubated with EnVision FLEX Peroxidase Block for 5 minutes with dispense volume of 300 μL (150 μL x 2 zones), then rinsed with FLEX wash buffer. (b) Slides are incubated with primary antibody (isotype control or TROP2 antibody at final concentration of 0.73 µg / mL in Dako Background-Reducing Antibody diluent) for 60 minutes with dispense volume of 300 μL (150 μL x 2 zones), then rinsed with wash buffer. (c) Slides are incubated with EnVision FLEX HRP for 30 minutes with dispense volume of 300 μL (150 μL x 2 zones), then rinsed with FLEX wash buffer, followed by incubation in FLEX wash buffer for 5 minutes. (d) Visualization is achieved with EnVision FLEX DAB+ for 10 minutes incubation with dispense volume of 300 μL (150 μL x 2 zones), then rinsed with wash buffer. (e) Slides are counterstained using EnVision FLEX Hematoxylin for 5 minutes with dispense volume of 300 μL (150 μL x 2 zones), then rinsed with deionized water. (f) Slides are rinsed with wash buffer and incubated for 5 minutes, then rinsed again with deionized water. 1.2.5 Finishing Run and Slide Mounting (a) Unload the slides from Autostainer. Dehydration and clear are performed manually with graded alcohols and xylene stations. (b) Slides are then cover slipped with mounting medium manually. 1.2.6 Data Analysis: Semi-quantitative Pathologist Evaluation Criteria (a) The percentage and intensity of TROP2 IHC expression will be assessed in tumor cells and read by a certified pathologist. TROP2 IHC staining predominantly shows membranous and cytosolic expression patterns. The percentage of tumor cells with membrane and / or cytoplasmic TROP2 expression will be recorded at each staining intensity (0, 1+, 2+, and 3+). (b) H-score will be used to present the TROP2 expression levels on tumor tissues. The H-score values range from 0 to 300 and are calculated by the following equation: H-score = (percentage of weak intensity x 1) + (percentage of moderate intensity x 2) + (percentage of strong intensity x 3).

[0206] 1.3 ResultPATENT Attorney Docket No.: G3004-01900PCT

[0207] The TROP2 IHC expression in human normal and tumor tissues was illustrated in Figure 1. Figure 1A indicated TROP2 expressed in human breast, lung, cervix, ovary, uterus, prostate, colon, esophagus, pancreas, larynx, stomach and bladder tumor tissues. Figure 1B further listed the calculated TROP2 H-score in every organ individually. Furthermore, recent reference also reported Trop 2 antigen was overexpressed in (a) 58% of oral squamous cell carcinoma; (b) 64% of lung adenocarcinoma, 75% of lung squamous cell carcinoma, and 18% of lung high-grade neuroendocrine tumor; (c) 80% of urothelial cancer and 71% of prostate cancer; (d) 58.6% of epithelial ovarian cancer and 71.8% of endometrial endometrioid carcinoma; (e) 82.5% of papillary thyroid carcinoma; (f) 80% of breast cancers, and 88% of TNBCs; (g) 55% of pancreatic cancers; (h) 56% of gastric carcinoma; and (i) 68.4% of colon cancer (Shutan Liao et al., (2021) Drug Dev Res, 82(8): 1096-1110). Example 2: TROP2 antibody and Topoisomerase inhibitor candidate selection

[0208] To determine the binding kinetics of TROP2 antibody to human TROP2, surface plasmon resonance (SPR) experiments were performed on an Biacore 8K instrument (GE Healthcare). TROP2 antibodies (10 nM) were covalently immobilized on the surface of Protein A sensor chip by using HBS-EP+ buffer, pH 7.4. Briefly, the Protein A chip is orientation-specific binding with antibodies (predominantly human) through Fc-region. The reference flow cell was treated without ligand. The kinetic experiment was performed by 2-fold serial dilutions to give a range of concentrations (0.7, 1.41, 2.81, 5.63, 11.25, 22.5, 45, and 90 nM) of TROP2 Ab in HBS-EP+ buffer, pH 7.4. Same experimental conditions (flow rate of 30 μL / min, contact time 150 seconds and dissociation time 600 seconds, regeneration contact time 30 seconds) were used for each cycle of measurement for analysis of association and dissociation rates. Regeneration of the chip was carried out by washing with glycine HCl, pH 1.5. The level of interaction on the sensor chip is represented as a change in Response Unit (RU). The data analysis was performed using BIA evaluation software with Langmuir fit model of 1:1 binding.

[0209] To evaluate the in vitro binding affinity of TROP2 antibodies to the TROP2 protein, we employed surface plasmon resonance (SPR) analysis using Biacore. The results revealed that R4702 exhibited a relatively high associated rate (7.45E+05 / Ms) and lower disassociated rate (1.50E-04 / s) with TROP2 protein than datopotamab and sacituzumab. Furthermore, the equilibrium dissociation constant (KD) values of R4702, datopotamab, and sacitzumab were at 2.01E-10 M, 6.43E-08 M, and 1.67E-09 M, respectively (Table 2). These results showed that R4702 has 320- and 8-fold better binding affinity to TROP2 protein than datopotamab and sacitzumab, respectively. The much higher affinity of R4702 compared to datopotamab is attributed to R4702 exhibiting a higher on-rate and a slower off-rate in the binding to TROP2 protein.PATENT Attorney Docket No.: G3004-01900PCT Table 2. Binding affinity of TROP2 antibodies based on SPR analysis TROP2 antibody KD (M) ka (1 / Ms) kd (1 / s) R4702 2.01E-10 7.45E+05 1.50E-04

[0210] To investigate the potential of topoisomerase 1 (TOP1) inhibitors as effective drug moiety for ADCs, the cytotoxicity of exatecan, deruxtecan (DXd), and SN-38 across different cancer cell types was evaluated. Exatecan demonstrated relative higher 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. In general, extecan displayed 2- to 5-fold better potency than others against the three different cell lines, the IC50was summarized in Table 3. Table 3. In-vitro cytotoxicity of TOP1 inhibitors IC50(nM)Example 3: Conjugation process of exemplary OBI-992 (TROP2 Antibody drug conjugate) 3.1 4-(N-Maleimidomethyl)-cyclohexane-1-carboxylate (MCCa) linker of R4702 conjugation

[0211] R4702 is an TROP2 monoclonal antibody which is as disclosed in WO2022 / 222992; Exatecan is a commercially available antineoplastic agent; 4-(N-Maleimidomethyl)-cyclohexane-1-carboxylate (MCCa) linker is modified from commercially available succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- carboxylate (SMCC). The entire chemical structure of OBI-992 (Ave. DAR=4) is indicated as follows:PATENT Attorney Docket No.: G3004-01900PCT Polyethylene glycol (PEG)

[0212] The Product N-PM-0017 was generated from the mixture of N-DT-0013 and N-PM-0018. The manufacturing scheme and process was listed as follows: OOOOO O NO OH OH O N N OTo a suspension of exatecan mesylate in DMF, N-PM-0015 and DIPEA were added at room temperature. The suspension became clear brown solution within 5 minutes. This mixture was stirred at room temperature for 20 hours. After reaction was completed, the reaction mixture was added to a stirring TBME over 30 minutes to get precipitate. After stirring for 30 minutes, the solids were collected by filtration and followed with high vacuum drying to obtain crude N-PM-0016. This crude product was used in next step without further purification. (b) Step 2: A stirring suspension of N-PM-0016 in DCM was cooled to -20 ⁰C. A -10 ⁰C pre-cooled TFA liquid wasPATENT Attorney Docket No.: G3004-01900PCT added to N-PM-0016 solution over 60 minutes. This mixture was stirred at -20 ⁰C for 10 hours. After reaction was completed, the reaction mixture was added to a stirring TBME over 30 minutes to get precipitate. After stirring for 30 minutes, the solids were collected by filtration and followed with high vacuum drying to obtain crude N-PM-0018. This crude product was used in next step without further purification. (c) Step 3: To a solution of N-DT-0013 in DMF, HATU and NMM was added. 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 at room temperature over 30 minutes. This mixture was stirred at room temperature for further 2 hours. After reaction was completed, the reaction mixture was added to a stirring TBME over 30 minutes to get precipitate. After stirring for 30 minutes, the solids were 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. 3.3 Conjugation process of OBI-992

[0213] The overall conjugation process of OBI-992 (R4702-N-PM-0017) is listed as follows:EDTA, pH 7.0) was cooled to 12-16 ⁰C. R4702 was treat with TCEP.HCl (2.29 mg; 0.00799 mmol) in reaction buffer (0.46 mL) for 2-6 hours at 12-16 ⁰C. In order to reduce the antibody solution, payload-linker N-PM-0017 (39.94 mg; 0.0183 mmol) within DMSO was added and conjugated for 1 hour at 12-16 ⁰C. After conjugation completed, the buffer of OBI-992 was changed to the storage buffer (20 mM Sodium acetate, pH 5.0 with 0.1% (w / w) polysorbate 80) via UF / DF dialysis membrane to achieve finalPATENT Attorney Docket No.: G3004-01900PCT concentration of OBI-992 (10.14 mg / mL, total 41.9 mL). The drug-to-antibody ratio (DAR) value of final ADC is 4.15 determined by hydrophilic interaction chromatography (HIC). Example 4: The cytotoxicity assay of OBI-992 in human cancer cell lines

[0214] 4.1 Cell line

[0215] Human prostate cancer DU145 cells were cultured in Eagle’s Minimum Essential Medium (Corning) with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). Human pancreatic cancer Capan-1 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM; Gibco) with 20% FBS and 1% P / S. Human ovary cancer ES2 cells were cultured in McCoy’s 5A (Gibco) with 5% FBS and 1% P / S. Human breast cancer MCF7 cells were cultured in Minimum Essential Medium (Gibco) with 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 (Gibco) with 10% FBS and 1% P / S. All above cell lines were cultured in a humidified incubator at 37 ^C with 5% CO2. Human breast cancer MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium (Gibco) with 10% FBS and 1% P / S in a humidified incubator at 37 ^C without CO2. All cell lines, except for MCF-7, were purchased from the American Type Culture Collection (ATCC).

[0216] 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 the anti- human TROP2 antibody on cells were calculated using QuantumTMSimply Cellular®(QSC) anti-Mouse IgG kit (Bangs Laboratories, Inc.) according to the manufacturer’s instructions. In brief, the QSC calibration microspheres coated with increasing amounts of capture antibody were labeled to saturation with the same antibody used to label cells. The mean fluorescence index (MFI) of the labeled calibration microspheres and cells were recorded and analyzed using QuickCal®analysis template (Bangs Laboratories, Inc.) to determine the Antibody Binding Capacity value of 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) following the manufacturer's instructions. Briefly, the cells treated with compounds were lysed using a 1:1 mixture of CellTiter-Glo®Reagent and culture media for 10 mins. After the incubation, the luminescence was recorded by SpectraMax M2 reader. The concentration of half maximal growth inhibitory (IC50) was calculated by GraphPad Prism 6.PATENT Attorney Docket No.: G3004-01900PCT

[0217] 4.3 Result OBI-992 is derived from conjugation of R4702 with extecan through a hydrophilic linker. To evaluate the correlation of OBI-992 cytotoxicity with TROP2 expression level in cancer cells, a diverse cancer cell lines were employed for the evaluation. The level of TROP2 expression was determined by flow cytometry (Figure 2A). The results revealed a positive correlation between OBI-992 IC50and TROP2 expression level, as indicated by spearman correlation (r=-0.8, p=0.0138) (Figure 2B). It could demonstrate the potent cancer-killing effect of OBI-992 among diverse cancer cell lines. Example 5: Demonstration of efficacy: Measurement of Anti-tumor activity of the exemplary TROP2 ADC (OBI-992) in BxPC-3 human pancreatic cancer cell-derived xenograft in BLAB / c nude mice

[0218] 5.1 Test Substances 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) Table 4. Study Design and sampling Route and Dose of Volume of Groups administration injection injection AnimalPATENT Attorney Docket No.: G3004-01900PCT

[0219] 5.2 Cell line: BxPC-3 cells (Highly TROP2 expressed; human pancreatic carcinoma)

[0220] 5.3 Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal Grouping: The mice were divided into 13 groups and each group contained 5 mice. A total of 65 mice were involved in the study.

[0221] 5.4 Equipment and Material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0222] 5.5 Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 5x106BxPC-3 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article: The first dosing day was denoted as Day 1 when average tumor volume reaches 150-200 mm3. All test articles (test item 1 to 6) or reference items (Sodium citrate solution) were intravenously administered to the mice on Day 1, 8, and 15 for G2 to G13. The injection was performed using insulin syringe with the dosage of 10 mg / kg or 3 mg / kg and the injection volume was 5 mL / kg. (c) Body weight measurement Measurement started from the next day of tumor inoculation. Animal body weight was measured and recorded three times per week. (d) Tumor growth inhibition rate calculationPATENT Attorney Docket No.: G3004-01900PCT Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 22). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (e) Blood sampling: On Day 0, 7, 14, blood samples were collected using submandibular bleeding. The collected blood sample was centrifuged 15 minutes at 4 °C and 1500 g to separate serum and pellet. The upper serum was collected and stored at -80 ^C. The study ended at Day 22. (f) Tumor resection Mice were sacrificed by CO2euthanasia at the end of the study. The connective tissue around tumor was resected and the tumor samples were frozen in liquid nitrogen immediately and stored at -80 ^C. (g) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0223] 5.6 Result A summary of group body weight was presented in Figure 3 and Table 5. There was no significant difference observed in the mean body weight between treatment groups and vehicle control during the study period. Table 5. Summary of body weight of individual mouse Body Weight (g) GroupAnimalIDD1 D3 D6 D8 D10 D13 D15 D17 D20 D22 7PATENT Attorney Docket No.: G3004-01900PCT 25* 17.36 17.29 17.34 17.44 17.57 17.70 17.51 17.57 18.14 18.02 Mean 18.04 18.28 18.19 18.10 18.25 18.38 18.40 18.44 19.15 18.92 SEM 041 043 044 045 042 045 031 043 044 038 4PATENT Attorney Docket No.: G3004-01900PCT 68 17.88 18.58 18.77 18.89 19.30 19.78 19.34 19.66 20.03 21.02 Mean 17.68 17.88 18.18 18.17 18.44 18.61 18.27 18.65 18.88 18.99 SEM 0.27 0.38 0.41 0.48 0.47 0.48 0.49 0.53 0.52 0.67ce n was remove rom te group ue to excuson o out ers, w c tumor was growngntramuscuary not subcutaneously. A summary of group tumor volume was presented in Figure 4 and Table 6. Tumor volume was measured three times a 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. Table 6. Summary of tumor volume of individual mouse and mean TGI% Tumor vol 3GroupAnimal ume (mm)ID. D1 D3 D6 D8 D10 D13 D15 D17 D20 D22TGI (%)30 19PATENT Attorney Docket No.: G3004-01900PCT 10 37 83.23 94.77 71.25 68.43 55.18 59.25 72.50 79.77 78.42 79.77 mg / kg 47 159.36 156.33 155.65 138.38 100.35 99.09 100.92 97.47 89.38 75.84 53 117.00 136.93 102.34 98.78 68.43 59.64 64.51 71.66 57.60 65.00 03 64 16 18 24 07 03 64PATENT Attorney Docket No.: G3004-01900PCT 70 109.33 126.85 133.12 138.92 168.78 183.75 210.94 184.00 178.96 176.40 25** 191.10 18.57 7.90 7.40 216.30 18.29 9.40 8.20 316.03 18.78 Mean 139.79 150.89 166.68 165.74 180.71 202.73 212.81 221.94 245.59 305.24 66 62y. **Mice #24 in GP11 was removed from the group due to exclusion of outliers, which tumor was growing intramuscularly not subcutaneously.

[0224] On Day 22, compared to the vehicle group, Figure 4A illustrated the inhibitory effect of the 10 mg / kg groups from high to low was 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 illustrated the inhibitory effect of the 3 mg / kg, R4702-MMAE and R4702-Exatecan remained potent and significant. R4702-MMAE showed the best 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 much less and insignificant antitumor effects compared to other treatment groups. In this study, all Anti-TROP2 ADCs exhibited remarkable tumor inhibition efficacy. R4702-MMAE showed the best antitumor effect followed by R4702-exatecan, DS-1062, IMMU-132, and R4702-SN38. Example 6: Demonstration of efficacy: Measurement of Anti-tumor activity of the exemplary TROP2 ADC (OBI-992) in DU-145 human prostate cancer cell-derived xenograft in BLAB / c nude mice

[0225] 6.1 Test Substances 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)PATENT Attorney Docket No.: G3004-01900PCT (d) R4702-MMAE-DAR4 (4.86 mg / mL) Table 7. Study Design and sampling Route and Dose of Volume of Groups administration injection injection Animal No.

[0227] 6.3 Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal Grouping: The mice were divided into 13 groups and each group contained 5 mice. A total of 65 mice were involved in the study.

[0228] 6.4 Equipment and Material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0229] 6.5 MethodPATENT Attorney Docket No.: G3004-01900PCT (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 5x106DU-145 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article: The first dosing day was denoted as Day 1 when average tumor volume reaches 100-150 mm3. All test articles (test item 1 to test item 4) were intravenously injected one shot to the mice on Day 1 for G2 to G13. The injection was performed using insulin syringe with the dosage of 10 mg / kg, 3 mg / kg, or 1 mg / kg and the injection volume was 5 mL / kg. The reference item (Sodium citrate solution) was administered for G1. (c) Body weight measurement Animal body weight was measured and recorded twice a week, starting from the next day after tumor inoculation. (d) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 22). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (e) Blood sampling: On Day 0 and Day 14, blood samples were collected using submandibular bleeding. On Day 23 at animal sacrifice, blood samples were collected using cardiac puncture. The collected blood sample was centrifuged 15 minutes at 4 °C and 1500 g to separate serum and pellet. The upper serum was collected and stored at -80 °C. The study ended on Day 25. (f) Tumor resection Mice were sacrificed by CO2euthanasia at the end of the study. The connective tissue around tumor was resected and the tumor samples were frozen in liquid nitrogen immediately and stored at -80 ^C. (g) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.PATENT Attorney Docket No.: G3004-01900PCT

[0230] 6.6 Result A summary of group body weight was presented in Figure 5 and Table 8. There was no significant difference observed in the mean body weight among groups G1 to G13 during the study period. Table 8. Summary of body weight of individual mouse GroupAnimalBody Weight (g) ID.D1 D4 D8 D11 D15 D18 D22 D25 4 1 2 1 4 1 1 1 1 1 1 2PATENT Attorney Docket No.: G3004-01900PCT Mean 19.36 19.51 19.74 20.10 20.43 20.71 20.88 21.18 SEM 0.33 0.34 0.38 0.20 0.21 0.31 0.29 0.42 SD 0.74 0.76 0.86 0.45 0.48 0.68 0.66 0.93PATENT Attorney Docket No.: G3004-01900PCT SEM 0.50 0.50 0.45 0.37 0.37 0.44 0.53 0.57 SD 1.13 1.12 1.02 0.82 0.83 0.97 1.17 1.27 *No.4 in GP1 was sacrificed on day 18 due to tumor size over 1500 mm3.A summary of group tumor volume was presented in Figure 6 and Table 9. Two animals were sacrificed prior to scheduled euthanasia due to large tumor size. One mouse of the vehicle group was sacrificed on Day 18 due to tumor volume exceeding 1500 mm3, and one of the DS-1062 (3 mg / kg) group was sacrificed on Day 22 due to ulcerous tumor. The mean tumor volume was recorded on Days 1, 4, 8, 11, 15, 18, 22, and 25. The study completed, and animals were sacrificed on Day 25. Table 9. Summary of tumor volume of individual mouse and mean TGI% GroupAnimal Tumor volume (mm3)ID. D1 D4 D8 D11 D15 D18 D22 D25TGI (%)2 5 3 7PATENT Attorney Docket No.: G3004-01900PCT Mean 154.49 181.92 169.47 162.10 199.85 212.07 273.06 412.37 SEM 11.89 14.19 32.56 55.94 102.86 140.01 188.71 279.53 SD 26.59 31.74 72.81 125.08 230.00 313.07 421.98 559.05 0 4 3 4 8 6PATENT Attorney Docket No.: G3004-01900PCT SEM 15.22 23.25 32.10 45.05 63.34 78.26 94.17 127.62 SD 34.04 51.99 71.78 100.74 141.62 175.00 210.57 285.36 61 175.88 213.66 183.32 153.08 168.67 195.52 254.58 287.76 8

[0231] All TROP2-targeted ADCs showed potent, statistically significant antitumor effect at 10 mg / kg on Day 25 (Figure 6A). Compared to vehicle control, 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 extent of tumor growth inhibition was dose- dependent from 1 to 10 mg / kg for tested ADCs. At 3 mg / kg (Figure 6B), the antitumor effect was strong and comparable for 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 reduced to TGI 57%, whereas the TGI remained relatively high for R4702-Exatecan-DAR8 (TGI 81%), R4702-Exatecan-DAR4 (TGI 78%) and DS-1062 (TGI 85%). The results indicated that R4702-Exatecan ADCs were better than or as effective as the benchmark DS-1062 in the TROP2 middle-level expression DU-145 xenograft model. Example 7: Demonstration of efficacy: Measurement of Anti-tumor activity of the exemplary TROP2 ADC (OBI-992) in NCI-N87 human gastric cancer cell-derived xenograft in BLAB / c nude mice

[0232] 7.1 Test Substances 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) Table 10. Study Design and sampling Route and Dose of Volume of AnimalPATENT Attorney Docket No.: G3004-01900PCT G4: DS-1062 (10 mg / kg) IV 10 5 5 G5: R4702-Exatecan-DAR4 (3 mg / kg) IV 3 5 5 R4 2 E DAR k I

[0234] 7.3 Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: Female (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal Grouping: The mice were divided into 13 groups and each group contained 5 mice. A total of 65 mice were involved in the study.

[0235] 7.4 Equipment and Material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0236] 7.5 Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 2.5x106NCI-N87 cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article: The first dosing day was denoted as Day 1 when average tumor volume reaches 100-150 mm3. All test articles (test item 1 to test item 3) were intravenously injected one shot to the mice on Day 1 for G2 to G13.PATENT Attorney Docket No.: G3004-01900PCT The injection was performed using insulin syringe with the dosage of 10 mg / kg, 3 mg / kg, 1 mg / kg or 0.3 mg / kg, and the injection volume was 5 mL / kg. The reference item (Sodium citrate solution) was administered for G1. (c) Body weight measurement Animal body weight was measured and recorded twice a week, starting from the next day after tumor inoculation. (d) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 22). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). (e) Blood sampling: On Day 0 and Day 14, blood samples were collected using submandibular bleeding. On Day 23 at animal sacrifice, blood samples were collected using cardiac puncture. The collected blood sample was centrifuged 15 minutes at 4 °C and 1500 g to separate serum and pellet. The upper serum was collected and stored at -80 °C. The study ended on Day 23. (f) Tumor resection Mice were sacrificed by CO2euthanasia at the end of the study. The connective tissue around tumor was resected and the tumor samples were frozen in liquid nitrogen immediately and stored at -80 ^C. (g) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0237] 7.6 Result A summary of group body weight was presented in Figure 7 and Table 11. There was no significant difference observed in the mean body weight among groups G1 to G13 during the study period. Table 11. Summary of body weight of individual mouse Body Weight (g)PATENT Attorney Docket No.: G3004-01900PCT GroupAnimalID. D1 D3 D6 D9 D13 D16 D20 D232 1712 1770 1757 1779 1789 1816 1877 1922PATENT Attorney Docket No.: G3004-01900PCT SEM 0.23 0.31 0.29 0.41 0.37 0.24 0.25 0.33 6 19.02 19.71 20.17 20.84 21.17 21.04 21.18 21.18 4 18.44 18.19 18.51 18.90 19.38 19.92 20.26 20.70A summary of group tumor volume was presented in Figure 8 and Table 12. The mean tumor volume was recorded on Days 1, 3, 6, 9, 13, 16, 20, and 23. The study completed, and animals were sacrificed on Day 23. Table 12. Summary of tumor volume of individual mouse and mean TGI% Tumo 3GroupAnimal r volume (mm )ID. D1 D3 D6 D9 D13 D16 D20 D23TGI (%)3 3 5PATENT Attorney Docket No.: G3004-01900PCT 9 162.07 184.99 215.55 191.40 171.30 188.31 164.85 192.84 33 139.85 135.36 108.00 82.43 57.47 37.17 50.26 63.98 R4702- 50 136.81 161.35 129.14 91.28 56.30 42.85 30.63 26.15 7 3 7 9 2 5

[0238] On Day 23, compared to vehicle control, a single dose of R4702-Exatecan-DAR8 at 10 mg / kg 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 noteworthy and statistically significant 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 significant antitumor effect (TGI: 86%, p<0.01), whereas the effect of R4702-Exatecan-PATENT Attorney Docket No.: G3004-01900PCT DAR4 (TGI: 49%, p>0.05) and DS-1062 (TGI: 53%, p>0.05) did not reach statistical significance. In this study, a single dose of R4702-Exatecan-DAR8, R4702-Exatecan-DAR4, or DS-1062 at 3 or 10 mg / kg exhibited significant, remarkable tumor growth inhibition in NCI-N87 xenograft tumor model. R4702- Exatecan-DAR8 was the most potent among three tested ADCs, with complete tumor regression observed at 10 mg / kg. Example 8: Demonstration of efficacy: Measurement of Anti-tumor activity of the exemplary TROP2 ADC (OBI-992) in NCI-H1975-C797S human lung cancer cell-derived xenograft in BLAB / c nude mice

[0239] 8.1 Test Substances 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) Table 13. Study Design and sampling Route and Dose of Volume of Groups administration injection injection Animal

[0240] 8.2 Cell line: NCI-H1975-C797S cells (TROP2 highly expressed; human lung carcinoma)

[0241] 8.3 Animal (a) Species: Mus musculus (b) Strain: CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) (c) Source: BioLasco Taiwan (d) Sex: FemalePATENT Attorney Docket No.: G3004-01900PCT (e) Age at initiation of study: 7 weeks (f) Body weight range at start of study: 15-25 g (g) Animal Grouping: The mice were divided into 9 groups and each group contained 6 mice. A total of 54 mice were involved in the study.

[0242] 8.4 Equipment and Material (a) Biosafety cabinet (NUAIRE / NU-620-400) (b) Electronic balance (CROMTECH / YP30002) (c) Isolated positive / negative pressure validated cage housing system (TECNIPLAST / Blue Line) (d) Vernier (METROLOGY / EC-9001V) (e) Matrigel (BD / Cat. No.: 356234)

[0243] 8.5 Method (a) Establishment of xenograft mouse model Subcutaneous inoculation of tumor cells: 1.0x107NCI-H1975-C797S cells were mixed with the equal volume of matrigel (volume ratio 1:1) (Corning, 354248, Lot No.: 0261002). Subcutaneous injection volume was 100 ^L / mouse. (b) Route and administration of test article: The first dosing day was denoted as Day 1 when average tumor volume reaches 100-150 mm3. All test articles (test item 1 to test item 4) were intravenously injected to the mice. The injection was performed using insulin syringe with the dosage of 10 mg / kg, 3 mg / kg, and the injection volume was 5 mL / kg. The reference item (Sodium citrate solution) was administered intravenously for vehicle group. Osimertinib (test item 5) was given by oral gavage. (c) Body weight measurement Animal body weight was measured and recorded twice a week, starting from the next day after tumor inoculation. (d) Tumor growth inhibition rate calculation Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%, where Ti and Ci indicate the mean tumor volume in the treatment groups and vehicle group at the end of the study (Day 16). Whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1).PATENT Attorney Docket No.: G3004-01900PCT (e) Blood sampling: On Day 0, blood samples were collected using submandibular bleeding. On day 16, blood samples were collected using cardiac puncture. The collected blood sample was centrifuged 15 minutes at 4 °C and 1500 g to separate serum and pellet. The upper serum was collected and stored at -80 °C. The study ended on Day 16. (f) Tumor resection Mice were sacrificed by CO2euthanasia at the end of the study. The connective tissue around tumor was resected and the tumor samples were frozen in liquid nitrogen immediately and stored at -80 ^C. (g) Statistical analysis Results were presented as mean and standard error of the mean (mean ± SEM). Comparisons of all data collected for each treatment group with vehicle group was calculated using Student’s t-test. p < 0.05 is considered significance.

[0244] 8.6 Result A summary of group body weight was presented in Figure 9 and Table 14. There was no significant difference observed in the mean body weight among groups G1 to G9 during the study period. Table 14. Summary of body weight of individual mouse A Body Weight (g) GroupnimalID.D1 D3 D7 D10 D14 D16 d*PATENT Attorney Docket No.: G3004-01900PCT 32 19.02 18.01 17.82 18.02 18.54 18.59 41 18.26 18.16 18.51 18.06 19.34 19.10 Mean 17.69 17.69 18.25 18.28 18.94 18.93PATENT Attorney Docket No.: G3004-01900PCT SEM 0.22 0.24 0.23 0.21 0.14 0.26 SD 0.54 0.58 0.57 0.51 0.35 0.64 *No.11 in GP1 was sacrificed on day 10 due to tumor size over 1500 mm3.e was recorded on Days 1, 3, 7, 10, 14, and 16. The study completed, and animals were sacrificed on Day 16.

[0245] Table 15. Summary of tumor volume of individual mouse and mean TGI% Tumor volume (mm3GroupAnimal) TGI (%) ID.D1 D3 D7 D10 D14 D16 16 6173 25952 46395 50535 72223 89965 2 01 7 5PATENT Attorney Docket No.: G3004-01900PCT SEM 20.64 39.55 48.29 117.29 171.24 158.29 SD 50.57 96.87 118.29 287.31 419.45 387.74 7 89.81 138.27 199.08 113.91 159.95 157.54 4 4 9 7

[0246] All TROP2-targeted ADCs, except for DS-1062, showed remarkable and statistically significant tumor inhibitory effect. Compared to vehicle control, a single dose of R4702-Exatecan-DAR8 at 10 mg / kg 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) remained strong antitumor effect, whereas DS-1062 showed an insignificant low response (TGI: 24%, p>0.05). A benchmark ADC IMMU-132 at 12.5 mg / kg with a different dosing regimen (twice a week for 2 weeks) showed a significant antitumor effect with 44% of TGI (P<0.05), which was less than the inhibitory response caused by R4702-Exatecan-DAR8 and R4702-Exatecan-DAR4 at 3 or 10 mg / kg. R4702-Exatecan-PATENT Attorney Docket No.: G3004-01900PCT DAR8 and R4702-Exatecan-DAR4 demonstrated much better antitumor effect than benchmarks DS-1062 and IMMU-132 in a human lung cancer xenograft model NCI-H1975-C797S, which carries three EGFR mutations and is resistant to TKI therapy. Example 9: Off-target effect of OBI-992

[0247] 9.1 Cell association of ADCs with PBLs The off-target association of TROP2 ADCs with peripheral blood leukocytes (PBLs) were assessed by ex- vivo assay. Normal human blood was purchased from Taipei Blood Center, Taiwan. Fluorescein-labeled ADCs were mixed with whole blood obtained from two healthy donors and incubated at 37 °C for four hours. After the 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, the three PBL populations, lymphocyte, monocyte, and granulocyte, were discriminated and identified by gating on FSC / SSC plot. Mean fluorescence intensity (MFI) of each population was measured to indicate the level of cell association.

[0248] 9.2 In vitro evaluation of ADC toxicity on differentiated neutrophils Bone marrow derived human CD34+stem cells were expanded in SFEM II medium with CC100 supplement and followed by three steps of neutrophil differentiations. The granulocyte marker, CD66b, was measured to monitor progression of neutrophil differentiation, revealing approximately 25%, 45% and 85% CD66b+cells post Step 1, 2, and 3 differentiation, respectively. For ADC toxicity, differentiated neutrophils were incubated with ADCs for six days, followed by CD66b and Propidium Iodide (PI) staining. Viable neutrophils were distinguished by being positive for CD66b and negative for PI (CD66b+ / PI-). Absolute cell counting was performed by referring to CountBright Beads (Invitrogen, Cat. No. C36950) that were added uniformly to each sample just before FACS analysis. Following the manufacturer’s instruction, viable CD66b+cell count of each sample was determined. The viable cell count was normalized to untreated control as presented with percentage of relative CD66b+ / PI- cells. The concentration of half maximal growth inhibitory (IC50) was calculated by GraphPad Prism 6.

[0249] 9.3 Result There are several reports that non-specific binding of mAb to Fc ^ receptors (Fc ^Rs) may cause off-target toxicity. To assess the binding ability of ADCs with immune cells, whole blood was incubated with fluorescein-labeled OBI-992 or Dato-DXd for four hours. The leukocyte population of lymphocyte, monocyte, and granulocyte were differentiated by flow cytometry. Limited binding signals were observed inPATENT Attorney Docket No.: G3004-01900PCT the lymphocyte population for both OBI-992 and Dato-DXd treatments. Conversely, the monocyte population treated with Dato-DXd exhibited a higher mean fluorescence intensity (MFI), ranging from 3- to 5-fold higher compared to OBI-992 (Figure 11A), suggesting a stronger association of Dato-DXd with monocytes. Furthermore, Dato-DXd, unlike OBI-992, exhibited dose-dependent binding to monocyte THP-1 cells (Figure 11B). Dato-DXd showed higher cytotoxicity against THP-1 cells, with an IC50 of 95 nM, 5- fold more toxic than OBI-992 (546 nM) (Figure 11C). Neutropenia is frequently observed in cancer patients treated with ADCs. Therefore, the viability of neutrophils (CD66b+) differentiated from hematopoietic stem cells was investigated by staining with propidium iodide (PI). The results showed that the neutrophils were more sensitive to Dato-DXd than OBI-992, with IC50 of 2.9 nM versus 93.4 nM (Figure 11D). These findings suggest that OBI-992 may have lower potential for off-target effect on immune cells compared to Dato-DXd. Example 10: In-vitro and in-vivo synthetic lethality between OBI-992 and PARPi

[0250] 10.1 In-vitro synergistic effect of OBI-992 Talazoparib was further evaluated with OBI-992 for potential combination. The potent synergistic effects were observed in different cancer cell lines (MDA-MB-231, NCI-N87, Capan-1) with the IC50value of talazoparib significantly reduced in the presence of OBI-992. In MDA-MB-231 cells, the IC50 value of talazoparib improved from 368 nM to 11 nM, representing a remarkable 32-fold enhancement in potency (Figure 12A). In addition, the potency enhancement for NCI-N87 and Capan-1 cells was 9- and 4-fold, respectively (Figure 12B and Figure 12C). These results suggest PARP inhibitors are promising candidates for combination with OBI-992 in cancer treatment.

[0251] 10.2 In-vivo synergistic effect of OBI-992

[0252] CAnN.Cg-Foxn1nu / CrlBltw (BALB / c nude) mice were used for xenograft study. C57BL / 6NCrl (B6) mice were used for syngeneic mouse model. Mice were housed in special pathogen-free (SPF) condition, up to five or six mice in Individually Ventilated Cages (IVC) with the sterile bedding in a controlled environment with temperature 22 ± 3 ^C, relative humidity 50 ± 20% and 12 / 12 hour light / dark cycle. The food (LabDiet 5010, PMI, USA) and water (sterile RO water) were provided ad libitum throughout the whole study period. Mice were acclimated for at least three days before the study initiation. After implantation of cancer cells, tumor size was measured by caliper and tumor volume was calculated by ellipsoid equation according to the records ((major axis × minor axis × minor axis) × (π / 6)). Tumor volumes were used to calculate tumor growth inhibition (TGI) rates according to the following formula: TGI (%) = [1 − (Ti − T1) / (Ci − C1)] × 100%. Ti and Ci indicate the mean tumor volume in the treatment groupsPATENT Attorney Docket No.: G3004-01900PCT and vehicle group at the end of the study, whereas T1 and C1 indicate the mean tumor volumes in the treatment group and vehicle group at the beginning of test item administration (Day 1). All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at National Laboratory Animal Center in Taiwan.

[0253] Capan-1 human pancreatic cancer cell-derived xenograft tumor in BALB / c nude mice were used to evaluate the synergistic effect of OBI-992 and PARP inhibitors (Olaparib and Talazoparib). Viable Capan-1 cells (1x107cells / mouse) were mixed with the same volume of Matrigel (BD) and subcutaneously injected into the right flank of female BALB / c nude mice (BioLasco Taiwan, 100 mL / mouse). Mice were grouped when the average tumor volume reached 100-150 mm3. OBI-992 was treated at suboptimal doses at 0.1 or 0.3 mg / kg to demonstrate the synergistic effect and to avoid potential overt toxicities when combined with PARP inhibitors. Olarparib (Combi-Blocks, Inc.) was treated at 80 mg / kg, and Talazoparib (AmBeed, Inc.) was treated as 0.1 mg / kg. OBI-992 were administered by intravenous injection as single dose on Day 1. Olaparib and Talazoparib were administered through oral gavage at five days on, two days off cycle. Tumor volume, body weight, mortality and animal behavior were monitored twice weekly till the study completion day (Day 22). During the study period, all mice were in good conditions, with no observed adverse effect. Figure 13A indicated the tumor growth inhibition (TGI) of OBI-992 only and OBI-992 + Talazoparib was 31-40% and 97-103%, individually (P<0.001). Similarly, Figure 13B also indicated the tumor growth inhibition (TGI) of OBI-992 only and OBI-992 + Olaparib was 31-40% and 83-98%, individually (P<0.001). These results also suggest PARP inhibitors are promising candidates for combination with OBI- 992 in cancer treatment.

[0254] MC38 mouse colon cancer with ectopic expression of human TROP2 (MC38 / hTROP2) were used to evaluate the synergistic effects of OBI-992 and PD-1 blockade. Viable MC38 / hTROP2 cells (1x106cells / mouse) mixed with the same volume of Matrigel (BD) were subcutaneously injected into the right flank of female B6 mice (BioLasco Taiwan, 100 mL / mouse). Tumor bearing mice were grouped when the average tumor volume reached 200-250 mm3. OBI-992 were administered at 3 mg / kg alone or in combination with anti-mPD-1 (Leinco Technologies, Inc.) at 5 mg / kg. OBI-992 were administered through intravenous injection as a single dose and anti-mPD-1 were administered by intravenous injection twice weekly. Tumor volume, body weight, mortality and animal behavior were monitored twice weekly till the study completion day (Day 11). During the study period, all mice were in good conditions, with no observed adverse effect. Figure 13C indicated the tumor growth inhibition (TGI) of OBI-992 + anti-mPD-1 was 96%, individually (P<0.05). This result also demonstrated the syngeneic effect of anti-PD-1 and OBI-992 in cancer treatment.PATENT Attorney Docket No.: G3004-01900PCT

[0255] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0256] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Claims

PATENT Attorney Docket No.: G3004-01900PCT Claims 1. An antibody-drug conjugate (ADC) comprising a drug moiety and an antibody or an antigen- binding fragment thereof that binds Tumor-associated calcium signal transducer 2 (TROP2), wherein the ADC has the formula (I): Ab-(L-D)n (I) wherein the drug moiety (D) is covalently linked to the antibody or the antigen-binding fragment thereof (Ab) via a linker (L); wherein the antibody is an anti-TROP2 antibody; and wherein n is an integer from 1 to 8. 2 The ADC of Claim 1, wherein the antibody is a monoclonal antibody, an antigen-binding fragment, a chimeric antibody, or a humanized antibody. 3 The ADC of Claim 2, wherein the antigen-binding fragment is a Fab, F(ab’)2, Fv, or scFv. 4 The ADC of Claim 1, wherein the anti-TROP2 antibody is hRS7, Hu2G10, hu4D3, MAAP-9001a, Pr1E11, R4702, datopotamb, or sacituzumab. 5 The ADC of Claim 1, wherein the drug moiety is exatecan, deruxtecan, SN-38, or MMAE. 6 The ADC of Claim 1, wherein the linker comprises a maleimide or a derivative thereof conjugated to a thio group of the antibody. 7 The ADC of Claim 6, wherein the linker is a 4-(N-Maleimidomethyl)-cyclohexane-1-carboxylate (MCCa). 8 The ADC of Claim 1, wherein the drug moiety is a chemotherapeutic agent, a photodynamic therapeutic agent, or a biological agent. 9 The ADC of Claim 8, wherein the photodynamic therapeutic agent is 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), Pyropheophorbidea (PPa), or Pheophorbide a (PhA). 10 The ADC of Claim 1, wherein the drug moiety is an anti-proliferative agent.PATENT Attorney Docket No.: G3004-01900PCT 11. The ADC of Claim 10, wherein the anti-proliferative agent is Exatecan, Irinotecan, Topotecan, Camptothecin, Rubitecan, MLN576, Exatecan, Belotecan, Seconeolitsine, SN-38, Genz-644282, Betulinic acid, β-Lapachone, Karenitecin, Gimatecan, Namitecan, Edotecarin, SW044248, LMP744, T-2513, Podocarpusflavone A, Indimitecan, Lurtotecan, TP3011 or 10- hydroxycamptothecin, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), mertansine (DM1), anthracycline, pyrrolobenzodiazepine, ^-amanitin, tubulysin, benzodiazepine, erlotinib, bortezomib, fulvestrant, sunitinib, letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, leucovorin, rapamycin, lapatinib, lonafarnib (SARASAR, SCH 66336), sorafenib, gefitinib, AG1478, AG1571, alkylating agent; alkyl sulfonate; aziridines; ethylenimine; methylamelamine; acetogenins; camptothecin; bryostatin; callystatin; CC-1065; cryptophycins; dolastatin; duocarmycin; eleutherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil; chlornaphazine; cholophosphamide; estramustine; ifosfamide; mechlorethamine; mechlorethamine oxide hydrochloride; melphalan; novembichin; phenesterine; prednimustine; trofosfamide; uracil mustard; carmustine; chlorozotocin; fotemustine; lomustine; nimustine; ranimustine; calicheamicin; dynemicin; clodronate; esperamicin; neocarzinostatin chromophore; aclacinomysins; actinomycin; authramycin; azaserine; bleomycins; cactinomycin; carabicin; caminomycin; carzinophilin; chromomycinis; dactinomycin; daunorubicin; detorubicin; 6-diazo-5-oxo-L-norleucine; doxorubicin; epirubicin; esorubicin; idarubicin; marcellomycin; mitomycin; mycophenolic acid; nogalamycin; olivomycins; peplomycin; potfiromycin; puromycin; quelamycin; rodorubicin; streptonigrin; streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; methotrexate; 5- fluorouracil (5-FU); denopterin; pteropterin; trimetrexate; fludarabine; 6-mercaptopurine; thiamiprine; thioguanine; ancitabine; azacitidine; 6-azauridine; carmofur; cytarabine; dideoxyuridine; doxifluridine; enocitabine; floxuridine; calusterone; dromostanolone propionate; epitiostanol; mepitiostane; testolactone; aminoglutethimide; mitotane; trilostane; frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine; ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecene; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;PATENT Attorney Docket No.: G3004-01900PCT pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoid; paclitaxel; doxetaxel; chloranbucil; 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 of Claim 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent, carrier, or excipient.

13. The pharmaceutical composition of Claim 12, further comprising an additional anti-cancer agent.

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

15. The method of Claim 14, wherein the cancer is a TROP2 expressing cancer selected from the group consisting of lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, and oral cancer.

16. The method of Claim 14, further comprising administering to the patient an additional anti-cancer agent.

17. The method of Claim 16, wherein a combination of the ADC and the additional anti-cancer agent provides a synergistic or additive effect in cancer treatment and enhanced therapeutic efficacy.

18. A method of inducing or enhancing an immune reaction in a patient in need thereof comprising: administering an effective amount of the pharmaceutical composition of Claim 12 and performing one or more of the following procedure selected from: (a) Administering the pharmaceutical composition two or more times; (b) Adjusting a time interval and / or a dosing amount regimen between two successive administrations; (c) Adjusting routes of administration and / or altering sites of administration; or (d) administering an additional anti-cancer agent.

19. The method of Claim 18, wherein the administration can be altered and / or supplemented by addition of immune response booster agents.PATENT Attorney Docket No.: G3004-01900PCT 20. The method of Claim 14 or 18, wherein the effective amount is from 0.001 ^g to 250 mg per kg body weight of the patient.

21. The method of Claim 18, wherein the method comprises administering an additional anti-cancer agent, and a combination of the pharmaceutical composition and the additional anti-cancer agent provides a synergistic or additive effect in inducing or enhancing the immune reaction.

22. Use of the ADC of Claim 1 in the manufacture of a medicament for use in combination with an effective amount of an additional agent selected from the group consisting of an anticancer agent, an immunosuppressant agent, and an anti-infectious agent for the treatment of lung cancer, breast cancer, head-and-neck cancer, esophagus cancer, stomach cancer, bladder cancer, pancreatic cancer, colorectal cancer, cervix cancer, endometrial cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, or oral cancer.

23. A method of selecting a patient for cancer therapy by imaging, wherein the method comprises: (a) Administering to the patient an effective amount of the ADC of Claim 1; and (b) Detecting a reporting signal of an imaging agent in the patient; wherein the imaging agent is a fluorophore, a dye, an MRI contrast agent or a radionuclide; and wherein the reporting signal is detected visually or instrumentally.

24. The method of Claim 23, wherein the patient has a detectable cancer, and wherein the method further detects a cancer metastasis.

25. An antibody-drug conjugate (ADC) which binds to TROP2, comprising: (a) an antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises : i. a first heavy chain complementarity determining region (HCDR1) having an amino acid sequence of SEQ ID NO: 1; ii. a second heavy chain complementarity determining region (HCDR2) having an amino acid sequence of SEQ ID NO: 2; iii. a third heavy chain complementarity determining region (HCDR3) having an amino acid sequence of SEQ ID NO: 3; wherein the light chain variable domain comprises: iv. a first light chain complementarity determining region (LCDR1) having an amino acidPATENT Attorney Docket No.: G3004-01900PCT sequence of SEQ ID NO: 4; v. a second light chain complementarity determining region (LCDR2) having an amino acid sequence of SEQ ID NO: 5; vi. a third light chain complementarity determining region (LCDR3) having an amino acid sequence of SEQ ID NO: 6; (b) a drug moiety; and (c) a linker. The ADC of Claim 25, wherein the antibody further comprises: (a) a heavy chain variable domain comprising an amino acid sequence 90% to 100% identical to the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable domain comprising an amino acid sequence 90% to 100% identical to the amino acid sequence of SEQ ID NO: 8.

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