Antibody-drug conjugates comprising anti-FOLR1 antibodies

Abstract

The present invention provides, inter alia, antibody-drug conjugates (ADCs) that specifically bind to folate receptor-I (FOLR1). Pharmaceutical compositions and methods for treating cancer are also disclosed.

Description

[0001] This application claims priority to international application PCT / CN2022 / 091860 filed on May 10, 2022, and priority to Chinese application No. 202310390683.5 filed on April 13, 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Throughout this application, various publications, patents and / or patent applications are cited. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entirety into this application in order to more fully describe the prior art to which this disclosure relates. Technical Field

[0003] The present disclosure relates to antibody-drug conjugates (ADCs) comprising anti-FOLR1 antibodies and methods of making and using the same. Summary of the invention

[0004] Antibody-drug conjugates (ADCs) allow targeted delivery of drug moieties to tumors and, in some embodiments, intracellular accumulation therein where systemic administration of the unconjugated drug may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005), Current Opinion in Pharmacology, Vol. 5: pp. 382-387). ADCs are targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to tumor cells expressing antigens (Teicher, BA (2009), Current Cancer Drug Targets, Vol. 9: pp. 982-1004), thereby improving the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008), The Cancer Jour., Vol. 14, No. 3, pp. 154-169; Chari, RV (2008), Acc. Chem. Res., Vol. 41: pp. 98-107).

[0005] The present disclosure provides ADCs comprising an anti-FOLR1 antibody coupled to a drug moiety via a linker moiety. In embodiments, the anti-FOLR1 antibody binds to cancer cells expressing FOLR1 and allows the ADC to be selectively taken up into the cancer cells. In embodiments, the ADCs provided herein selectively deliver an effective amount of the drug moiety to tumor tissue and reduce nonspecific toxicity associated with the associated ADC. The ADC compounds described herein include those having anticancer activity.

[0006] Folate receptor 1 (FOLR1), also known as folate receptor-α (FRα) or folate binding protein (UniProtP15328), is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein with strong binding affinity for folic acid and reduced folic acid derivatives (Leung et al. (2013), Clin. Biochem., Vol. 46: pp. 1462-1468). FOLR 1 has important functions related to cell proliferation and survival (Kelemen LE (2006), Int. J. Cancer, Vol. 119 No. 2: 2430250), and it mediates the delivery of the physiological folate 5-methyltetrahydrofolate to the interior of cells. The expression of FOLR1 on normal tissues is restricted to the apical membrane of epithelial cells in the proximal renal tubules, alveolar pneumocytes of the lung, bladder, testis, choroid plexus, and thyroid (Weitman SD et al. (1992), Cancer Res., Vol. 52: 3396-3401; Antony AC (1996), Ann. Rev. Nutr., Vol. 16: 501-521; Kalli KR et al. (2008), Gynecol. Oncol., Vol. 108: 619-626). FOLR1 is overexpressed in epithelial tumors, including ovarian, uterine, breast, endometrial, pancreatic, kidney, lung, colorectal, and brain tumors. This expression pattern of FOLR1 makes it an ideal target for FOLR1-directed cancer therapy.

[0007] Improved methods for immunomodulation of folate receptor alpha (FOLR1) and downstream signaling processes activated by folate receptor alpha (FOLR1) are needed. In addition, given the specific expression of folate receptor alpha (FOLR1) in cancer-transformed cells of different tissue origins and its lower expression in non-cancerous tissues, improved therapeutic agents are needed that can specifically target cells and tissues that overexpress folate receptor alpha (FOLR1). Antibodies conjugated to FOLR1 can be used to deliver therapeutic or diagnostic payload moieties to target cells expressing folate receptor alpha to treat such diseases. Therefore, antibody-drug conjugates (ADCs) in which the drug is conjugated to an anti-FOLR1 antibody can provide highly targeted and potent anti-tumor activity.

[0008] In one aspect, provided herein are antibody-drug conjugates (ADCs) comprising monoclonal antibodies. In another aspect, provided herein are antibody-drug conjugates (ADCs) comprising anti-FOLR1 antibodies. In another aspect, provided herein are methods of using the ADCs disclosed herein to treat cancers (such as cancers expressing FOLR1).

[0009] In an embodiment, the present disclosure provides an antibody-drug conjugate (ADC) having an IgG antibody that binds to a FOLR1 target coupled at a cysteine ​​site of the IgG antibody. In an embodiment, the present disclosure provides an antibody-drug conjugate (ADC) having an IgG antibody that binds to a FOLR1 target coupled at a lysine site of the IgG antibody. The present disclosure also provides a method for treating epithelial tumors, including ovarian, uterine, breast, endometrial, pancreatic, kidney, lung, colorectal and brain tumors, the method comprising providing an effective amount of FOLR1 ADC.

[0010] In one aspect, provided herein is an antibody-drug conjugate (ADC) of formula (I): or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-FOLR1 antibody; m is an integer from 1 to 8; L 1 is a linker that binds to the anti-FOLR1 antibody; L 2 is a bond, -C(O)-, -NH-, an amino acid unit, –(CH2CH2O) n –, –(CH2) n -, -(4-aminobenzyloxycarbonyl)-, -(C(O)CH2CH2NH)-, or any combination thereof; wherein n is an integer from 1 to 24; and D is a drug moiety.

[0011] In one aspect, provided herein is a method of treating a cancer expressing FOLR1 in a subject in need thereof, the method comprising administering to the subject an ADC described herein (included in an aspect, embodiment, table, example or claim) or a pharmaceutically acceptable salt thereof.

[0012] In one aspect, provided herein is a method of treating a FOLR1-expressing cancer in a subject in need thereof, the method comprising administering an ADC described herein (included in an aspect, embodiment, table, example, or claim) or a pharmaceutically acceptable salt thereof, and further comprising administering a therapeutically effective amount of one or more additional active agents. In embodiments, the additional active agent is a VEGF inhibitor.

[0013] In any of the embodiments disclosed herein, the monoclonal antibody can be an anti-FOLR1 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A-Figure 1C Shown are the results of in vitro efficacy studies of 151-C-Lock-D5 and 151-K-Lock-D5 and corresponding controls IgG-C-Lock-D5 and IgG-K-Lock-D5 using the following cells: Figure 1AIGROV1 (highly expressing FOLR1) cells; Figure 1B SKOV-3 (low FOLR1 expression) cells; and Figure 1C A549 is FOLR1 negative (negative control).

[0015] Figure 2A Shown are the results of an in vivo efficacy study of IGROV1 xenografts in BALB / c athymic nude mice treated with 151-K-Lock-D5 (3 mg / kg or 6 mg / kg), 151-C-Lock-D5 (1.5 mg / kg, 3 mg / kg or 6 mg / kg) or control IgG-C-Lock-D5 (6 mg / kg). Figure 2B It shows that Figure 2A Tumor volumes of mice on day 46 after the treatment. Figure 2C The results of in vivo toxicity studies in mice are shown ( Figure 2A The figure shows the body weight changes of mice treated with ADC.

[0016] Figure 3A Shown are the results of an in vivo efficacy study of SKOV-3 xenografts in BALB / c athymic nude mice on days 15 and 29 after treatment with two doses of 151-K-Lock-D5 (3 mg / kg or 6 mg / kg per dose), 151-C-Lock-D5 (1.5 mg / kg, 3 mg / kg or 6 mg / kg per dose), and control IgG-K-Lock-D5 (6 mg / kg per dose). Figure 3B It shows that Figure 3A Tumor volumes of mice on day 49 after the treatment. Figure 3C The results of in vivo toxicity studies in mice are shown ( Figure 3A The figure shows the body weight changes of mice treated with ADC.

[0017] Figure 4A The in vivo anti-tumor efficacy of the LU-01-1618 PDX model in BALB / c athymic nude mice after treatment with a single dose of 151-K-Lock-D5 (1.5 mg / kg), 151-K-Lock-D5 (3 mg / kg), 151-K-Lock-D5 (6 mg / kg), 151-K-Lock-D5 (10 mg / kg), isotype ADC (10 mg / kg) and vehicle is shown. Figure 4B The results of in vivo toxicity studies in mice are shown ( Figure 4A The figure shows the weight changes of mice.

[0018] Figure 5Shown are the results of in vitro efficacy studies of 151-C-Lock-D5 and 151-GGFG-Dxd and negative control 151-mAb using SKOV-3 (lowly expressing FOLR1) cells.

[0019] Fig. 6A Shown are the results of an in vivo efficacy study of SKOV-3 xenografts in BALB / c athymic nude mice on days 15 and 29 after treatment with two doses of 151-C-Lock-D5 (3 mg / kg or 6 mg / kg per dose), 151-VC-MMAE (3 mg / kg or 6 mg / kg per dose), and control IgG-VC-MMAE (6 mg / kg per dose). Figure 6B The results of in vivo toxicity studies in mice are shown ( Fig. 6A The figure shows the body weight changes of mice treated with ADC.

[0020] Fig. 7A Shown are the results of an in vivo efficacy study of OVCAR-3 xenografts in BALB / c athymic nude mice treated with a single dose of 151-K-Lock-D5 (5 mg / kg), bevacizumab (5 mg / kg), 151-K-Lock-D5 / bevacizumab (5 mg / kg each, 151-K-Lock-D5 administered intravenously and bevacizumab administered intraperitoneally), and a control isotype ADC (10 mg / kg). Figure 7B It shows that Fig. 7A Tumor volumes of mice on day 48 after the treatment. Figure 7C The results of in vivo toxicity studies in mice are shown ( Fig. 7A The figure shows the body weight changes of mice treated with ADC. DETAILED DESCRIPTION definition:

[0021] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms related to the technology of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques provided herein are generally carried out according to conventional procedures well-known in the art and as described in various general and more specific references cited and discussed herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992).Many basic texts describe standard antibody production procedures, including Borrebaeck (ed.), Antibody Engineering, 2nd ed., Freeman and Company, NY, 1995; McCafferty et al., Antibody Engineering, A Practical Approach, IRL at Oxford Press, Oxford, England, 1996; and Paul (1995), Antibody Engineering Protocols, Humana Press, Towata, NJ, 1995; Paul (ed.), Fundamental Immunology, Raven Press, NY, 1993; Coligan (1991), Current Protocols in Immunology, Wiley / Greene, NY; Harlow and Lane (1989), Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY; Stites et al. (eds.), Basic and Clinical Immunology (4th ed.), Lange Medical Publications, Los Angeles, CA. Altos, Calif. and references cited therein; Coding Monoclonal Antibodies: Principles and Practice (2nd Edition), Academic Press, New York, NY, 1986; and Kohler and Milstein, Nature, Vol. 256: pp. 495-497, 1975. All references cited herein are incorporated herein by reference in their entirety. Enzymatic reactions and enrichment / purification techniques are also well known and performed according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. The nomenclature, laboratory procedures, and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, drug preparation, formulation, and delivery, and treatment of patients.

[0022] The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0023] Unless otherwise required by the context herein, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as use of any word in the singular, include plural referents unless expressly and unambiguously limited to one referent.

[0024] It should be understood that alternatives (eg, "or") used herein refer to one or both or any combination of the alternatives.

[0025] The term "and / or" as used herein refers to the specific disclosure of each of the specific features or components, with or without another feature or component. For example, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0026] "Any combination thereof" refers to any two or more or all of the aforementioned elements, and the combination also includes those in which the elements are repeated. Thus, "X, Y, Z, or any combination thereof" encompasses X and Y; Y and Z; X and Z; X, Y and Z, and other combinations in which one or more of X, Y, and Z appear more than once.

[0027] As used herein, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, "about" or "approximately" may mean within one or more than one standard deviation in each practice in this field. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ± 10%) or more, depending on the limitations of the measurement system. For example, about 5 mg may include any numerical value between 4.5 mg and 5.5 mg. In addition, particularly for biological systems or processes, the term may mean a value of up to an order of magnitude or up to 5 times. When a particular value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within the acceptable error range of the particular value or composition. In an embodiment, "about" includes a specified value.

[0028] In the present disclosure, "comprise / comprising", "containing", "having", etc. may have the meanings ascribed to them in U.S. patent law, and may mean "include / including", etc. "Consisting essentially of" or "consisting essentially of" also have the meanings ascribed to them in U.S. patent law, and the term is open-ended, allowing for the presence of more than those listed, as long as the basic or novel features of those listed are not changed by the presence of more than those listed, but excluding prior art embodiments.

[0029] The terms "polypeptide", "peptide" and "protein" as used herein, and other related terms, are used interchangeably and refer to polymers of amino acid residues, wherein in embodiments, the polymer may be coupled to a portion that is not composed of amino acids. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. "Fusion protein" refers to a chimeric protein containing sequences from two or more separate proteins, for example, these proteins are recombinantly expressed, synthesized or coupled to form a single part. Polypeptides include mature molecules that have been cleaved. These terms cover natural and artificial proteins, protein fragments and polypeptide analogs of protein sequences (such as mutants, variants, chimeric proteins and fusion proteins), as well as proteins that are covalently or non-covalently modified after translation or otherwise. Two or more polypeptides (e.g., 3 polypeptide chains) can be associated with each other by covalent and / or non-covalent association to form a multimeric polypeptide complex (e.g., a multispecific antigen-binding protein complex). The association of polypeptide chains may also include peptide folding. Thus, a polypeptide complex may be a dimer, trimer, tetramer or higher order complex, depending on the number of polypeptide chains forming the complex.

[0030] As used herein, the terms "cancer", "cancer" and "tumor" are used interchangeably and in the singular or plural to refer to cells that have undergone malignant transformation that causes them to have a pathological effect on the host organism. In embodiments, the cancer is a cancer that overexpresses FOLR1. Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells, in vitro cultures derived from cancer cells, and cell lines. When referring to cancer types that usually manifest as solid tumors, "clinically detectable" tumors are tumors that are detectable based on tumor masses, for example, by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, or palpation during physical examination; and / or tumors that are detectable due to the expression of one or more cancer-specific antigens in samples that can be obtained from patients. The tumor can be a hematopoietic (or blood or hematological or blood-related) cancer, for example, a cancer derived from blood cells or immune cells, which can be referred to as a "liquid tumor". Specific examples of hematological neoplasm-based clinical conditions include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS, and Waldenstrom's macroglobulinemia; lymphomas, such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, and the like.

[0031] Cancer can be any cancer in which there is an abnormal number of mother cells or unwanted cell proliferation, or any cancer diagnosed as a hematological cancer, including both lymphocytic malignancies and myeloid malignancies. Myeloid malignancies include but are not limited to acute myeloid (or myeloid or myeloid or myeloblastic) leukemia (undifferentiated or differentiated), acute promyelocytic (or promyelocytic or promyelocytic or promyelocytic) leukemia, acute myelomonocytic (or myelomonocytic) leukemia, acute monocytic (or mononuclear) leukemia, erythroleukemia and megakaryocyte (or megakaryoblastic) leukemia. These leukemias can be collectively referred to as acute myeloid (or myeloid or myeloid) leukemia (AML). Myeloproliferative diseases (MPDs) are also included in myeloproliferative diseases, including but not limited to chronic myeloid (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocythemia), and polycythemia vera (PCV). Myeloproliferative diseases also include myelodysplasia (or myelodysplastic syndrome or MDS), and myelofibrosis (MFS) with or without idiopathic myeloid metaplasia. Myelodysplasia can be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT).

[0032] Hematopoietic cancers also include lymphocytic malignancies, which can affect the lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal sites. Lymphocytic cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin lymphoma (B-NHL). B-NHL can be low-grade (or indolent), intermediate-grade (or aggressive), or high-grade (very aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL), including nodal MZL, extranodal MZL, splenic MZL, and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemia, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt lymphoma (BL), Burkitt-like lymphoma, small noncleaved cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV-associated (or AIDS-associated) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and giant lymphadenopathy. NHL may also include T-cell non-Hodgkin lymphoma (T-NHL), which includes, but is not limited to, T-cell non-Hodgkin lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphopathy (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sezary syndrome.

[0033] Hematopoietic cancers also include Hodgkin lymphoma (or disease), which includes classical Hodgkin lymphoma, nodular sclerosis Hodgkin lymphoma, mixed cell Hodgkin lymphoma, lymphocyte-dominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphocyte-reduced Hodgkin lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers, such as multiple myeloma (MM), including smoldering MM; monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS); plasmacytoma (bone, extramedullary); lymphoplasmacytic lymphoma (LPL); Waldenstrom's macroglobulinemia; plasma cell leukemia; and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes, and natural killer cells. Tissues comprising hematopoietic cells are referred to herein as "hematopoietic cell tissues" and include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissue associated with mucosa (such as gut-associated lymphoid tissue), tonsils, Peyer's patches and appendix, as well as lymphoid tissue associated with other mucosa, such as bronchial mucosa.

[0034] Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease and non-Hodgkin's lymphoma. Exemplary cancers that can be treated with the compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer and uterine cancer. Additional examples include thyroid cancer, bile duct cancer, pancreatic cancer, skin melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, invasive breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, pancreatic endocrine or exocrine cancer, medullary thyroid cancer, medullary thyroid cancer, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0035] In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include epithelial tumors, including ovarian tumors, uterine tumors, breast tumors, endometrial tumors, pancreatic tumors, kidney tumors, lung tumors, colorectal tumors, and brain tumors. In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include serous and endometrioid epithelial ovarian cancer, endometrial adenocarcinoma, adenocarcinoma subtypes of non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC).

[0036] "Advanced" cancer is a cancer that has spread beyond the site or organ of origin due to local invasion or metastasis. The term "advanced" cancer includes both locally advanced and metastatic disease. "Metastatic" cancer refers to a cancer that has spread from one part of the body to another part of the body. "Refractory" cancer is a cancer that progresses even if anti-tumor treatments such as chemotherapy are administered to the cancer patient. An example of a refractory cancer is a platinum-refractory cancer. "Recurrent" cancer is a cancer that has regrowth at the original site or at a distant site after responding to an initial therapy.

[0037] As used herein, "antibody" and related terms refer to an intact immunoglobulin or an antigen-binding portion thereof that specifically binds to an antigen. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding portions particularly include Fab, Fab', F(ab')2, Fv, domain antibodies (dAb) and complementary determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, tribodies, tetrabodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0038] Antibodies include recombinantly produced antibodies and antigen-binding portions. Antibodies include non-human antibodies, chimeric antibodies, humanized antibodies and fully human antibodies. Antibodies include monospecific antibodies and multispecific (e.g., bispecific, trispecific and higher specific) antibodies. Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers and heavy chain dimers. Antibodies include F(ab')2 fragments, Fab' fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragments (scFv), camelized antibodies, affibodies, disulfide bond-connected Fv (sdFv), anti-idiotypic antibodies (anti-Id) and miniantibodies. Antibodies include monoclonal colonies and polyclonal colonies. Anti-FOLR1 antibodies are described herein.

[0039] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody constituting a population is identical and / or binds to the same epitope, except for example containing naturally occurring mutations or possible variant antibodies produced during the production process of a monoclonal antibody preparation, such variants are generally present in trace amounts. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that an antibody is a feature obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibodies to be produced by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies as described herein.

[0040] As used herein, "epitope" and related terms refer to the portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or antigen binding portion thereof). An epitope may comprise two or more portions of an antigen that are bound by an antigen binding protein. An epitope may comprise a non-contiguous portion of an antigen or two or more non-contiguous portions of an antigen (e.g., amino acid residues that are not contiguous in the primary sequence of the antigen, but are sufficiently close to each other to be bound by an antigen binding protein in the context of the tertiary and quaternary structure of the antigen). Typically, the variable regions of an antibody, particularly the CDRs, interact with the epitope. Anti-FOLR1 antibodies and antigen binding proteins thereof that bind to an epitope of a FOLR1 polypeptide are described herein.

[0041] As used herein, "antibody fragment," "antibody portion," "antigen-binding fragment of an antibody," or "antigen-binding portion of an antibody" and other related terms refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAbs; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); a polypeptide containing at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. Antigen-binding portions of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs) and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer antigen-binding properties to the antibody fragment. Antigen-binding fragments of anti-FOLR1 antibodies are described herein.

[0042] Antigen binding proteins may have the structure of, for example, immunoglobulins. In one embodiment, "immunoglobulins" refer to tetrameric molecules. Each tetrameric molecule consists of two pairs of identical polypeptide chains, each pair of polypeptide chains having a "light" chain (about 25 kDa) and a "heavy" chain (about 50 kDa-70 kDa). The N-terminal of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are classified as κ or λ light chains. In an embodiment, the light chain is a κ light chain. In an embodiment, the light chain is a λ light chain. The heavy chain is divided into μ, δ, γ, α or ε heavy chains, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology, Chapter 7 (Paul, W. ed., 2nd ed., Raven Press, NY (1989)) (incorporated herein by reference in its entirety for all purposes). The variable region of each light chain / heavy chain pair forms an antibody binding site, such that a complete immunoglobulin has two antigen binding sites. In one embodiment, the antigen binding protein can be a synthetic molecule having a structure that is different from a tetrameric immunoglobulin molecule but still binds to a target antigen or binds to two or more target antigens. For example, a synthetic antigen binding protein can comprise an antibody fragment, 1-6 or more polypeptide chains, an asymmetric assembly of polypeptides, or other synthetic molecules. The terms "variable heavy chain", "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain, including Fv, scFv, dsFv or Fab; while the term "variable light chain", "V L" or "VL" refers to the variable region of an immunoglobulin light chain, including Fv, scFv, dsFv or Fab. "Variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman, ed.) and Co., p. 91 (2007). ) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, the VH or VL domain can be used to separate antibodies that bind to a specific antigen from antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol., Vol. 150: pp. 880-887 (1993); Clarkson et al., Nature, Vol. 352: pp. 624-628 (1991). Described herein are antigen-binding proteins with immunoglobulin-like properties that specifically bind to FOLR1.

[0043] Examples of antibody functional fragments include, but are not limited to, complete antibody molecules, antibody fragments such as Fv, single-chain Fv (scFv), complementarity determining regions (CDR), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2', and any combination of them or any other functional parts of immunoglobulin peptides that can bind to the target antigen (see, for example, FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th edition, 2001). As will be appreciated by those skilled in the art, various antibody fragments can be obtained by a variety of methods, such as digesting complete antibodies with enzymes such as pepsin; or synthesizing them de novo. Antibody fragments are generally synthesized de novo by chemical methods or by using recombinant DNA methods. Therefore, as used herein, the term "antibody" includes antibodies produced by modifying whole antibodies. Fragments, or antibody fragments synthesized de novo using recombinant DNA methods (e.g., single-chain Fv), or antibody fragments identified using phage display libraries (see, e.g., McCafferty et al. (1990), Nature, Vol. 348: p. 552). The term "antibody" also includes bivalent or bispecific molecules, diabodies, tribodies and tetrabodies. Bivalent and bispecific molecules are described, for example, in Kostelny et al. (1992), J. Immunol., Vol. 148: p. 1547; Pack and Pluckthun (1992), Biochemistry, Vol. 31: p. 1579; Hollinger et al. (1993), PNAS. USA, Vol. 90: p. 6444; Gruber et al. (1994), J. Immunol., Vol. 152: p. 5368; Zhu et al. (1997), Protein Sci., Vol. 6: p. 781; Hu et al. (1996), Cancer Res., Vol. 56: p. 3055; Adams et al. (1993), Cancer Res., Vol. 53: p. 4026; and McCartney et al. (1995), Protein Eng., Vol. 8: p. 301.

[0044] As used herein, the terms "antigen binding protein", "antigen binding domain", "antigen binding region" or "antigen binding site" and related terms refer to a protein comprising a portion that binds to an antigen and an optional scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., antigen binding portions of antibodies), antibody derivatives, and antibody analogs. The antigen binding protein may comprise an alternative protein scaffold or artificial scaffold, for example, with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising introduced mutations, for example, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Vol. 53, No. 1: pp. 121-129; Roque et al., 2004, Biotechnol. Prog., Vol. 20: pp. 639-654. Additionally, peptide antibody mimetics ("PAMs") can be used, as well as antibody mimetic-based scaffolds utilizing fibronectin components as scaffolds.Antigen binding proteins that bind FOLR1 are described herein.

[0045] In one embodiment, the dissociation constant (K D ) can be measured using the BIACORE surface plasmon resonance (SPR) assay. Surface plasmon resonance refers to an optical phenomenon that allows real-time interactions to be analyzed by detecting changes in protein concentration within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0046] As used throughout this specification, "specific binding" with respect to an anti-FOLR1 antigen binding protein means that the antigen binding protein binds to human FOLR1 (h FOLR1) and does not bind to other human proteins or does not bind significantly to other human proteins. However, this term does not exclude the fact that the antigen binding protein of the invention may also cross-react with other forms of FOLR1, such as primate FOLR1. In an embodiment, if the antibody is expressed at 10 -5 M or lower, or 10 -6 M or lower, or 10 -7 M or lower, or 10 -8 M or lower, or 10 -9 M or lower, or 10 -10 Dissociation constant K M or lower DBinding to antigen, the antibody specifically binds to the target antigen.

[0047] Unless otherwise indicated, as used herein, the term "FOLR1" refers to any native FOLR1 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)) and rodents (e.g., mice and rats). FOLR1 is also known as "human folate receptor 1," "folate receptor alpha (FR-α)" and "FRα." FOLR1 is a single-chain membrane protein capable of binding folate and its derivatives. The term encompasses "full-length," unprocessed FOLR1 as well as any form of FOLR1 produced as a result of processing in cells. The term also encompasses naturally occurring variants of FOLR1, such as splice variants, allelic variants and isoforms. Human FOLR 1 sequences are known and include, for example, publicly available sequences (including isoforms) at UniProtKB Accession No. P 15328. The amino acid sequence of an exemplary human FOLR1 protein is shown in SEQ ID NO: 9.

[0048] The term "FOLR1 -expressing cancer" refers to a cancer comprising cells that express FOLR on the surface of the cells.

[0049] The term "increased expression" or "overexpression" of FOLR1 in a particular tumor, tissue or cell sample means that FOLR1 (FOLR1 polypeptide or nucleic acid encoding such polypeptide) is present at a level higher than that present in healthy or non-diseased (natural, wild-type) tissues or cells of the same type or origin. Such increased expression or overexpression may be caused by, for example, mutation, gene amplification, increased transcription, increased translation or increased protein stability.

[0050] The terms "anti-FOLR1 antibody" and "antibody that binds to FOLR1" refer to an antibody that is capable of binding to FOLR1 with sufficient affinity such that the antibody can be used as a therapeutic agent targeting FOLR1. In one embodiment, the extent of binding of the anti-FOLR1 antibody to an unrelated, non-FOLR1 protein is less than about 10% of the binding of the antibody to FOLR1, as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that binds to FOLR1 is ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13M). In certain embodiments, an anti-FOLR1 antibody binds to a FOLR1 epitope that is conserved among FOLR1 from different species.

[0051] The term "chimeric antibody" and related terms used herein refer to an antibody containing one or more regions from a first antibody and an antibody containing one or more regions from one or more other antibodies. In one embodiment, one or more CDRs in the CDR are derived from human antibodies. In another embodiment, all CDRs in the CDR are derived from human antibodies. In another embodiment, CDRs from more than one human antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody may include CDR1 from the light chain of the first human antibody, CDR2 and CDR3 from the light chain of the second human antibody, and CDRs from the heavy chain of the third antibody. In another example, CDRs are derived from different species such as humans and mice, or humans and rabbits, or humans and goats. It will be appreciated by those skilled in the art that other combinations are also possible.

[0052] In addition, the framework region can be derived from one of the same antibodies, from one or more different antibodies such as human antibodies, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy chain and / or light chain is identical, homologous, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical, homologous, or derived from an antibody from another species or belonging to another antibody class or subclass. Fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to a target antigen) are also included. Chimeric antibodies can be prepared from portions of any of the anti-FOLR1 antibodies described herein.

[0053] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0054] As used herein, the term "Fc" or "Fc region" refers to the portion of the constant region of the heavy chain of an antibody that begins in or after the hinge region and ends at the C-terminus of the heavy chain. The Fc region comprises at least a portion of the CH and CH3 regions, and may or may not comprise a portion of the hinge region. Two polypeptide chains, each carrying half of the Fc region, may dimerize to form the Fc region. The Fc region may bind to some proteins of the Fc cell surface receptor and the immune complement system. The Fc region exhibits effector functions, including complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent phagocytosis (ADP), opsonization and / or cell binding, any one activity or any combination of two or more activities. The Fc region may bind to an Fc receptor, including FcγRI (e.g., CD64), FcγRII (e.g., CD32) and / or FcγRIII (e.g., CD16a).

[0055] "Humanized antibody" refers to an antibody having an antibody sequence different from that of a non-human species antibody by one or more amino acid substitutions, deletions and / or additions, so that when a humanized antibody is applied to a human subject, compared to a non-human species antibody, the humanized antibody is less likely to induce an immune response, and / or induces a less severe immune response. In one embodiment, certain amino acid mutations in the framework and constant domain of the heavy chain and / or light chain of a non-human species antibody are used to produce a humanized antibody. In another embodiment, the constant domains from a human antibody are fused with the variable domains of a non-human species. In another embodiment, when a non-human antibody is applied to a human subject, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the possible immunogenicity of the non-human antibody, wherein the amino acid residues changed are not critical for the immunospecific binding of the antibody to its antigen, or the changes made to the amino acid sequence are conservative changes, so that the binding of a humanized antibody to an antigen is not significantly worse than that of a non-human antibody to an antigen. Examples of how to make humanized antibodies can be found in US Pat. Nos. 6,054,297, 5,886,152, and 5,877,293.

[0056] The term "human antibody" refers to an antibody having one or more variable regions and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable domains and constant domains are derived from human immunoglobulin sequences (e.g., fully human antibodies). These antibodies can be prepared in a variety of ways, examples of which are described below: including by recombinant methodology or by immunizing mice with an antigen of interest, the mice being genetically modified to express antibodies derived from human heavy and / or light chain encoding genes. Fully human anti-FOLR1 antibodies and antigen-binding proteins thereof are described herein. This definition of a human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues.

[0057] The term "isolated" means "artificially" changed from its natural state, has been changed or removed from its original environment, or both. When the term "isolated" is applied to nucleic acids or proteins, it means that the nucleic acids or proteins are substantially free of other cellular components associated with them in the natural state. For example, nucleic acids or proteins can be in a homogeneous state and can be dry or aqueous solutions. Purity and homogeneity are generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high performance liquid chromatography or mass spectrometry. Proteins that are the main substances present in a preparation are substantially purified. For example, polynucleotides or polypeptides naturally present in living organisms are not "isolated", but the same polynucleotides or polypeptides separated from coexisting substances in their natural state are "isolated", including but not limited to when such polynucleotides or polypeptides are introduced back into cells, even if the cells are the same species or type as the cells from which the polynucleotides or polypeptides were isolated.

[0058] "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is a hypervariable domain of an immunoglobulin heavy chain and light chain. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" may refer to all three heavy chain CDRs or all three light chain CDRs (or both all heavy chain and all light chain CDRs, if appropriate).

[0059] CDRs provide most of the contact residues for antibody binding to antigens or epitopes. The CDRs of interest of the present invention are derived from donor antibody variable heavy and light chain sequences and include analogs of naturally occurring CDRs that also share or retain the same antigen binding specificity and / or neutralization ability as the donor antibodies from which they are derived.

[0060] The CDR sequences of an antibody can be determined by the Kabat numbering system (Kabat et al., Sequences of proteins of Immunological Interest NIH, 1987); alternatively these sequences can be determined using the Chothia numbering system (Al-Lazikani et al. (1997), JMB, Vol. 273: pp. 927-948), the contact definition method (MacCallum RM, Martin ACR, and Thornton JM (1996), Journal of Molecular Biology, Vol. 262 No. 5: pp. 732-745), or any other well-established method known to those of skill in the art for numbering residues in antibodies and determining CDRs.

[0061] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlap region can be determined using at least two of the Kabat, Chothia, AbM and contact methods to provide a "minimum binding unit". The minimum binding unit can be a sub-portion of a CDR.

[0062] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be determined by conventional methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0063] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such alterations.

[0064] As used herein, the terms "variant" polypeptide and "variant" of a polypeptide refer to a polypeptide comprising an amino acid sequence having one or more amino acid residues inserted into, substituted into, and / or deleted from an amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, a variant polynucleotide comprises a nucleotide sequence having one or more nucleotides inserted into, substituted into, and / or deleted from a nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0065] As used herein, the term "domain" refers to a folded protein structure that has a tertiary structure that is independent of the rest of the protein. Typically, a domain is responsible for discrete functional properties of a protein, and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and / or the function of the domain. An "antibody single variable domain" is a folded polypeptide domain comprising a characteristic sequence of an antibody variable domain. Therefore, it includes complete antibody variable domains and modified variable domains (e.g., wherein one or more loops have been replaced by sequences characteristic of non-antibody variable domains); or antibody variable domains that have been truncated or comprise N-terminal or C-terminal extensions; and folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain.

[0066] As used herein, the term "cytotoxic agent" or "payload" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other chimeric agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0067] A "chemotherapeutic agent" is a compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins (particularly bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachol; colchicine; betulinic acid (betulinic); camptothecin (including synthetic analogs topotecan CPT-11 (irinotecan, ), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; callisthenic acid; CC-1065 (including its synthetic analogs adolesin, carzelesin, and biszelesin); podophyllotoxin; podophyllic acid; teniposide; scutellarin (particularly scutellarin 1 and scutellarin 8); dolastatin; auristatin; dolostatin; duocarmycin (including its synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongestatin; nitrogen mustard; mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nebiquinol, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem Intl. Ed. Engl., Vol. 33: pp. 183-186 (1994)); danemycins, including danemycin A; esperamicins;and neocarzinostatin chromophores and related chromoprotein enediyne antiobiotic chromophores, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carmomycin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), acid), nogamycin, olivomycin, peplomycin, porfibrinocin, puromycin, quelamycin, rhodorubicin, streptomycin, streptozotocin, tuberculocidin, ubenimex, zoloft, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludalafil guanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, propionate, cyclothiodine, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansines, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triiminoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine Dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ without Cremophor, an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and docetaxel ( Rorer, Antony, France); chloranbucil; gemcitabine 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Oxaliplatin; leucovorin; vinorelbine Mitoxantrone (novantrone); edatrexate (edatrexate); daunorubicin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine A pharmaceutically acceptable salt, acid or derivative of any of the above substances; and a combination of two or more of the above substances, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; CVP, an abbreviation for the combination therapy of cyclophosphamide, vincristine and prednisolone; and FOLFOX, an abbreviation for a treatment regimen utilizing oxaliplatin (ELOXATIN™) in combination with 5-FU and folinic acid.

[0068] "Antibody-drug conjugate" or "ADC" is an antibody coupled to one or more heterologous molecules, including but not limited to a cytotoxic agent. The antibody can be any antibody described herein. The cytotoxic agent can be any cytotoxic agent described herein. The antibody can be directly connected to the cytotoxic agent by a covalent bond, or the antibody can be indirectly connected to the cytotoxic agent by a linker. Typically, the linker is covalently bonded to the antibody and also covalently bonded to the cytotoxic agent. Such a linker can be a cleavable linker, for example, cleavable under certain pH conditions (pH sensitive linker), cleavable by a protease (protease sensitive linker), or cleavable in the presence of glutathione (glutathione sensitive linker). In some examples, the linker comprises a protease cleavage site, which may contain 2-5 amino acid residues that can be recognized and / or cleaved by a suitable protease. Such a peptide may comprise naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof. In one example, the peptide linker may be a dipeptide linker. Examples include valine-citrulline (val-cit) linkers, phenylalanine-lysine (phe-lys) linkers, or maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linkers. Alternatively, the linker may be non-cleavable, such as a linker comprising an optionally substituted alkane or thioether. In some examples, the linker may include a functional group that can form a covalent bond with the antibody. Exemplary functional groups include, but are not limited to, maleimide groups, iodoacetamide groups, vinyl sulfone groups, acrylate groups, acrylamide groups, acrylonitrile groups, or methacrylate groups. The term "antibody-drug conjugate" or "ADC" refers to a conjugate in which at least one cytotoxic agent is a therapeutic moiety such as a drug.

[0069] As used herein, the term "coupled" when referring to two parts means that the two parts are bonded, wherein the bond or bonds connecting the two parts can be covalent or non-covalent. In an embodiment, the two parts are covalently bonded to each other (e.g., directly or through a covalently bonded intermediate). In an embodiment, the two parts are non-covalently bonded (e.g., through ionic bonds, van der Waals bonds / interactions, hydrogen bonds, polar bonds, or combinations or mixtures thereof).

[0070] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human. In certain embodiments, a subject is an adult, a teenager, a child, or an infant. In some embodiments, the term "individual" or "patient" is intended to be used interchangeably with "subject."

[0071] "Percentage (%) of amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and, if necessary, introducing gaps to achieve maximum percentage sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math., Vol. 2: 482, by means of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol., Vol. 48: 443, by means of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, Vol. 88: 2444, or by means of a computer program using said algorithm (e.g., EMBOSS Needle or EMBOSS Water, available from www.ebi.ac.uk / Tools / psa / ). Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for maximum comparison on the total length of the sequence being compared. As used herein, "sequence identity percentage" or "percentage (%) [sequence] identity" is determined by comparing the sequences of two best local comparisons on a comparison window limited by the length of the local comparison between the two sequences. (It can also be considered that this is homology percentage or "percentage (%) homology".) Compared with the reference sequence, the amino acid sequence in the comparison window can include additions or deletions (for example, gaps or overhangs) for the best comparison of the two sequences. The local comparison between the two sequences only includes that each sequence is considered to be sufficiently similar according to the standard depending on the algorithm (for example, EMBOSS Water) used for comparison. "Identical" or "identity" percentage means that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or specified region). The identity percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100.The best alignment for the sequence compared can be by the local homology algorithm of Smith and Waterman (Add.APL.Math., Vol. 2: Page 482, 1981), by the global homology alignment algorithm of Needleman and Wunsch (J.Mol.Biol., Vol. 48: Page 443, 1970), by the similarity search method of Pearson and Lipman (Proc.Natl.Acad.Sci.USA, Vol. 85: Page 2444, 1988), or by inspection. As another example, GAP and BESTFIT can be used to determine the best alignment of two sequences identified for comparison. Usually, the default value 5.00 of the gap weight and the default value 0.30 of the gap weight length are used.

[0072] The comparison of sequences and the determination of the percent identity between two polypeptide sequences or between two polynucleotide sequences can be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences can be determined by comparing sequences using the GAP computer program (part of the GCG Wisconsin software package, version 10.3 (Accelrys, San Diego, Calif.)) using default parameters. A statement about a test sequence such as "comprising a sequence having at least X% identity to Y" means that, when aligned with a sequence Y as described above, the test sequence comprises a residue having at least X% identity to the residues of Y.

[0073] In one embodiment, the amino acid sequence of the test antibody may be similar but not identical to any of the amino acid sequences of the polypeptides that constitute the multispecific antigen-binding protein complexes described herein. The similarity between the test antibody and the polypeptide may be at least 95%, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the polypeptides that constitute the multispecific antigen-binding protein complexes described herein. In one embodiment, similar polypeptides may contain amino acid substitutions within the heavy chain and / or light chain. In one embodiment, the amino acid substitutions include one or more conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, e.g., Pearson (1994), Methods Mol. Biol., Vol. 24: pp. 307-331, which is incorporated herein by reference in its entirety. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine.

[0074] Antibodies can be obtained from sources such as serum or plasma, which contain immunoglobulins with different antigen specificities. If these antibodies are subjected to affinity purification, antibodies with specific antigen specificity can be enriched. This enriched preparation of antibodies is usually made of less than about 10% of antibodies with specific binding activity to a specific antigen. Subjecting these preparations to several rounds of affinity purification can increase the proportion of antibodies with specific binding activity to an antigen. Antibodies prepared in this way are generally referred to as "monospecific". Monospecific antibody preparations can be made of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 99.9% of antibodies with specific binding activity to a specific antigen. Antibodies can be produced using recombinant nucleic acid technology as described below.

[0075] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0076] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may include mutations. Mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0077] The term "pharmaceutically acceptable salt" is intended to include salts of active compounds prepared with relatively nontoxic acids or bases, depending on the specific substituents present on the compounds described herein. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired pure base or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired pure acid or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and those derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid, and the like (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, Vol. 66: pp. 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functional groups that allow the compounds to be converted into either base addition salts or acid addition salts.

[0078] Therefore, the compounds of the present disclosure may exist as salts, such as salts with pharmaceutically acceptable acids. The present disclosure includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, mesylates, nitrates, maleates, acetates, citrates, fumarates, propionates, tartrates (e.g., (+)-tartrates, (-)-tartrates or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts formed with amino acids such as glutamic acid, and quaternary ammonium salts (e.g., iodomethane, iodoethane, etc.). These salts can be prepared by methods known to those skilled in the art.

[0079] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0080] In addition to salt forms, the present disclosure provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide compounds of the present disclosure. Prodrugs of the compounds described herein may be converted in vivo after administration. In addition, prodrugs may be converted into compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.

[0081] Some compounds of the present disclosure can exist in non-solvated form and solvated form (including hydrated form). Generally speaking, the solvated form is equivalent to the non-solvated form and is included in the scope of the present disclosure. Some compounds of the present disclosure can exist in multiple crystals or amorphous forms. Generally speaking, all physical forms are equivalent for the intended use of the present disclosure and are intended to be within the scope of the present disclosure.

[0082] "Pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate administration of an active agent to a subject and facilitate absorption of the active agent by the subject, and that may be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions, lactated Ringer's solutions, physiological sucrose, physiological glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (e.g., Ringer's solutions), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants, etc. Such preparations may be sterilized and, if desired, mixed with adjuvants that do not react deleteriously with the compounds of the present disclosure, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, colorants, and / or aromatic substances, etc. Those skilled in the art will recognize that other pharmaceutical excipients may also be used in the present disclosure.

[0083] The term "pharmaceutical formulation" refers to a preparation which is in such form as to render the biological activity of the active ingredient contained therein effective, and which contains no additional components which would be unacceptably toxic to a subject to which the formulation would be administered.

[0084] The term "administering / administered" and grammatical variations refer to the physical introduction of a medicament into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of preparations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. In some embodiments, the preparation is by non-parenteral route, such as oral administration. Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical administration. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0085] Administration "in combination" with one or more other therapeutic agents includes administration simultaneously (in parallel) or sequentially in any order. Combination therapy can provide "synergy" and is "synergistic", that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. A synergistic effect is obtained when the active ingredients meet the following conditions: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered sequentially, alternatingly or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be obtained when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. The effect produced by a synergistic combination is greater than the additive effect of the individual components of the combination.

[0086] An "effective amount" of an agent, such as a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0087] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.Chemical structures and formulae described herein are constructed according to standard rules of chemical valency known in the chemical art.

[0088] The description of the compounds of the present disclosure is limited to the principles of chemical bonding known to those skilled in the art. Therefore, when a group may be substituted by one or more of a plurality of substituents, such substituents are selected so as to comply with the principles of chemical bonding and to provide compounds that are not inherently unstable and / or that are known to those of ordinary skill in the art to be unstable under environmental conditions such as aqueous, neutral, and several known physiological conditions. For example, heterocycloalkyl or heteroaryl groups are connected to the rest of the molecule via ring heteroatoms in accordance with the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0089] Where substituent groups are designated by their conventional chemical formula, written left to right, they also encompass chemically identical substituents that would result from writing the structure right to left, for example -CH2O- is equivalent to -OCH2-.

[0090] The term "saccharide" means a carbohydrate (or sugar). In an embodiment, the saccharide is a monosaccharide. In an embodiment, the saccharide is a polysaccharide. The most basic unit of a saccharide is a carbohydrate monomer. Its general formula is C n H 2n O n The term "sugar derivative" means a sugar molecule that has been modified with a substituent other than a hydroxyl group. Examples include glycosylamines, sugar phosphates, and sugar esters. Other sugar derivatives include, for example, β-D-glucuronyl, D-galactosyl, and D-glucosyl.

[0091] The term "charged group" means a chemical group that carries a positive or negative charge, such as, for example, phosphate, phosphonate, sulfate, sulfonate, nitrate, carboxylate, carbonate, etc. In some embodiments, the charged group is at least 50% ionized in at least one aqueous solution having a pH in the range of 5 to 9. In some embodiments, the charged group is an anionic charged group.

[0092] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight (i.e., unbranched) or branched carbon chain (or carbons), or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include monovalent, divalent, and polyvalent groups. An alkyl group may contain a specified number of carbons (e.g., C1-C 10 Meaning one to ten carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, methyl, such as homologues and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologues and isomers. Alkoxy is an alkyl group connected to the rest of the molecule by an oxygen linker (-O-). The alkyl portion may be an alkenyl portion. The alkyl portion may be an alkynyl portion. The alkyl portion may be fully saturated. Alkenyl groups may contain, in addition to the one or more double bonds, more than one double bond and / or one or more triple bonds. Alkynyl groups may contain, in addition to the one or more triple bonds, more than one triple bond and / or one or more double bonds.

[0093] Unless otherwise stated, the term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkyl group, such as, but not limited to, -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, with those having 10 or fewer carbon atoms being preferred herein. "Lower alkyl" or "lower alkylene" is a short chain alkyl or alkylene group typically having eight or fewer carbon atoms. Unless otherwise indicated, the term "alkenylene" by itself or as part of another substituent means a divalent radical derived from an alkene.

[0094] Unless otherwise indicated, the term "heteroalkyl" by itself or in combination with another term means a stable straight or branched chain, or any combination thereof, comprising at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, or S), and wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatom (e.g., O, N, S, Si, or P) may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3 and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. The heteroalkyl moiety may contain one heteroatom (e.g., O, N, S, Si or P). The heteroalkyl moiety may contain two optionally different heteroatoms (e.g., O, N, S, Si or P). The heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P). Unless otherwise stated, the term "heteroalkenyl" by itself or in combination with another term means a heteroalkyl group comprising at least one double bond. The heteroalkenyl group may optionally include more than one double bond and / or one or more triple bonds in addition to the one or more double bonds. Unless otherwise stated, the term "heteroalkynyl" by itself or in combination with another term means a heteroalkyl group comprising at least one triple bond. The heteroalkynyl group may optionally include more than one triple bond and / or one or more double bonds in addition to the one or more triple bonds.

[0095] Similarly, unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms may also occupy one or both ends of the chain (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). In addition, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the chemical formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups as used herein include those groups that are attached to the rest of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR' and / or -S02R'. When referring to "heteroalkyl" followed by a listing of specific heteroalkyl groups such as -NR'R", etc., it should be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, specific heteroalkyl groups are listed to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups, such as -NR'R", etc.

[0096] Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms mean cyclic forms of "alkyl" and "heteroalkyl", respectively. Cycloalkyl and heterocycloalkyl are not aromatic. In addition, for heterocycloalkyl, heteroatoms may occupy the position where the heterocycle is connected to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene" by themselves or as part of another substituent mean a divalent radical derived from cycloalkyl and heterocycloalkyl, respectively.

[0097] In an embodiment, the term "cycloalkyl" means a monocyclic, bicyclic or polycyclic cycloalkyl ring system. In an embodiment, the monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, wherein such groups can be saturated or unsaturated, but not aromatic. In an embodiment, the cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl. The bicyclic cycloalkyl ring system is a bridged monocyclic or fused bicyclic. In an embodiment, the bridged monocyclic contains a monocyclic cycloalkyl ring in which two non-adjacent carbon atoms of the monocyclic ring are connected by an alkylene bridge (i.e., in the form of (CH2) between one and three additional carbon atoms) . w In some embodiments, the bicyclic cycloalkyl ring system is connected to the parent molecular moiety by any carbon atom contained in the bicyclic cycloalkyl ring. In some embodiments, the bicyclic cycloalkyl ring system is connected to the parent molecular moiety by any carbon atom contained in the bicyclic cycloalkyl ring. In some embodiments, the cycloalkyl group is optionally substituted by one or two groups that are independently oxo or thia. In an embodiment, the fused bicyclic cycloalkyl is a 5 or 6-membered monocyclic cycloalkyl ring fused to a benzene ring, a 5 or 6-membered monocyclic cycloalkyl, a 5 or 6-membered monocyclic cycloalkenyl, a 5 or 6-membered monocyclic heterocyclyl, or a 5 or 6-membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups independently being oxy or thia. In an embodiment, the polycyclic cycloalkyl ring system is a monocyclic cycloalkyl ring (base ring) fused to any of the following: (i) a ring system selected from bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl; or (ii) two other ring systems independently selected from phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. In an embodiment, the polycyclic cycloalkyl is connected to the parent molecular moiety through any carbon atom contained in the base ring. In an embodiment, the polycyclic cycloalkyl ring system is a monocyclic cycloalkyl ring (base ring) fused to any of the following: (i) one ring system selected from bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl and bicyclic heterocyclyl; or (ii) two other ring systems independently selected from phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl and monocyclic heterocyclyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, tetradecahydrophenanthryl, perhydrophenothiazin-1-yl and perhydrophenoxazin-1-yl.

[0098] In an embodiment, cycloalkyl is cycloalkenyl. The term "cycloalkenyl" is used according to its usual common meaning. In an embodiment, cycloalkenyl is a monocyclic, bicyclic or polycyclic cycloalkenyl ring system. In an embodiment, a monocyclic cycloalkenyl ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, wherein such a group is unsaturated (i.e., contains at least one cyclic carbon-carbon double bond), but is not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In an embodiment, a bicyclic cycloalkenyl ring is a bridged monocyclic or fused bicyclic. In an embodiment, a bridged monocyclic contains a monocyclic cycloalkenyl ring, wherein two non-adjacent carbon atoms of the monocyclic ring are connected by an alkylene bridge (i.e., in the form of (CH2) between one and three additional carbon atoms) . w In some embodiments, the bicyclic cycloalkenyl group is connected to the parent molecular moiety by any carbon atom contained in the monocyclic cycloalkenyl ring. In some embodiments, the cycloalkenyl group is optionally substituted by one or two groups that are independently oxo or thia. In an embodiment, the polycyclic cycloalkenyl ring contains a monocyclic cycloalkenyl ring (base ring) fused to any of the following: (i) one ring system selected from bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl; or (ii) two ring systems independently selected from phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. In an embodiment, the polycyclic cycloalkenyl is attached to the parent molecular moiety via any carbon atom contained within the base ring. In an embodiment, the polycyclic cycloalkenyl ring contains a monocyclic cycloalkenyl ring (base ring) fused to any of the following: (i) one ring system selected from bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl; or (ii) two ring systems independently selected from phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl.

[0099] In an embodiment, heterocycloalkyl is a heterocyclic radical. As used herein, the term "heterocyclic radical" means a monocyclic, bicyclic or polycyclic heterocycle. The heterocyclic radical monocyclic heterocycle is a 3, 4, 5, 6 or 7-membered ring containing at least one heteroatom independently selected from O, N and S, wherein the ring is saturated or unsaturated, but not aromatic. The 3 or 4-membered ring contains 1 heteroatom selected from O, N and S. The 5-membered ring may contain zero or one double bond and one, two or three heteroatoms selected from O, N and S. The 6 or 7-membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from O, N and S. The heterocyclic radical monocyclic heterocycle is connected to the parent molecular moiety by any carbon atom or any nitrogen atom contained in the heterocyclic radical monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiamorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithiopyranyl. Heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused with phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocycle or monocyclic heteroaryl.Heterocyclyl bicyclic heterocycle is connected to the parent molecular part by any carbon atom or any nitrogen atom contained in the monocyclic heterocyclic part of the bicyclic ring system.Representative examples of bicyclic heterocyclic radicals include but are not limited to 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indol-1-yl, indol-2-yl, indol-3-yl, 2,3-dihydrobenzothiophene-2-yl, decahydroquinolyl, decahydroisoquinolyl, octahydro-1H-indolyl and octahydrobenzofuranyl.In an embodiment, the heterocyclyl group is optionally substituted by one or two groups independently being oxo or thia. In certain embodiments, the bicyclic heterocyclic radical is a 5 or 6-membered monocyclic heterocyclic radical ring fused to a benzene ring, a 5 or 6-membered monocyclic cycloalkyl, a 5 or 6-membered monocyclic cycloalkenyl, a 5 or 6-membered monocyclic heterocyclic radical, or a 5 or 6-membered monocyclic heteroaryl, wherein the bicyclic heterocyclic radical is optionally substituted by one or two groups independently being oxy or thia. The polycyclic heterocyclic radical ring system is a monocyclic heterocyclic radical ring (base ring) fused to any of the following: (i) a ring system selected from bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl and bicyclic heterocyclic radical; or (ii) two other ring systems independently selected from phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl and monocyclic or bicyclic heterocyclic radical. The polycyclic heterocyclic radical is connected to the parent molecular moiety by any carbon atom or nitrogen atom contained in the base ring.In an embodiment, the polycyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (base ring) fused to any of: (i) one ring system selected from bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl and bicyclic heterocyclyl; or (ii) two other ring systems independently selected from phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl and monocyclic heterocyclyl. Examples of polycyclic heterocyclyl groups include, but are not limited to, 10H-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl, 1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

[0100] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" includes but is not limited to fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0101] Unless otherwise indicated, the term "acyl" means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0102] Unless otherwise indicated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which may be a monocyclic ring or polycyclic rings (preferably 1 to 3 rings) fused together (i.e., fused ring aryl) or covalently linked. A fused ring aryl refers to multiple rings fused together, wherein at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom such as N, O or S, wherein the nitrogen atom and the sulfur atom are optionally oxidized, and the nitrogen atom is optionally quaternized. Therefore, the term "heteroaryl" includes fused ring heteroaryl groups (i.e., multiple rings fused together, wherein at least one of the fused rings is an aromatic heterocycle). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, one of which has 6 members and the other has 6 members, and at least one of which is a heteroaryl ring. And, a 6,5-fused ring heteroarylene refers to two rings fused together, one of which has 6 members and the other has 5 members, and at least one of which is a heteroaryl ring. A heteroaryl group can be attached to the rest of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, thienyl, pyridyl, pyrimidinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothienyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazole 4-Pyridyl, 2-Pyrimidyl, 4-Pyrimidyl, 5-Benzothiazolyl, 2-Benzimidazolyl, 5-Indolyl, 1-Isoquinolyl, 5-Isoquinolyl, 2-Quinoxalinyl, 5-Quinolinyl, 3-Quinolinyl, and 6-Quinolinyl. Substituents for each of the above mentioned aryl and heteroaryl ring systems are selected from the group consisting of acceptable substituents described below. "Arylene" and "heteroarylene" by themselves or as part of another substituent mean a divalent radical derived from aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0103] A fused ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. A fused ring heterocycloalkyl-aryl, a fused ring heterocycloalkyl-heteroaryl, a fused ring heterocycloalkyl-cycloalkyl or a fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein.

[0104] Spirocycle is two or more rings, wherein adjacent rings are connected by a single atom. Each ring in the spirocycle can be the same or different. Each ring in the spirocycle can be substituted or unsubstituted, and can have a substituent different from each other ring in a group of spirocycles. The possible substituents of each ring in the spirocycle are possible substituents of the same ring when it is not a part of the spirocycle (for example, substituents of cycloalkyl or heterocycloalkyl rings). The spirocycle can be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or substituted or unsubstituted heterocycloalkylene, and each ring in the spirocyclic group can be any ring just listed above, including all rings with one type (for example, all rings are substituted heterocycloalkylene, wherein each ring can be the same or different substituted heterocycloalkylene). When referring to the spirocyclic ring system, heterocyclic spirocycle means a spirocycle in which at least one ring is a heterocycle and each ring can be different rings. When referring to the spirocyclic ring system, the substituted spirocycle means that at least one ring is substituted, and each substituent can be optionally different.

[0105] symbol (Wavy line) indicates the point at which a chemical moiety is attached to the rest of the molecule or formula.

[0106] As used herein, the term "oxy" means oxygen double-bonded to a carbon atom.

[0107] As used herein, the term "alkylsulfonyl" refers to a moiety having the formula -S(O2)-R', wherein R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specific number of carbon atoms (eg, "C1-C4 alkylsulfonyl").

[0108] The term "alkylenaryl" is an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In an embodiment, the alkylenaryl group has the formula:

[0109] The alkylenearyl moiety may be substituted with substituents (e.g., halogen, oxo, -N, -CF, -CCl, -CBr, -CI, -CN, -CHO, -OH, -NH, -COOH, -CONH, -NO, -SH, -SOCH, -SOH, -OSOH, -SONH, NHNH, ONH, NHC(O)NHNH, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted 2 to 5 membered heteroalkyl) on the alkylene moiety or the arylene linker (e.g., at carbon 2, 3, 4, or 6). In an embodiment, the alkylenearyl is unsubstituted.

[0110] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the group. Preferred substituents for each type of group are provided below.

[0111] Substituents for alkyl and heteroalkyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be one or more groups selected from, but not limited to, the following groups: -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R ", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R"')=NR"", -NR-C(NR' R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', NR'NR"R"', ONR'R", NR'C(O)NR"NR"'R"", - CN, -NO2, -NR'SO2R", -NR'C(O)R", -NR'C(O)-OR", -NR'OR", the number is in the range of zero to (2m'+1), where m' is the total number of carbon atoms in the group. R, R', R", R'' and R"" each preferably independently refers to hydrogen; substituted or unsubstituted heteroalkyl; substituted or unsubstituted cycloalkyl; substituted or unsubstituted heterocycloalkyl; substituted or unsubstituted aryl (e.g., substituted by 1-3 halogens); aryl); substituted or unsubstituted heteroaryl; substituted or unsubstituted alkyl, alkoxy or thioalkoxy group; or arylalkyl group. When the compounds described herein contain more than one R group, for example, each of the R groups is independently selected, just as when there are more than one R', R", R"' and R"" groups, each of these groups is independently selected. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example, -NR'R" includes but is not limited to 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0112] Similar to the substituents described for the alkyl groups, the substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', NR'NR"R"', ONR'R", NR'C(O)NR"NR"'R"', -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, -NR'SO2R", -NR'C(O)R", -NR'C(O)-OR", -NR'OR", in a number ranging from zero to the total number of open valences on the aromatic ring system; and wherein R', R", R"' and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When the compounds described herein contain more than one R group, for example, each of the R groups is independently selected, just as each of these groups is independently selected when more than one R', R", R"' and R"" groups are present.

[0113] Substituents of the ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene or heteroarylene) may be depicted as substituents on the ring, rather than substituents on specific atoms of the ring (commonly referred to as floating substituents). In this case, the substituent may be attached to any atom in the ring atoms (in compliance with the rules of chemical valence), and in the case of fused or spiro rings, the substituent described as being associated with one member of the fused or spiro ring (floating substituent on a monocyclic ring) may be a substituent on any of the fused or spiro rings (floating substituent on a polycyclic ring). When the substituent is attached to the ring rather than a specific atom (floating substituent), and the subscript of the substituent is an integer greater than one, multiple substituents may be on the same atom, the same ring, different atoms, different fused rings, different spiro rings, and each substituent may be optionally different. When the point of attachment of the ring to the rest of the molecule is not limited to a single atom (floating substituent), the point of attachment may be any atom of the ring, and in the case of fused or spiro rings, it may be any atom of any of the fused or spiro rings while complying with the rules of chemical valence. When a ring, fused ring, or spiro ring contains one or more ring heteroatoms and the ring, fused ring, or spiro ring is shown to have one or more floating substituents (including but not limited to points of attachment to the rest of the molecule), the floating substituent may be bonded to the heteroatom. When a ring heteroatom is shown to be bonded to one or more hydrogens in a structure or formula having a floating substituent (e.g., two bonds in a ring nitrogen are bonded to ring atoms and a third bond is bonded to a hydrogen), when the heteroatom is bonded to a floating substituent, it is understood that the substituent will replace the hydrogen while respecting the rules of chemical valence.

[0114] Two or more substituents may be optionally connected to form an aryl, heteroaryl, cycloalkyl or heterocycloalkyl group. Such so-called ring-forming substituents are usually but not necessarily always connected to the cyclic base structure. In one embodiment, the ring-forming substituent is connected to the adjacent members of the base structure. For example, two ring-forming substituents are connected to the adjacent members of the cyclic base structure to form a condensed ring structure. In another embodiment, the ring-forming substituent is connected to a single member of the base structure. For example, two ring-forming substituents are connected to a single member of the cyclic base structure to form a spiro ring structure. In another embodiment, the ring-forming substituent is connected to non-adjacent members of the base structure.

[0115] Two substituents on adjacent atoms of an aromatic or heteroaromatic ring may optionally form a substituent of the formula -TC(O)-(CRR') p -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and p is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of the aromatic or heteroaromatic ring may be optionally replaced by a substituent of the formula -A-(CH2) r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 4. One of the single bonds of the new ring thus formed may be optionally replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aromatic or heteroaromatic ring may be optionally replaced by a substituent of the formula -(CRR') s -X'-(C"R"R"') d -, wherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2- or -S(O)2NR'-. The substituents R, R', R'' and R'' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0116] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0117] As used herein, "substituent group" means a group selected from the following moieties: (A) oxy, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, NHNH2, ONH2, NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -O CF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (B) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), which is substituted with at least one substituent selected from: (i) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, NHNH2, ONH2, NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -O CF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (ii) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), which is substituted with at least one substituent selected from: (a) oxy, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, NHNH2, ONH2, NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -O CF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (b) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), substituted with at least one substituent selected from the group consisting of oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, NHN H2, ONH2, NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl 2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl or phenyl) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0118] As used herein, "size-limited substituent" or "size-limited substituent group" means a group selected from all substituents described above for "substituent group", wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 The aryl group is an aryl group, and each substituted or unsubstituted heteroaryl group is a substituted or unsubstituted 5- to 10-membered heteroaryl group.

[0119] As used herein, "lower substituent" or "lower substituent group" means a group selected from all substituents described above for the "substituent group", wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 6-membered heteroaryl.

[0120] In some embodiments, each substituted group described in the compounds herein is substituted by at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene described in the compounds herein is substituted by at least one substituent group. In other embodiments, at least one or all of these groups are substituted by at least one size-restricted substituent group. In other embodiments, at least one or all of these groups are substituted by at least one low-level substituent group.

[0121] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl group may be a substituted or unsubstituted C1-C 20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C 20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 The arylene group and / or each substituted or unsubstituted heteroarylene group is a substituted or unsubstituted 5- to 10-membered heteroarylene group.

[0122] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10In some embodiments, each substituted or unsubstituted alkylene group is a substituted or unsubstituted C1-C8 alkylene group, each substituted or unsubstituted heteroalkylene group is a substituted or unsubstituted 2-8 membered heteroalkylene group, each substituted or unsubstituted cycloalkylene group is a substituted or unsubstituted C3-C7 cycloalkylene group, each substituted or unsubstituted heterocycloalkylene group is a substituted or unsubstituted 3-7 membered heterocycloalkylene group, each substituted or unsubstituted arylene group is a substituted or unsubstituted C6-C8 alkylene group. 10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 9-membered heteroarylene. In some embodiments, the compound is a chemical species listed in the Examples section, Figures, or Tables below.

[0123] In embodiments, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and / or unsubstituted heteroarylene, respectively). In embodiments, the substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is substituted (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene, respectively).

[0124] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene and / or substituted heteroarylene) is substituted with at least one substituent group, wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In an embodiment, if the substituted moiety is substituted with a plurality of substituent groups, each substituent is different.

[0125] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one size-restricted substituent group, wherein if the substituted moiety is substituted with multiple size-restricted substituent groups, each size-restricted substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with multiple size-restricted substituent groups, each size-restricted substituent group is different.

[0126] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one lower substituent group, wherein if the substituted moiety is substituted with multiple lower substituent groups, each lower substituent group may optionally be different. In an embodiment, if the substituted moiety is substituted with multiple lower substituent groups, each lower substituent is different.

[0127] In an embodiment, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent group, size-restricted substituent group, or low-level substituent group; wherein if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-restricted substituent groups, and low-level substituent groups, each substituent group, size-restricted substituent group, and / or low-level substituent group may optionally be different. In an embodiment, if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-restricted substituent groups, and low-level substituent groups, each substituent group, size-restricted substituent group, and / or low-level substituent group is different.

[0128] Certain compounds of the present disclosure have asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms (which may be defined as (R)- or (S)-, or as (D)- or (L)-amino acids, according to absolute stereochemistry) and individual isomers are included within the scope of the present disclosure. The compounds of the present disclosure do not include those known in the art to be too unstable to be synthesized and / or separated. The present disclosure is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-isomers, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise indicated, these compounds are intended to include both E and Z geometric isomers.

[0129] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ in the structural arrangement or configuration of the atoms.

[0130] As used herein, the term "tautomer" refers to one structural isomer of two or more structural isomers that exist in equilibrium and that is readily converted from one isomeric form to another.

[0131] It will be apparent to those skilled in the art that certain compounds of the present disclosure may exist in tautomeric forms and all such tautomeric forms of the compounds are within the scope of the present disclosure.

[0132] Unless otherwise stated, structures depicted herein are also intended to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the present disclosure.

[0133] It should be noted that throughout the application, alternatives are written in Markush groups, e.g., each amino acid position contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered individually, thereby comprising another embodiment, and the Markush group is not to be understood as a single unit.

[0134] "Linker" or "linker reagent" are used interchangeably and refer to a chemical moiety comprising a covalent bond or chain of atoms that covalently connects an antibody to a drug moiety. In various embodiments, the linker comprises a divalent group. In various embodiments, the linker may comprise one or more amino acid residues.

[0135] An "amino acid unit" has the formula Where R0 is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, —CH2CH2SCH3, —CH2CONH2, —CH2COOH, —CH2CH2CONH2, —CH2CH2COOH, —(CH2)3NHC(═NH)NH2, —(CH2)3NH2, —(CH2)3NHCOCH3, —(CH2)3NHCHO, —(CH2)4NHC(═NH)NH2, —(CH2)4NH2, —(CH2)4NHCOCH3, —(CH2)4NHCHO, —(CH2)3NHCONH2, —(CH2)4NHCONH2, —CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl or cyclohexyl. In various embodiments, the amino acid unit includes not only naturally occurring amino acids, but also minor amino acids and non-naturally occurring amino acid analogs, such as citrulline, norleucine, selenomethionine, β-alanine, etc. The amino acid unit can be represented by its amino acid standard three-letter code (e.g., Ala, Cys, Asp, Glu, Val, Phe, Lys, etc.).

[0136] As used herein, the terms "bioconjugate" and "bioconjugate linker" refer to the resulting association between atoms or molecules of a "bioconjugate reactive group" or "bioconjugate reactive portion". The association can be direct or indirect. For example, the coupling between a first bioconjugate reactive group (e.g., -NH2, -C(O)OH, -N-hydroxysuccinimide or -maleimide) and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amino acid containing an amine side chain or carboxylate) provided herein can be direct, such as coupling via a covalent bond or a linker (e.g., a first linker or a second linker); or indirect, such as coupling via a non-covalent bond (e.g., electrostatic interaction (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interaction (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interaction, etc.). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e., association of two bioconjugate reactive groups), including but not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd Edition, John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982. In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently linked to the second bioconjugate reactive group (e.g., sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacyl moiety) is covalently linked to the second bioconjugate reactive group (e.g., sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently linked to the second bioconjugate reactive group (e.g., sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -N-hydroxysuccinimide moiety) is covalently linked to the second bioconjugate reactive group (e.g., amine). In embodiments, the first bioconjugate reactive group (e.g., fluorophenyl ester moiety) reacts with the second bioconjugate reactive group (e.g., amine) to form a covalent bond.In embodiments, a first bioconjugate reactive group (eg, a -sulfo-N-hydroxysuccinimide moiety) reacts with a second bioconjugate reactive group (eg, an amine) to form a covalent bond.

[0137] Useful bioconjugate reactive moieties for use in the bioconjugation chemistry herein include, for example: (a) carboxyl groups and their various derivatives, including but not limited to N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acyl halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl esters, alkenyl esters, alkynyl esters, and aromatic esters; (b) Hydroxyl groups, which can be converted into esters, ethers, aldehydes, etc. (c) haloalkyl groups, where the halide ion can subsequently be displaced by a nucleophilic group such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion or an alkoxide ion, resulting in a new group covalently attached at the site of the halogen atom; (d) dienophile groups, such as, for example, maleimido or maleimide groups, which are able to participate in a Diels-Alder reaction; (e) an aldehyde or ketone group, so that subsequent derivatization may occur by formation of a carbonyl derivative such as, for example, an imine, hydrazone, semicarbazone or oxime, or by a mechanism such as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, e.g. to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or reacted with maleimides; (h) amine or sulfhydryl groups (e.g., as present in cysteine), which may be, for example, acylated, alkylated or oxidized; (i) olefins, which may undergo, for example, cycloaddition, acylation, Michael addition, etc.; (j) epoxides, which can react with, for example, amines and hydroxy compounds; (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (l) metal silicon oxide bonding; and (m) The metal is bonded to a reactive phosphorus group (eg, a phosphine) to form, for example, a phosphodiester bond. (n) Azides, which are coupled to alkynes using copper-catalyzed cycloaddition click chemistry. (o) The biotin conjugate can react with avidin or streptavidin to form an avidin-biotin complex or a streptavidin-biotin complex.

[0138] The bioconjugate reactive groups can be selected so that they do not participate in or interfere with the chemical stability of the conjugates described herein. Alternatively, the reactive functional groups can be protected from participating in cross-linking reactions due to the presence of protecting groups. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond such as maleimide with a sulfhydryl group.

[0139] "Analog" or "analogs" are used according to their usual ordinary meaning within chemistry and biology, and refer to compounds that are structurally similar to another compound (i.e., the so-called "reference" compound) but differ in composition (e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or in the replacement of one functional group by another, or in the absolute stereochemistry of one or more chiral centers of the reference compound). Thus, an analog is a compound that is similar or comparable to a reference compound in function and appearance, but differs from the reference compound in structure or origin.

[0140] As used herein, common organismal and cell type abbreviations are defined below: Ac Acetyl Ala Alanine Asn Asparagine aq. water-based β-Ala β-Alanine ℃ Temperature in degrees Celsius Citrulline DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DIEA Diisopropylethylamine DMF N,N'-Dimethylformamide EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Et Ethyl Eq equivalent g Glycine h hour HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOBt N-Hydroxybenzotriazole HPLC High Performance Liquid Chromatography LC / MS Liquid chromatography-mass spectrometry LYM lymphocytes Lys Me Methyl mg milligram MeOH Methanol mL milliliters μL / μL microliter MONO monocytes mol mole mmol millimole μmol / umol micromole MS NHS N-Hydroxysuccinimide NEUT Neutrophil PABC p-aminobenzyloxycarbonyl Phe Phenylalanine PLT Platelets RP-HPLC Reverse Phase HPLC rt room temperature Ser Serine t-Bu tert-butyl Tert, Uncle T TFA Trifluoroacetic acid Thr Threonine Val Composition Antibody-drug conjugates

[0141] In one aspect, provided herein is an antibody-drug conjugate (ADC) comprising a monoclonal antibody (Ab), a drug moiety (D), and a linker moiety covalently linking the monoclonal antibody to the drug moiety.

[0142] In another aspect, provided herein is an antibody-drug conjugate (ADC) of formula (I): or a pharmaceutically acceptable salt thereof, wherein: Ab is a monoclonal antibody; m is an integer from 1 to 8; L 1 A linker for binding to a monoclonal antibody; L 2 Contains or is a bond, -C(O)-, -NH-, an amino acid unit, –(CH2CH2O) n –, –(CH2) n – -(4-aminobenzyloxycarbonyl)-, -(C(O)CH2CH2NH)-, or any combination thereof, wherein n is an integer from 1 to 24; and D is the drug moiety.

[0143] In another aspect, provided herein is an antibody-drug conjugate (ADC) of formula (I): or a pharmaceutically acceptable salt thereof, wherein: Ab is anti-FOLR1 antibody; m is an integer from 1 to 8; L 1 A linker that binds to an anti-FOLR1 antibody; L 2 Contains or is a bond, -C(O)-, -NH-, an amino acid unit, –(CH2CH2O) n –, –(CH2) n – -(4-aminobenzyloxycarbonyl)-, -(C(O)CH2CH2NH)-, or any combination thereof, wherein n is an integer from 1 to 24; and D is the drug moiety.

[0144] In an embodiment, m is an integer from 1 to 8. In an embodiment, m is an integer from 2 to 8. In an embodiment, m is an integer from 2 to 4. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8.

[0145] In embodiments, n is an integer from 1 to 24. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9. In embodiments, n is 10. In embodiments, n is 11. In embodiments, n is 12. In embodiments, n is 13. In embodiments, n is 14. In embodiments, n is 15. In embodiments, n is 16. In embodiments, n is 17. In embodiments, n is 18. In embodiments, n is 19. In embodiments, n is 20. In embodiments, n is 21. In embodiments, n is 22. In embodiments, n is 23. In embodiments, n is 24.

[0146] In an embodiment, Ab is a monoclonal antibody. In an embodiment, the monoclonal antibody is an anti-FOLR1 antibody.

[0147] In an embodiment, L 1 is a linker that binds to an anti-FOLR1 antibody. 1 is a linker that binds to one or two sulfur or nitrogen atoms on the anti-FOLR1 antibody. 1is a linker that binds to a sulfur atom on an anti-FOLR1 antibody. 1 is a linker that binds to two sulfur atoms on the anti-FOLR1 antibody. 1 is a linker that binds to a nitrogen atom on the anti-FOLR1 antibody. 1 It is a linker that binds to two nitrogen atoms on the anti-FOLR1 antibody.

[0148] In an embodiment, L 1 is a linker that binds to a cysteine ​​molecule on the anti-FOLR1 antibody. 1 is a linker that binds to two cysteine ​​molecules on the anti-FOLR1 antibody. 1 is a linker that binds to a lysine molecule on the anti-FOLR1 antibody. 1 It is a linker that binds to two lysine molecules on the anti-FOLR1 antibody.

[0149] In an embodiment, L 1 for

[0150] In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for In an embodiment, L 1 for

[0151] When L 1 for When L 1 for When , the two olefinic carbons shown at the bottom of the structure can each bind to a separate sulfur of the anti-FOLR1 antibody.

[0152] When L 1 for When , the carbonyl group binds to the amine of lysine of the anti-FOLR1 antibody.

[0153] In an embodiment, D is:

[0154] In an embodiment, D is: R 1 is H or –C1-C8 alkyl; R 3 H, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR 3A 、-NR 3A R 3B 、-(CH2) v OR 6 、-C(O)NHSO2R 7 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 4 H, halogen, -OR 4A 、-NR 4A R 4B , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; Z 1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; Z 2 is substituted aryl, substituted heteroaryl, substituted cycloalkyl or substituted heterocycloalkyl; R 6 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO(CH2CH2O) w CH2CH2Y, -CONH(CH2CH2O) w CH2CH2Y, A charged group or a saccharide derivative, wherein: v is an integer from 1 to 24; w is an integer from 1 to 24; Y is -NH2, -OH, -COOH or -OCH3; R 7are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; R 10 is -OH, -OCH3 or -COOH; Each R 3A , R 3B , R 4A and R 4B is independently H, or substituted or unsubstituted alkyl.

[0155] In an embodiment, L 2 Contains or is a bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -(4-aminobenzyloxycarbonyl)-, -Gly-, -Ser-, -Thr-, -Ala-, -β-Ala-, -citrulline-(Cit), -(CH2) n –, –(CH2CH2O) n – or any combination thereof.

[0156] In an embodiment, L 2 Contains or is a bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -(4-aminobenzyloxycarbonyl)-, -(CH2) n –, –(CH2CH2O) n – or any combination thereof.

[0157] In an embodiment, L 2 Contains or is a bond, -C(O)-, -NH-, -Gly-, -Ser-, -Thr-, -Ala-, -β-Ala-, -Cit-, –(CH2) n –, –(CH2CH2O) n – or any combination thereof.

[0158] In an embodiment, L 2 Contains or is a bond, -C(O)-, -NH-, Val, Gly, Lys, Cit, –(CH2) n –, –(CH2CH2O) n – or any combination thereof.

[0159] In an embodiment, L 2 Contains or is a bond, -C(O)-, Val, Gly, Cit, –(CH2) n – or any combination thereof.

[0160] In an embodiment, L 2 Contains or is In an embodiment, L2 Contains or is In an embodiment, L 2 Contains or is In an embodiment, L 2 Contains or is In an embodiment, L 2 Contains or is In an embodiment, L 2 Contains or is In an embodiment, L 2 comprises or is -C(O)-(CH2)5-. In an embodiment, L 2 Contains or is In an embodiment, L 2 Contains or is In an embodiment, L 2 Contains or is

[0161] In an embodiment, L 2 In an embodiment, L 2 In the embodiment, L 2 In the embodiment, L 2 In the embodiment, L 2 In one embodiment, L 2 In one embodiment, L 2 comprises or is -(4-aminobenzyloxycarbonyl)-. In an embodiment, L 2 Containing or being –(CH2) n –. In the embodiment, L 2 Contains or is –(CH2CH2O) n –. In the embodiment, L 2 In the embodiment, L 2 In one embodiment, L 2 In one embodiment, L 2 In one embodiment, L 2 In one embodiment, L 2 Contains or is -Cit-.

[0162] In an embodiment, -L 1 -L 2 -yes

[0163] In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes The two CH2 moieties shown on the left side of the structure can each be bound to a separate sulfur of the anti-FOLR1 antibody. 1 -L 2 -yes In embodiments, the two CH2 moieties shown on the left side of the structure can each be bound to a separate sulfur of the anti-FOLR1 antibody. -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes The two olefinic carbons shown at the bottom of the structure can each be bound to a separate sulfur of the anti-FOLR1 antibody. 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes The two CH2 moieties shown on the left side of the structure can each be bound to a separate sulfur of the anti-FOLR1 antibody. 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes In an embodiment, -L 1 -L 2 -yes

[0164] In an embodiment, R 1is H. In an embodiment, R 1 It is a –C1-C8 alkyl group.

[0165] In an embodiment, R 1 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl or hexyl. 1 is methyl. In an embodiment, R 1 In an embodiment, R 1 is propyl. In an embodiment, R 1 is isopropyl. In an embodiment, R 1 In an embodiment, R 1 is isobutyl. In an embodiment, R 1 is tert-butyl. In an embodiment, R 1 In an embodiment, R 1 It is hexyl.

[0166] In an embodiment, R 3 H, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR 3A 、-NR 3A R 3B 、-(CH2) v OR 6 、-C(O)NHSO2R 7 , substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted or unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl).

[0167] In an embodiment, R 3 H, -OR 3A 、-(CH2) v OR 6 、-C(O)NHSO2R 7 , substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl).

[0168] In an embodiment, R 3 H, -OR 3A、-(CH2) v OR 6 、-C(O)NHSO2R 7 , substituted (eg, substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted C1-C6 alkyl.

[0169] In an embodiment, R 3 is an alkyl group (e.g., a C1-C8 alkyl group, a C1-C6 alkyl group, or a C1-C4 alkyl group) that is substituted (e.g., substituted with at least one substituent group, a size-limiting substituent group, or a lower substituent group). In an embodiment, R 3 is an unsubstituted alkyl group (e.g., a C1-C8 alkyl group, a C1-C6 alkyl group, or a C1-C4 alkyl group). 3 is a substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) heteroalkyl (e.g., a 2- to 8-membered heteroalkyl, a 2- to 6-membered heteroalkyl, or a 2- to 4-membered heteroalkyl). 3 is an unsubstituted heteroalkyl group (eg, a 2- to 8-membered heteroalkyl group, a 2- to 6-membered heteroalkyl group, or a 2- to 4-membered heteroalkyl group).

[0170] In an embodiment, R 3 Methyl, ethyl, propyl, butyl, –CH2OH, -CH2CH2OH, -CH2N3, -CH2CH2N3, -CH2OCH3, -CH2OCH2CH3, -CH2CH2OCH3, -CH2CH2OCH2CH3 In an embodiment, R 3 In the embodiment, R 3 In the embodiment, R 3 In the embodiment, R 3 In the embodiment, R 3 is -CH2OH. In an embodiment, R 3 is -CH2CH2OH. In an embodiment, R 3 is -CH2N3. In an embodiment, R 3 is -CH2CH2N3. In an embodiment, R 3 is -CH2OCH3. In an embodiment, R 3 is -CH2OCH2CH3. In an embodiment, R 3 is -CH2CH2OCH3. In an embodiment, R 3 is -CH2CH2OCH2CH3. In an embodiment, R 3 In the embodiment, R3 is H. In an embodiment, R 3 for In an embodiment, R 3 for

[0171] In an embodiment, R 3 Methyl, –CH2OH, -CH2N3. In an embodiment, R 3 for Or -CH2N3.

[0172] In embodiments, v is an integer from 1 to 24. In embodiments, v is 1. In embodiments, v is 2. In embodiments, v is 3. In embodiments, v is 4. In embodiments, v is 5. In embodiments, v is 6. In embodiments, v is 7. In embodiments, v is 8. In embodiments, v is 9. In embodiments, v is 10. In embodiments, v is 11. In embodiments, v is 12. In embodiments, v is 13. In embodiments, v is 14. In embodiments, v is 15. In embodiments, v is 16. In embodiments, v is 17. In embodiments, v is 18. In embodiments, v is 19. In embodiments, v is 20. In embodiments, v is 21. In embodiments, v is 22. In embodiments, v is 23. In embodiments, v is 24.

[0173] In an embodiment, R 4 H, halogen, -OR 4A 、-NR 4A R 4B , substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted or unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl).

[0174] In an embodiment, R 4 H, -OR 4A , substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), or substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl).

[0175] In an embodiment, R 4is an alkyl group (e.g., a C1-C8 alkyl group, a C1-C6 alkyl group, or a C1-C4 alkyl group) that is substituted (e.g., substituted with at least one substituent group, a size-limiting substituent group, or a lower substituent group). In an embodiment, R 4 is an unsubstituted alkyl group (e.g., a C1-C8 alkyl group, a C1-C6 alkyl group, or a C1-C4 alkyl group). 4 is a substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) heteroalkyl (e.g., a 2- to 8-membered heteroalkyl, a 2- to 6-membered heteroalkyl, or a 2- to 4-membered heteroalkyl). 4 is an unsubstituted heteroalkyl group (eg, a 2- to 8-membered heteroalkyl group, a 2- to 6-membered heteroalkyl group, or a 2- to 4-membered heteroalkyl group).

[0176] In an embodiment, R 4 is H, -OH, methyl, ethyl, propyl or butyl. 4 In the embodiment, R 4 In the embodiment, R 4 In the embodiment, R 4 In the embodiment, R 4 is H. In an embodiment, R 4 It is -OH.

[0177] In an embodiment, R 4 It is H or -OH.

[0178] In an embodiment, Z 1 is substituted (e.g., substituted by a substituent group, a size-limiting substituent group, or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In an embodiment, Z 1 is a cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl) that is substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group). In an embodiment, Z 1 is an unsubstituted cycloalkyl group (e.g., a C3-C8 cycloalkyl group, a C3-C6 cycloalkyl group, or a C5-C6 cycloalkyl group). 1 is substituted (e.g., substituted by a substituent group, a size-limiting substituent group, or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., a 3- to 8-membered heterocycloalkyl, a 3- to 6-membered heterocycloalkyl, or a 5- to 6-membered heterocycloalkyl). In an embodiment, Z 1is a substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) heterocycloalkyl (e.g., a 3- to 8-membered heterocycloalkyl, a 3- to 6-membered heterocycloalkyl, or a 5- to 6-membered heterocycloalkyl). 1 is unsubstituted heterocycloalkyl (e.g., 3 to 8-membered heterocycloalkyl, 3 to 6-membered heterocycloalkyl, or 5 to 6-membered heterocycloalkyl). 1 is substituted (e.g., substituted by a substituent group, a size-limiting substituent group, or a lower substituent group) or unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 In an embodiment, Z 1 is substituted (eg, substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) aryl (eg, C6-C 10 Aryl, C 10 In an embodiment, Z 1 is an unsubstituted aryl group (e.g., C6-C 10 Aryl, C 10 In an embodiment, Z 1 is substituted (e.g., substituted by a substituent group, a size-limiting substituent group, or a lower substituent group) or unsubstituted heteroaryl (e.g., a 5- to 10-membered heteroaryl, a 5- to 9-membered heteroaryl, or a 5- to 6-membered heteroaryl). In an embodiment, Z 1 is a substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) heteroaryl (e.g., a 5- to 10-membered heteroaryl, a 5- to 9-membered heteroaryl, or a 5- to 6-membered heteroaryl). In an embodiment, Z 1 is an unsubstituted heteroaryl group (eg, a 5- to 10-membered heteroaryl group, a 5- to 9-membered heteroaryl group, or a 5- to 6-membered heteroaryl group).

[0179] In an embodiment, Z 1 for Wherein each X is independently Cl, Br, I or F; each R' is independently -CH3, -CH2CH3 or -CH2CH2CH3; and q is an integer from 1 to 5.

[0180] In an embodiment, q is 1. In an embodiment, q is 2. In an embodiment, q is 3. In an embodiment, q is 4. In an embodiment, q is 5.

[0181] In an embodiment, X is Cl. In an embodiment, X is Br. In an embodiment, X is I. In an embodiment, X is F.

[0182] In an embodiment, R' is -CH3. In an embodiment, R' is -CH2CH3. In an embodiment, R' is -CH2CH2CH3.

[0183] In an embodiment, Z 1 for In an embodiment, Z 1 for In an embodiment, Z 1 for In an embodiment, Z 1 for

[0184] In an embodiment, Z 2 is a cycloalkyl group (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl) that is substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group). In an embodiment, Z 2 is a substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) heterocycloalkyl (e.g., a 3- to 8-membered heterocycloalkyl, a 3- to 6-membered heterocycloalkyl, or a 5- to 6-membered heterocycloalkyl). 2 is substituted (eg, substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) aryl (eg, C6-C 10 Aryl, C 10 In an embodiment, Z 2 is a heteroaryl group (eg, a 5- to 10-membered heteroaryl group, a 5- to 9-membered heteroaryl group, or a 5- to 6-membered heteroaryl group) that is substituted (eg, substituted with a substituent group, a size-limiting substituent group, or a lower substituent group).

[0185] In an embodiment, Z 2 for in Each G is independently Cl, Br, I, F, -CH3, -CH2CH3, -CH2CH2CH3, -OCH3, -OCH2CH3, -OH or -NH2; and p is an integer from 0 to 4.

[0186] In an embodiment, p is 0. In an embodiment, p is 1. In an embodiment, p is 2. In an embodiment, p is 3. In an embodiment, p is 4.

[0187] In an embodiment, G is Cl. In an embodiment, G is Br. In an embodiment, G is I. In an embodiment, G is F. In an embodiment, G is -CH3. In an embodiment, G is -CH2CH3. In an embodiment, G is -CH2CH2CH3. In an embodiment, G is -OCH3. In an embodiment, G is -OCH2CH3. In an embodiment, G is -OH. In an embodiment, G is -NH2.

[0188] In an embodiment, Z 2 for In an implementation scheme, Z 2 for In an embodiment, Z 2 for In an embodiment, Z 2 for

[0189] In an embodiment, R 6 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO(CH2CH2O) w CH2CH2Y, -CONH(CH2CH2O) w CH2CH2Y, A charged group or a sugar derivative, w is an integer from 1 to 24; Y is -NH2, -OH, -COOH or -OCH3; R 10 It is -OH, -OCH3 or -COOH.

[0190] In an embodiment, R 6 is H, or substituted (e.g., substituted by at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g., substituted by at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted (e.g., substituted by at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), substituted (e.g., substituted by at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C 10 Aryl, C 10aryl or phenyl), substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent) or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), or a saccharide derivative.

[0191] In an embodiment, R 6 is H, substituted (e.g., substituted by a substituent group, a size-limiting substituent group, or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8-membered heterocycloalkyl, 3 to 6-membered heterocycloalkyl, or 5 to 6-membered heterocycloalkyl). In an embodiment, R 6 is a substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) heterocycloalkyl (e.g., a 3- to 8-membered heterocycloalkyl, a 3- to 6-membered heterocycloalkyl, or a 5- to 6-membered heterocycloalkyl). In an embodiment, R 6 is an unsubstituted heterocycloalkyl group (eg, a 3- to 8-membered heterocycloalkyl group, a 3- to 6-membered heterocycloalkyl group, or a 5- to 6-membered heterocycloalkyl group).

[0192] In an embodiment, R 6 is H or substituted (eg, substituted with a substituent group, a size-limiting substituent group, or a lower substituent group) heterocycloalkyl (eg, 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl).

[0193] In an embodiment, R 6 H or

[0194] In an embodiment, R 6 -CO(CH2CH2O) w CH2CH2Y or -CONH(CH2CH2O) w CH2CH2Y, wherein w is an integer from 1 to 24 and Y is -NH2, -OH, -COOH or -OCH3. In an embodiment, R 6 -CO(CH2CH2O) w CH2CH2NH2. In an embodiment, R 6 -CO(CH2CH2O) w CH2CH2OH. In an embodiment, R 6 -CO(CH2CH2O) w CH2CH2COOH. In an embodiment, R 6 -CO(CH2CH2O) w CH2CH2OCH3. In an embodiment, R 6 for -CONH(CH2CH2O) wCH2CH2NH2. In an embodiment, R 6 -CONH(CH2CH2O) w CH2CH2OH. In an embodiment, R 6 -CONH(CH2CH2O) w CH2CH2COOH. In an embodiment, R 6 -CONH(CH2CH2O) w CH2CH2OCH3.

[0195] In embodiments, w is an integer from 1 to 24. In embodiments, w is 1. In embodiments, w is 2. In embodiments, w is 3. In embodiments, w is 4. In embodiments, w is 5. In embodiments, w is 6. In embodiments, w is 7. In embodiments, w is 8. In embodiments, w is 9. In embodiments, w is 10. In embodiments, w is 11. In embodiments, w is 12. In embodiments, w is 13. In embodiments, w is 14. In embodiments, w is 15. In embodiments, w is 16. In embodiments, w is 17. In embodiments, w is 18. In embodiments, w is 19. In embodiments, w is 20. In embodiments, w is 21. In embodiments, w is 22. In embodiments, w is 23. In embodiments, w is 24.

[0196] In an embodiment, Y is -NH2, -OH, -COOH or -OCH3. In an embodiment, Y is -NH2. In an embodiment, Y is -OH. In an embodiment, Y is -COOH. In an embodiment, Y is -OCH3.

[0197] In an embodiment, R 6 for In an embodiment, R 6 for In an embodiment, R 6 for In an embodiment, R 6 for

[0198] In an embodiment, R 6 is a saccharide derivative. In an embodiment, R 6 for In an embodiment, R 6 for In an embodiment, R 6 for

[0199] In an embodiment, R 7is H, substituted or unsubstituted alkyl, or substituted or unsubstituted cycloalkyl.

[0200] In an embodiment, R 7 is H, or substituted (e.g., substituted by at least one substituent group, size-limiting substituent group or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), or substituted (e.g., substituted by at least one substituent group, size-limiting substituent group or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl or C5-C6 cycloalkyl).

[0201] In an embodiment, R 7 is H, substituted (e.g., substituted by a substituent group, a size-limiting substituent group, or a lower substituent group) or unsubstituted cycloalkyl (e.g., a 3- to 8-membered cycloalkyl, a 3- to 6-membered cycloalkyl, or a 5- to 6-membered cycloalkyl). In an embodiment, R 7 is a cycloalkyl group (e.g., a 3- to 8-membered cycloalkyl group, a 3- to 6-membered cycloalkyl group, or a 5- to 6-membered cycloalkyl group) that is substituted (e.g., substituted with a substituent group, a size-limiting substituent group, or a lower substituent group). In an embodiment, R 7 is an unsubstituted cycloalkyl group (eg, a 3- to 8-membered cycloalkyl group, a 3- to 6-membered cycloalkyl group, or a 5- to 6-membered cycloalkyl group).

[0202] In an embodiment, R 7 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. In an embodiment, R 7 In an embodiment, R 7 In an embodiment, R 7 In the embodiment, R 7 In the embodiment, R 7 It is cycloheptyl.

[0203] In an embodiment, each R 3A , R 3B , R 4A and R 4B and R is independently H, or a substituted or unsubstituted alkyl group (eg, a C1-C8 alkyl group, a C1-C6 alkyl group, or a C1-C4 alkyl group).

[0204] In an embodiment, each R 3A , R 3B , R 4A and R 4Bis independently H, or substituted (e.g., substituted by at least one substituent group, size-limiting substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In an embodiment, each R 3A , R 3B , R 4A and R 4B is independently H. In an embodiment, each R 3A , R 3B , R 4A and R 4B is independently substituted (e.g., substituted with at least one substituent group, size-limiting substituent group, or lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). 3A , R 3B , R 4A and R 4B and are independently unsubstituted alkyl groups (eg, C1-C8 alkyl groups, C1-C6 alkyl groups, or C1-C4 alkyl groups).

[0205] In an embodiment, each R 3A , R 3B , R 4A and R 4B R is independently H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl or pentyl. 3A , R 3B , R 4A and R 4B is independently H. In an embodiment, each R 3A , R 3B , R 4A and R 4B is independently methyl. In an embodiment, each R 3A , R 3B , R 4A and R 4B is independently ethyl. In an embodiment, each R 3A , R 3B , R 4A and R 4B In an embodiment, each R 3A , R 3B , R 4A and R 4B is independently isopropyl. In an embodiment, each R 3A , R 3B , R 4A and R 4B In an embodiment, each R 3A, R 3B , R 4A and R 4B is independently isobutyl. In an embodiment, each R 3A , R 3B , R 4A and R 4B is independently tert-butyl. 3A , R 3B , R 4A and R 4B are independently pentyl.

[0206] In an embodiment, D is:

[0207] In an embodiment, D is:

[0208] In embodiments, the ADC (e.g., anti-FOLR1 ADC) is: or a pharmaceutically acceptable salt thereof, wherein m is an integer from 1 to 8. Drug loading

[0209] The drug loading is represented by m, which is the average number of drug moieties (i.e., D) per monoclonal antibody in the antibody-drug conjugate (ADC) of formula (I) and its variants. The drug loading may be in the range of 1 to 20 drug moieties per antibody. The ADC of formula (I) and any embodiment, variant or aspect thereof includes a collection of antibodies coupled to 1 to 20 drug moieties, such as 1 to 8. The average number of drug moieties per antibody in the ADC preparation from the coupling reaction can be characterized by conventional means such as mass spectrometry, ELISA assays and HPLC. The quantitative distribution of the ADC can also be determined based on m. In some cases, the separation, purification and characterization of homogeneous ADCs in which m is a specific value from ADCs with other drug loadings can be achieved by means such as reverse phase HPLC or electrophoresis. In an embodiment, the monoclonal antibody is an anti-FOLR1 antibody. In an embodiment, the average number of drug moieties (i.e., D) per anti-FOLR1 antibody may be in the range of 1 to 20 drug moieties, such as 1 to 8 drug moieties per antibody.

[0210] For some ADCs, m may be limited by the number of attachment sites on the antibody. For example, when the attachment is a cysteine ​​thiol, as in some of the exemplary embodiments described herein, the antibody may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups through which the linker can be attached. In embodiments, the average drug loading of the ADC is in the range of 1 to about 8 or about 2 to about 8. In embodiments, L 1 Capable of forming a covalent bond with the thiol group of a free cysteine ​​in an IgG antibody. 1 Capable of forming covalent bonds with the amine groups of lysine in IgG antibodies.

[0211] In an embodiment, during the coupling reaction, less than the theoretical maximum of the drug moiety is coupled to the antibody. Typically, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be connected to the drug moiety; in fact, most of the cysteine ​​thiol residues in antibodies exist as disulfide bridges. In an embodiment, the antibody can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups. In an embodiment, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0212] The loading capacity (drug / antibody ratio or "dar") of the ADC can be controlled in various ways, for example by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) limiting the reducing conditions used for cysteine ​​thiol modification.

[0213] In an embodiment, provided herein is an antibody-drug conjugate wherein the linker moiety reacts with a cysteine ​​or lysine on the antibody in a controlled manner. In an embodiment, for most conjugates, the drug-antibody ratio (DAR) for conjugation to antibody lysine is 2. In an embodiment, for most conjugates, the drug-antibody ratio (DAR) for conjugation to antibody cysteine ​​is 4.

[0214] It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific to the antibody and specific to the drug. Individual ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006), Prot. Engr. Design & Selection, Vol. 19, No. 7: pp. 299-307; Hamblett et al. (2004), Clin. Cancer Res., Vol. 10: pp. 7063-7070; Hamblett, KJ et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC et al., "Controlling the location of drug attachment in antibody-drug conjugates," conjugates", Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004). In embodiments, homogeneous ADCs with a single loading value can be separated from the conjugated mixture by electrophoresis or chromatography. In embodiments, the improvement of lysine selectivity can result in a less heterogeneous mixture (U.S. Pat. No. 9,981,046). Anti-FOLR1 Antibodies The disclosure of WO2020 / 016661 is incorporated herein by reference in its entirety. i. Exemplary Antibodies and Antibody Sequences

[0215] In an embodiment, the ADC comprises an antibody that binds to FOLR1.FOLR1 has been reported to be overexpressed in tumors of epithelial origin, including ovarian, uterine, breast, endometrial, pancreatic, kidney, lung, colorectal, and brain tumors.

[0216] In embodiments, an anti-FOLR1 antibody provided herein comprises a cysteine. In embodiments, an anti-FOLR1 antibody is conjugated to a drug via the sulfur of a cysteine ​​residue. In embodiments, an anti-FOLR1 antibody is conjugated to a drug via the sulfur of two cysteine ​​residues.

[0217] In embodiments, an anti-FOLR1 antibody provided herein comprises a lysine. In embodiments, an anti-FOLR1 antibody is conjugated to a drug via the amine of a lysine residue. In embodiments, an anti-FOLR1 antibody is conjugated to a drug via the amines of two lysine residues.

[0218] In embodiments, the ADC provided herein comprises an anti-FOLR1 antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a light chain complementarity determining region 1 (CDR1), a light chain CDR2, and a light chain CDR3, and the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3.

[0219] In an embodiment, the ADC provided herein comprises an anti-FOLR1 antibody comprising at least one, two, three, four, five or six CDRs selected from the following: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VLCDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least one CDR selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VHCDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least two CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least three CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6.In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least four CDRs selected from the group consisting of: (a) a VLCDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least five CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VHCDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least six CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6.

[0220] In an embodiment, the ADC provided herein comprises an anti-FOLR1 antibody comprising at least one, two, three, four, five or six CDRs selected from: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VLCDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least one CDR selected from: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least two CDRs selected from: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least three CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17.In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least four CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least five CDRs selected from the group consisting of: (a) a VLCDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least six CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17.

[0221] In embodiments, the ADC provided herein comprises an anti-FOLR1 antibody comprising at least one, two, three, four, five or six CDRs selected from: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VLCDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least one CDR selected from: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least two CDRs selected from: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least three CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VLCDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22.In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least four CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least five CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising at least six CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22.

[0222] In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising a CDR selected from the group consisting of: (a) a VLCDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising two CDRs selected from the following: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VHCDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising three CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising four CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6.In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising five CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising six CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6.

[0223] In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising a CDR selected from the group consisting of: (a) a VLCDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising two CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising three CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising four CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17.In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising five CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising six CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17.

[0224] In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising a CDR selected from the group consisting of: (a) a VLCDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising two CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising three CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising four CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22.In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising five CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising six CDRs selected from the group consisting of: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22.

[0225] In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR1 comprising the sequence of SEQ ID NO: 1; a VL CDR2 comprising the sequence of SEQ ID NO: 2; a VL CDR3 comprising the sequence of SEQ ID NO: 3; a VH CDR1 comprising the sequence of SEQ ID NO: 4; a VH CDR2 comprising the sequence of SEQ ID NO: 5; and a VH CDR3 comprising the sequence of SEQ ID NO: 6. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR1 comprising the sequence of SEQ ID NO: 1. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR2 comprising the sequence of SEQ ID NO: 2. In an embodiment, an anti-FOLR1 antibody comprises: a VLCDR3 comprising the sequence of SEQ ID NO: 3. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR1 comprising the sequence of SEQ ID NO: 4. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR2 comprising the sequence of SEQ ID NO: 5. In an embodiment, the anti-FOLR1 antibody comprises: a VH CDR3 comprising the sequence of SEQ ID NO:6.

[0226] In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR1 comprising the sequence of SEQ ID NO: 12; a VLCDR2 comprising the sequence of SEQ ID NO: 13; a VL CDR3 comprising the sequence of SEQ ID NO: 14; a VH CDR1 comprising the sequence of SEQ ID NO: 15; a VH CDR2 comprising the sequence of SEQ ID NO: 16; and a VH CDR3 comprising the sequence of SEQ ID NO: 17. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR1 comprising the sequence of SEQ ID NO: 12. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR2 comprising the sequence of SEQ ID NO: 13. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR3 comprising the sequence of SEQ ID NO: 14. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR1 comprising the sequence of SEQ ID NO: 15. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR2 comprising the sequence of SEQ ID NO: 16. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR3 comprising the sequence of SEQ ID NO:17.

[0227] In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR1 comprising the sequence of SEQ ID NO: 12; a VLCDR2 comprising the sequence of SEQ ID NO: 18; a VL CDR3 comprising the sequence of SEQ ID NO: 19; a VH CDR1 comprising the sequence of SEQ ID NO: 20; a VH CDR2 comprising the sequence of SEQ ID NO: 21; and a VH CDR3 comprising the sequence of SEQ ID NO: 22. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR1 comprising the sequence of SEQ ID NO: 12. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR2 comprising the sequence of SEQ ID NO: 18. In an embodiment, an anti-FOLR1 antibody comprises: a VL CDR3 comprising the sequence of SEQ ID NO: 19. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR1 comprising the sequence of SEQ ID NO: 20. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR2 comprising the sequence of SEQ ID NO: 21. In an embodiment, an anti-FOLR1 antibody comprises: a VH CDR3 comprising the sequence of SEQ ID NO:22.

[0228] In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising: a light chain CDR1 having an amino acid sequence of SEQ ID NO: 1; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 2; a light chain CDR3 having an amino acid sequence of SEQ ID NO: 3; a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 4; a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 5; and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0229] In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising: a light chain CDR1 having an amino acid sequence of SEQ ID NO: 12; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 13; a light chain CDR3 having an amino acid sequence of SEQ ID NO: 14; a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 15; a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 16; and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 17.

[0230] In an embodiment, the ADC comprises an anti-FOLR1 antibody comprising: a light chain CDR1 having an amino acid sequence of SEQ ID NO: 12; a light chain CDR2 having an amino acid sequence of SEQ ID NO: 18; a light chain CDR3 having an amino acid sequence of SEQ ID NO: 19; a heavy chain CDR1 having an amino acid sequence of SEQ ID NO: 20; a heavy chain CDR2 having an amino acid sequence of SEQ ID NO: 21; and a heavy chain CDR3 having an amino acid sequence of SEQ ID NO: 22.

[0231] In embodiments, the anti-FOLR1 antibody comprises: a VL having a sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 30. In embodiments, the anti-FOLR1 antibody comprises: a VL having a sequence of SEQ ID NO: 7. In embodiments, the VL sequence having at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 30 contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-FOLR1 antibody comprising the sequence retains the ability to bind to FOLR1. In an embodiment, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:30. In an embodiment, a total of 1 to 5 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:30. In an embodiment, the substitution, insertion or deletion occurs in a region outside of the CDRs (i.e., in the FRs). In an embodiment, the anti-FOLR1 antibody comprises the VL sequence of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:30, and comprises a post-translational modification of that sequence.

[0232] In embodiments, the anti-FOLR1 antibody comprises: a VH having a sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28. In embodiments, the anti-FOLR1 antibody comprises: a VH having a sequence of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28. In embodiments, the VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-FOLR1 antibody comprising the sequence retains the ability to bind to FOLR1. In embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 have been substituted, inserted and / or deleted. In an embodiment, a total of 1 to 5 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28. In an embodiment, the substitution, insertion or deletion occurs in a region outside of the CDRs (i.e., in the FRs). In an embodiment, an anti-FOLR1 antibody comprises the VH sequence of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, and comprises a post-translational modification of that sequence.

[0233] In embodiments, the anti-FOLR1 antibody is an IgG antibody. In embodiments, the anti-FOLR1 antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In embodiments, the anti-FOLR1 antibody is an IgG1 or IgG4 antibody. In embodiments, the anti-FOLR1 antibody is an IgG1 antibody.

[0234] In embodiments, the anti-FOLR1 antibody binds to human FOLR1. In embodiments, human FOLR1 has the amino acid sequence of SEQ ID NO:9.

[0235] In any of the above embodiments, the anti-FOLR1 antibody is humanized. In an embodiment, the anti-FOLR1 antibody comprises the CDRs as in any of the above embodiments, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In an embodiment, the humanized anti-FOLR1 antibody comprises: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 2; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 3; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 4; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 5; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 6.

[0236] In any of the above embodiments, the anti-FOLR1 antibody is humanized. In an embodiment, the anti-FOLR1 antibody comprises the CDRs as in any of the above embodiments, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In an embodiment, the humanized anti-FOLR1 antibody comprises: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 13; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 15; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 16; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 17.

[0237] In any of the above embodiments, the anti-FOLR1 antibody is humanized. In an embodiment, the anti-FOLR1 antibody comprises the CDRs as in any of the above embodiments, and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In an embodiment, the humanized anti-FOLR1 antibody comprises: (a) a VL CDR1 comprising the sequence of SEQ ID NO: 12; (b) a VL CDR2 comprising the sequence of SEQ ID NO: 18; (c) a VL CDR3 comprising the sequence of SEQ ID NO: 19; (d) a VH CDR1 comprising the sequence of SEQ ID NO: 20; (e) a VH CDR2 comprising the sequence of SEQ ID NO: 21; and (f) a VH CDR3 comprising the sequence of SEQ ID NO: 22.

[0238] In an embodiment, the anti-FOLR1 antibody comprises: a VL having the sequence of SEQ ID NO:7; a VH having the sequence of SEQ ID NO:8; a light chain constant region having the sequence of SEQ ID NO:10; and a heavy chain constant region having the sequence of SEQ ID NO:11.

[0239] In an embodiment, the anti-FOLR1 antibody is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-FOLR1 antibody is an antibody fragment, such as a Fv, Fab', scFv, diabody or F(ab')2 fragment. In another embodiment, the antibody is a substantially full-length antibody, such as an IgG1 antibody or other antibody class or isotype as defined herein. ii. Antibody affinity

[0240] In embodiments, the anti-FOLR1 antibodies provided herein bind to human FOLR1 with an affinity of ≤10 nM or ≤5 nM or ≤4 nM or ≤3 nM or ≤2 nM. In embodiments, the anti-FOLR1 antibodies bind to human FOLR1 with an affinity of ≥0.0001 nM or ≥0.001 nM or ≥0.01 nM. Binding affinity can be determined using standard assays known to those of skill in the art. For example, a standard Scatchard analysis can be used to determine whether an anti-FOLR1 antibody "binds with an affinity of ≤10 nM or ≤5 nM or ≤4 nM or ≤3 nM or ≤2 nM" (see, e.g., Munson et al., Anal Biochem, Vol. 107: pp. 220-239, 1980).

[0241] In embodiments, the dissociation constant (Kd) of an anti-FOLR1 antibody provided herein is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM, and optionally ≥10 -13 M (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0242] In an embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab form of the antibody of interest and its antigen as described in the assay below. The solution binding affinity of the Fab for the antigen is determined by quantifying the binding affinity of the Fab for the antigen in the presence of a series of titrations of unlabeled antigen with a minimum concentration of ( 125I) Fab is balanced with labeled antigen, and then the bound antigen is captured and measured using an ELISA plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol., Vol. 293: pp. 865-881 (1999)). In order to establish the conditions for the assay, Multiwell plates (Thermo Scientific) were coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (wt / vol) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res., Vol. 57: pp. 4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period of time (e.g., up to about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and the plate is washed with 0.1% polysorbate 20. The plates were washed eight times with PBS solution of 1% PBS. When the plates were dry, 150 μL / well scintillation fluid (MICROSCINT-20™; Packard) was added and the plates were counted for tens of minutes on a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that provided less than or equal to 20% of maximum binding were selected for competitive binding assays.

[0243] According to another embodiment, Kd is determined using surface plasmon resonance using -2000 or -3000 (BIAcore, Inc., Piscataway, NJ), at 25 ° C, with fixed antigen CM5 chip, measured with about 10 response units (RU). Briefly, according to the supplier's instructions, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen was diluted to 5 μg / mL (about 0.2 μM) with 10 mM sodium acetate at pH 4.8, and then injected at a flow rate of 5 μL / min to obtain about 10 response units (RU) of coupled protein. After the antigen was injected, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20TM) surfactant at 25°C at a flow rate of approximately 25 μL / min. The association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model ( The equilibrium dissociation constant (Kd) was calculated by simultaneously fitting the association and dissociation sensorgrams using the ELISA software version 3.2. The equilibrium dissociation constant (Kd) was calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol., Vol. 293: 865-881 (1999). If the on-rate determined by the surface plasmon resonance assay described above exceeds 106 M -1 s -1 , then the association rate can be determined using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer, such as a flow-cut configuration spectrophotometer (Aviv Instruments) or a 8000 series SLM-AMINCO TM spectrophotometer with a stirred cuvette (ThermoSpectronic). iii. Antibody fragments

[0244] In an embodiment, the anti-FOLR1 antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med., Vol. 9: pp. 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having extended in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0245] Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med., Vol. 9: pp. 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med., Vol. 9: pp. 129-134 (2003).

[0246] Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 Bl).

[0247] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein. iv. Chimeric and humanized antibodies

[0248] In an embodiment, the anti-FOLR1 antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, Vol. 81: pp. 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0249] In embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or a portion thereof) are derived from non-human antibodies, and FR (or a portion thereof) are derived from human antibody sequences. Humanized antibodies also optionally include at least a portion of a human constant region. In embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0250] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro and Fransson, Front. Biosci., Vol. 13: pp. 1619-1633 (2008); and are also described in, e.g., Riechmann et al., Nature, Vol. 332: pp. 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA, Vol. 86: pp. 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods, Vol. 36: pp. 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol., Vol. 28: pp. 489-49 8 (1991) (describing “surface resurfacing”); Dall'Acqua et al., Methods, Vol. 36: pp. 43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods, Vol. 36: pp. 61-68 (2005) and Klimka et al., Br. J. Cancer, Vol. 83: pp. 252-260 (2000) (describing the “guided selection” method of FR shuffling).

[0251] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol., Vol. 151: p. 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular light chain variable region subgroup or a particular heavy chain variable region subgroup (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, Vol. 89: p. 4285 (1992); and Presta et al., J. Immunol., Vol. 151: p. 2623 (1993)). 1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., Vol. 13: pp. 1619-1633 (2008)); and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem., Vol. 272: pp. 10678-10684 (1997) and Rosok et al., J. Biol. Chem., Vol. 271: pp. 22611-22618 (1996)). v. Human Antibodies

[0252] In an embodiment, the anti-FOLR1 antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol., Vol. 5: pp. 368-374 (2001) and Lonberg, Curr. Opin. Immunol., Vol. 20: pp. 450-459 (2008).

[0253] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech., Vol. 23: pp. 1117-1125 (2005). See also, for example, the description of XENOMOUSE TM U.S. Patents 6,075,181 and 6,150,584; Description US Patent 5,770,429; describes KM US Patent 7,041,870 and Description of Technology Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0254] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor, J. Immunol., Vol. 133: p. 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., Vol. 147: p. 86 (1991).) Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, Vol. 103: pp. 3557-3562 (2006). Additional methods include, for example, methods described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, Vol. 26, No. 4: pp. 265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (tri-hybridoma) is also described in Vollmers and Brandlein, Histology and Histopathology, Vol. 20, No. 3: pp. 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, Vol. 27, No. 3: pp. 185-191 (2005).

[0255] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The technology for selecting human antibodies from antibody libraries is described below. vi. Antibodies from libraries

[0256] In an embodiment, the anti-FOLR1 antibodies provided herein are derived from an antibody library. Antibodies can be isolated by screening combinatorial libraries for antibodies with a desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed in, e.g., Hoogenboom et al., Methods in Molecular Biology, Vol. 178: pp. 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are also described in, e.g., McCafferty et al., Nature, Vol. 348: pp. 552-554; Clackson et al., Nature, Vol. 352: pp. 624-628 (1991); Marks et al., J. Mol. Biol., Vol. 222: pp. 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology, Vol. 248: pp. 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol., Vol. 338, No. 2: pp. 299-310 (2004); Lee et al., J. Mol. Biol., Vol. 340, No. 5: pp. 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA, Vol. 101, No. 34: pp. 12467-12472 (2004); and Lee et al., J. Immunol. Methods, Vol. 284, No. 1-2: pp. 119-132 (2004).

[0257] In the phage display method, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then the antigen-binding phage can be screened, as described in Winter et al., Ann. Rev. Immunol., Vol. 12: pp. 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunization provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a primary library (e.g., obtained from a human) can be cloned without any immunization to provide a single source of antibodies to a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J, Vol. 12: pp. 725-734 (1993). Finally, the primary library can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences for encoding highly variable CDR3 regions and rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., Vol. 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373 and U.S. Pat. Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0258] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. vii. Multispecific Antibodies

[0259] In embodiments, the anti-FOLR1 antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In embodiments, one of the binding specificities is for FOLR1 and the other binding specificity is for any other antigen. In embodiments, bispecific antibodies can bind to two different epitopes of FOLR1. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing FOLR1. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0260] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, Milstein and Cuello, Nature, Vol. 305: p. 537 (1983); WO 93 / 08829; and Traunecker et al., EMBO J., Vol. 10: p. 3655 (1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by the following methods: Engineered electrostatic manipulation effects for preparing antibody Fc-heterodimer molecules (WO 93 / 08829; and Traunecker et al., EMBO J., Vol. 10: p. 3655 (1991)). 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980; and Brennan et al., Science, Vol. 229: p. 81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., Vol. 148 No. 5: pp. 1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (see See, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90: 6444-6448 (1993); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., Vol. 152: 5368 (1994)); and the preparation of trispecific antibodies as described, e.g., in Tutt et al., J. Immunol., Vol. 147: 60 (1991).

[0261] Also included herein are engineered antibodies (including "octopus antibodies") having three or more functional antigen binding sites (see, e.g., US 2006 / 0025576 A1).

[0262] The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs" which comprise an antigen binding site that binds to FOLR1 as well as another different antigen. viii. Antibody Variants

[0263] In embodiments, amino acid sequence variants of antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution may be performed to obtain the final construct, provided that the final construct has the desired properties (e.g., antigen binding). a) Substitution, insertion and deletion variants

[0264] In embodiments, the anti-FOLR1 antibodies provided herein have one or more amino acid substitutions. Sites of interest for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1, and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and products having the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC) can be screened. Table 1. Exemplary amino acid substitutions. Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aroma: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0265] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) in biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently produced, for example, using affinity maturation techniques based on phage display (such as those described herein). In short, one or more HVR residues are mutated, the variant antibodies are displayed on phage, and screened for specific biological activities (e.g., binding affinity).

[0266] Changes (e.g., substitutions) can be made in HVR, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo high frequency mutations during the somatic maturation process) (see, e.g., Chowdhury, Methods Mol. Biol., Vol. 207: pp. 179-196 (2008)) and / or SDR (a-CDR), and the resulting variant VH or VL binding affinity is tested. Affinity maturation by constructing a secondary library and reselecting from the secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology, Vol. 178: pp. 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ (2001).) In an embodiment of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues that participate in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0267] In an embodiment, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside HVR "hot spots" or SDRs. In the embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two or three amino acid substitutions.

[0268] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989), Science, Vol. 244: pp. 1081-1085. In this method, a target residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at the amino acid position, indicating functional sensitivity to the initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0269] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody to an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody. b) Glycosylation variants

[0270] In embodiments, the anti-FOLR1 antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation.Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0271] When an antibody comprises an Fc region, the sugars attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region via an N-bond. See, e.g., Wright et al., TIBTECH, Vol. 15: pp. 26-32 (1997). Oligosaccharides may include a variety of sugars, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In an embodiment, the oligosaccharides in the antibody may be modified to produce antibody variants with certain improved properties.

[0272] In one embodiment, an antibody variant is provided, which has a sugar structure that lacks fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297 within the sugar chain, as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US 2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US 2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol., Vol. 336: pp. 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng., Vol. 87: p. 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys., Vol. 249: 533-545 (1986); U.S. Patent Application US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng., Vol. 87: p. 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., Vol. 94 No. 4: pp. 680-688 (2006); and WO2003 / 085107).

[0273] Antibody variants also have bisected oligosaccharides, for example, biantennary oligosaccharides in which the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). c) Fc region variants

[0274] In an embodiment, one or more amino acid modifications can be introduced into the Fc region of an anti-FLOR1 antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0275] In the embodiments, antibody variants with some but not all effector functions are envisioned, which make them ideal candidates for applications in which the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) are non-essential or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to determine the reduction / consumption of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., Vol. 9: pp. 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, Vol. 83: pp. 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, Vol. 82: pp. 1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med., Vol. 166: pp. 1351-1361 (1987)). Alternatively, non-radioactive assays may be employed (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA, Vol. 95: 652-656 (1998). C1q binding assays may also be performed to determine that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISA experiments in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods, Vol. 202: p. 163 (1996); Cragg, MS et al., Blood, Vol. 101: p. 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood, Vol. 103: p. 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol., Vol. 18 No. 12: p. 1759-1769 (2006)).

[0276] Antibodies with reduced effector function include those in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0277] Certain antibody variants with enhanced or reduced binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem., Vol. 9, No. 2: pp. 6591-6604 (2001).)

[0278] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., Vol. 117: p. 587 (1976) and Kim et al., J. Immunol., Vol. 24: p. 249 (1994)) are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more amino acid substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0279] See also Duncan and Winter, Nature, Vol. 322: 738-740 (1988); U.S. Patent 5,648,260; U.S. Patent 5,624,821; and WO 94 / 29351 for other examples of Fc region variants. ix. Antibody derivatives

[0280] In embodiments, the anti-FOLR1 antibodies provided herein may be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc. x. Recombinant Methods and Compositions

[0281] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. Those skilled in the art will be familiar with suitable host cells for antibody expression. Exemplary host cells include eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0282] To recombinantly produce an anti-FOLR1 antibody, nucleic acid encoding an antibody, such as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0283] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Human Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli). After expression, the antibody can be separated from the bacterial cell paste of the soluble fraction and can be further purified.

[0284] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech., Vol. 22: pp. 1409-1414 (2004); and Li et al., Nat. Biotech., Vol. 24: pp. 210-215 (2006).

[0285] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0286] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES producing antibodies in transgenic plants). TM technology).

[0287] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney cell CV1 lines transformed by SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol., Vol. 36: p. 59 (1977)); baby hamster kidney cells (BHK); mouse supporting cells (TM4 cells, as described, for example, in Mather, Biol. Reprod., Vol. 23: p. 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals N.Y. Acad. Sci., Vol. 383: 44-68 (1982); MRC 5 cells; and FS4 cells. Other mammalian host cell lines that may be used include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, Vol. 77: p. 4216 (1980)); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003); Dhara, VG et al., BioDrugs, Vol. 32: pp. 571-584 (2018); Kunert, R. and Reinhart, D., Applied microbiology and biotechnology, Vol. 100 No. 8: pp. 3451-3461 (2016). xi. Determination

[0288] The anti-FOLR1 antibodies described herein can be identified, screened or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0289] In an embodiment, for example, by known methods such as ELISA, Antibodies were tested for antigen binding activity by FACS or Western blotting.

[0290] In another embodiment, competition assays can be used to identify antibodies that compete with any of the antibodies described herein for binding to FOLR1. In embodiments, such competing antibodies bind to the same epitope (e.g., a linear epitope or a conformational epitope) as the epitope bound by an antibody described herein. Detailed exemplary methods for locating epitopes bound by antibodies are provided in Morris (1996), "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0291] In an exemplary competition assay, immobilized FOLR1 is incubated in a solution comprising a first labeled antibody that binds to FOLR1 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to FOLR1. The second antibody may be present in the hybridoma supernatant. As a control, immobilized FOLR1 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to FOLR1, excess unbound antibody is removed and the amount of label associated with immobilized FOLR1 is measured. If the amount of label associated with immobilized FOLR1 in the test sample is greatly reduced compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to FOLR1. In embodiments, immobilized FOLR1 is present on the surface of a cell or in a membrane preparation obtained from a cell expressing FLOR1 on its surface. See, Harlow and Lane (1988), Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Methods for preparing antibody-drug conjugates

[0292] ADCs of formula (I) can be prepared by several routes utilizing organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of an antibody with a linker reagent (B, B' or B") to form Ab-B, Ab-B' or Ab-B" via a covalent bond, followed by binding to a drug moiety D or a drug-linker molecule DL 2reaction; and (2) the nucleophilic group of the drug portion D and the divalent linker reagent (L 2 and / or B, B' or B") react via covalent bonds to form DL 2 ,DL 2 -B, DL 2 -B' or DL 2 -B", followed by reaction with a nucleophilic group of an antibody or reduced antibody. Several such methods are described in Agarwal et al. (2015), Bioconjugate Chem., Vol. 26: pp. 176-192.

[0293] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, these electrophilic groups include: (i) active esters, such as NHS esters, HOBt esters, haloformates and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls and maleimide groups. In addition to NHS esters, non-limiting exemplary functional groups for coupling to antibody lysines may include pentafluorophenyl, tetrafluorophenyl, tetrafluorobenzenesulfonate, nitrophenyl, isocyanate, isothiocyanate and sulfonyl chloride.

[0294] In another embodiment, the antibody can be coupled to a "receptor" (such as streptavidin) for tumor pretargeting, where the antibody-receptor conjugate is administered to the patient, followed by the use of a clearing agent to remove unbound conjugate from the circulation, followed by administration of a "ligand" (e.g., avidin) coupled to a cytotoxic agent (e.g., a drug or radionucleotide).

[0295] Some antibodies have reducible interchain disulfide bonds, i.e., cysteine ​​bridges. By treating with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), antibodies can be made reactive with linker reagent couplings, so that antibodies are completely or partially reduced. Therefore, in theory, each cysteine ​​bridge will form two reactive thiol nucleophilic groups. By modifying lysine residues, for example, by reacting lysine residues with 2-iminothiolane (Trout's reagent), amines are converted into thiols, and other nucleophilic groups can be introduced into antibodies. Reactive thiol groups can also be introduced into antibodies by introducing one, two, three, four or more cysteine ​​residues (e.g., by preparing variant antibodies comprising one or more non-natural cysteine ​​amino acid residues). Non-limiting examples of functional groups that can react with reactive thiols include, but are not limited to, maleimide, pyridyldithio, bromoacetyl, iodoacetyl, bromobenzyl, iodobenzyl, and 4-(cyanoethynyl)benzoyl.

[0296] In an embodiment, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to produce reactive cysteine ​​thiol groups. The interchain cysteine ​​residues can then be alkylated, for example, using maleimide. Alternatively, the interchain cysteine ​​residues can be bridged alkylated, for example, using a bissulfone linker or propargyl dibromomaleimide, followed by Cu-click connection. In an embodiment, the antibody can be coupled via a lysine amino acid. This coupling can be a one-step coupling or a two-step coupling. In an embodiment, the one-step coupling requires the -amino group of the lysine residue to be coupled to a drug-linker molecule (DL-linker) containing an amine reactive group via an amide bond. 2 ,DL 2 -B, DL 2 -B' or DL 2 -B"). In an embodiment, the amine-reactive group is an activated ester. In an embodiment, the antibody can be coupled via a two-step coupling. The two-step coupling requires a first step in which a bifunctional reagent containing both amine and thiol-reactive functional groups reacts with the lysine-amino group. In a second step, a drug-linker molecule (DL 2 ,DL 2 -B, DL 2 -B' or DL 2 -B”) to a sulfhydryl-reactive group of a bifunctional reagent. Several examples are provided by Jain et al. (2015), Pharm. Res., Vol. 32: pp. 3526-3540. In an embodiment, the first step may involve functionalizing the antibody with an azide, followed by coupling with an alkyne-modified linker or drug-linker molecule (DL 2 ,DL 2 -B, DL 2 -B' or DL2 -B”) to perform click chemistry. In embodiments, the first step may involve functionalizing the antibody with an alkyne, followed by ligation with an azide-modified linker or drug-linker molecule (DL 2 ,DL 2 -B, DL 2 -B' or DL 2 -B”) for click chemistry reaction. In embodiments, the first step may involve functionalizing the antibody with an aldehyde, followed by ligation with an alkoxyamine or hydrazine-modified linker or drug-linker molecule (DL 2 ,DL 2 -B, DL 2 -B' or DL 2 -B”) for click chemistry reaction. In embodiments, the first step may involve functionalizing the antibody with tetrazine, followed by ligation with a trans-cyclooctene or cyclopropene-modified linker or drug-linker molecule (DL 2 ,DL 2 -B, DL 2 -B' or DL 2 -B”) for click chemistry reaction. In embodiments, the first step may involve functionalizing the antibody with trans-cyclooctene or cyclopropene, followed by ligation with a tetrazine-modified linker or drug-linker molecule (DL 2 ,DL 2 -B, DL 2 -B' or DL 2 -B”) for click chemistry reactions. Some examples are described in Pickens et al. (2018), Bioconjug. Chem., Vol. 29: pp. 686-701; Li et al. (2018), MAbs, Vol. 10: pp. 712-719; and Chio et al. (2020), Methods Mol. Biol., Vol. 2078: pp. 83-97.

[0297] In an embodiment, the ADC of formula (I) can be prepared by combining an anti-FOLR1 antibody (Ab) with a molecule of formula (PI): or a pharmaceutically acceptable salt thereof, wherein: B is a reactive moiety capable of forming a bond with the anti-FOLR1 antibody.

[0298] In an embodiment, B is a reactive moiety capable of forming bonds with two thiol groups of an anti-FOLR1 antibody. In an embodiment, B is

[0299] In an embodiment, the ADC of formula (I) can be prepared by combining an anti-FOLR1 antibody (Ab) with a molecule of formula (P-II): or a pharmaceutically acceptable salt thereof, wherein: B' is a reactive moiety capable of forming a bond with the anti-FOLR1 antibody.

[0300] In an embodiment, B' is a reactive moiety capable of forming a bond with a sulfhydryl group of an anti-FOLR1 antibody. In an embodiment, B' is

[0301] In an embodiment, the ADC of formula (I) can be prepared by combining an anti-FOLR1 antibody (Ab) with a molecule of formula (P-III): or a pharmaceutically acceptable salt thereof, wherein: B" is a reactive moiety capable of forming a bond with an anti-FOLR1 antibody (this reaction is described in Example 3 of US Patent 10,590,165).

[0302] In an embodiment, B" is a reactive moiety capable of forming a bond with an amine group of an anti-FOLR1 antibody. In an embodiment, B" is

[0303] In an embodiment, PI, P-II or P-III is a compound selected from:

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] or a pharmaceutically acceptable salt thereof. Pharmaceutical composition

[0311] In one aspect, provided herein is a pharmaceutical composition comprising an ADC as described herein (including embodiments) and a pharmaceutically acceptable carrier. In embodiments, a therapeutically effective amount of an ADC as described herein is included.

[0312] In embodiments, the pharmaceutical composition is formulated as a tablet, powder, capsule, pill, cachet, or lozenge as described herein. The pharmaceutical composition may be formulated as a tablet, capsule, pill, cachet, or lozenge for oral administration. The pharmaceutical composition may be formulated for dissolution into a solution for administration by techniques such as, for example, intravenous administration. The pharmaceutical composition may be formulated for oral administration, suppository administration, topical administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, subcutaneous administration, implantation, transdermal administration, or transmucosal administration as described herein.

[0313] ADC and its pharmaceutical composition are particularly useful for parenteral administration, i.e., subcutaneous (sc), intrathecal, intraperitoneal, intramuscular (im) or intravenous (iv) administration. In an embodiment, ADC and its pharmaceutical composition are administered intravenously or subcutaneously.

[0314] The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusters and buffers, etc. The concentration of the antigen binding protein of the present invention in such a pharmaceutical preparation can vary widely, i.e., from less than about 0.5% by weight, usually equal to or at least about 1% by weight to as much as about 15% by weight or 20% by weight, and will be selected primarily based on fluid volume, viscosity, etc. according to the specific mode of administration selected.

[0315] Actual methods for preparing parenterally administrable compositions are well known or apparent to those skilled in the art and are described in greater detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. For the preparation of intravenously administrable antigen-binding protein formulations of the invention, see Lasmar U and Parkins D, "The formulation of Biopharmaceutical products", Pharma. Sci. Tech. today, pp. 129-137, Vol. 3 (April 3, 2000); Wang, W, "Instability, stabilisation and formulation of liquid protein pharmaceuticals", Int. J. Pharm, Vol. 185 (1999): pp. 129-188; Stability of Protein Pharmaceuticals Part A and B, Ahern TJ, Manning MC, eds., New York, NY: Plenum Press (1992); Akers, MJ, "Excipient-Drug interactions in Parenteral Formulations", J. Pharm Sci, Vol. 91 (2002): pp. 2283-2300; Imamura, K et al., "Effects of types of sugar on stabilization of Protein in the dried state", J Pharm Sci, Vol. 92 (2003): pp. 266-274; Izutsu, Kkojima, S., "Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying", J. Pharm. Pharmacol, Vol. 54 (2002): pp. 1033-1039; Johnson, R, "Mannitol-sucrose mixtures-versatile formulations for protein peroxidise 19g19n", J. Pharm.Sci, Vol. 91 (2002): pp. 914-922; and Ha, E, Wang W, Wang Yj, "Peroxide formation in polysorbate 80 and protein stability", J. Pharm Sci, Vol. 91: pp. 2252-2264 (2002), the entire contents of which are incorporated herein by reference and are particularly preferred by the reader. .

[0316] In an embodiment, the pharmaceutical composition may include an optical isomer, diastereomer, enantiomer, isoform, polymorph, hydrate, solvate or product or a pharmaceutically acceptable salt of a compound described herein. As described above, the compound described herein (including its pharmaceutically acceptable salt) contained in the pharmaceutical composition may be covalently attached to the carrier portion. In an embodiment, the compound described herein (including its pharmaceutically acceptable salt) contained in the pharmaceutical composition is non-covalently attached to the carrier portion. The combination of covalently attached and non-covalently attached compounds described herein may be in the pharmaceutical composition herein. How to use

[0317] In one aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering an effective amount of an antibody-drug conjugate (ADC) comprising an IgG antibody, a conjugated linker moiety (L 1 ) and a drug portion, the coupling linker portion is bound to the thiol of the cysteine ​​residue of the IgG antibody or to the amine of the lysine residue, and the drug portion is bound to L 1 Or another optional connector L 2 Covalent Binding. In embodiments, the IgG antibody binds to FOLR1.

[0318] In one aspect, the ADC provided herein is used in a method for inhibiting the proliferation of cells expressing FOLR1, the method comprising contacting the cell with the ADC, for example, exposing the cell to the ADC under conditions that allow the anti-FOLR1 antibody of the ADC to bind on the cell surface, thereby inhibiting the proliferation of the cell. In embodiments, the method is an in vitro or in vivo method. In embodiments, the cell is a B cell. In embodiments, the cell is a cancer cell. In embodiments, the cell is a multiple myeloma cell. In any of these embodiments, the cell can be a mammalian cell, such as a human cell.

[0319] In vitro cell proliferation inhibition can be performed using CellTiter-Glo TMThe assay is performed using a luminescent cell viability assay reagent, which is commercially available from Promega (Madison, WI). The assay determines the number of viable cells in culture based on the quantification of the ATP present, which is indicative of metabolically active cells. See, Crouch et al. (1993), J. Immunol., Meth., Vol. 160: pp. 81-88; U.S. Patent 6,602,677. The assay can be performed in a 96-well or 384-well format, making it suitable for automated high-throughput screening (HTS). See, Cree et al. (1995), AntiCancer Drugs, Vol. 6: pp. 398-404. The assay procedure involves adding a single reagent ( Reagents). This results in cell lysis and the generation of a luminescent signal produced by a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is proportional to the number of viable cells present in the culture. The data can be recorded by a luminometer or CCD camera imaging device. The luminescent output is expressed as relative light units (RLU).

[0320] In another aspect, an ADC for use as a medicament is provided. In a further aspect, an ADC for use in a method of treatment is provided. In another aspect, provided herein is a method of treating a disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition of an ADC as described herein.

[0321] In embodiments, the disease is cancer. In embodiments, cancer is associated with overexpression of FOLR1. In embodiments, provided herein is an ADC for use in a method of treating an individual with a cancer expressing FOLR1, the method comprising administering an effective amount of the ADC to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual. In embodiments, the additional therapeutic agent is a VEGF inhibitor. In embodiments, the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor. In embodiments, the VEGF inhibitor is an anti-VEGF antibody. In embodiments, the VEGF inhibitor is a small molecule VEGF inhibitor. In embodiments, the anti-VEGF antibody is bevacizumab or ramucirumab. In embodiments, the anti-VEGF antibody is bevacizumab. In embodiments, the anti-VEGF antibody is ramucirumab. In embodiments, the small molecule VEGF inhibitor is sunitinib, sorafenib, axitinib, pazopanib or regorafenib. In embodiments, the small molecule VEGF inhibitor is sunitinib. In embodiments, the small molecule VEGF inhibitor is sorafenib. In embodiments, the small molecule VEGF inhibitor is axitinib. In an embodiment, the small molecule VEGF inhibitor is pazopanib. In an embodiment, the small molecule VEGF inhibitor is regorafenib.

[0322] In another aspect, the disclosure provides for the use of an ADC in the manufacture or preparation of a medicament. In an embodiment, the medicament is used to treat a cancer expressing FOLR1. In another embodiment, the medicament is used in a method of treating a cancer expressing FOLR1, the method comprising administering an effective amount of the medicament to an individual suffering from a cancer expressing FOLR1. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0323] In embodiments, the present disclosure provides the use of an ADC as described herein (including in embodiments) in combination with an additional therapeutic agent in the manufacture or preparation of a medicament for treating a cancer expressing FOLR1. In embodiments, the present disclosure provides the use of an ADC as described herein (including in embodiments) in combination with a VEGF inhibitor in the manufacture or preparation of a medicament for treating a cancer expressing FOLR1. In embodiments, the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor. In embodiments, the VEGF inhibitor is an anti-VEGF antibody. In embodiments, the VEGF inhibitor is a small molecule VEGF inhibitor. In embodiments, the anti-VEGF antibody is bevacizumab or ramucirumab. In embodiments, the anti-VEGF antibody is bevacizumab. In embodiments, the anti-VEGF antibody is ramucirumab. In embodiments, the small molecule VEGF inhibitor is sunitinib, sorafenib, axitinib, pazopanib or regorafenib. In embodiments, the small molecule VEGF inhibitor is sunitinib. In embodiments, the small molecule VEGF inhibitor is sorafenib. In embodiments, the small molecule VEGF inhibitor is axitinib. In embodiments, the small molecule VEGF inhibitor is pazopanib. In embodiments, the small molecule VEGF inhibitor is regorafenib. In embodiments, the ADC described herein is ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6. In embodiments, the ADC is ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6. In embodiments, the ADC is ADC-1. In embodiments, the ADC is ADC-2. In embodiments, the ADC is ADC-3. In embodiments, the ADC is ADC-4. In embodiments, the ADC is ADC-5. In embodiments, the ADC is ADC-6.

[0324] In embodiments, the methods provided herein are used to treat cancer in a mammal. In embodiments, the methods provided herein are used to treat cancer in a human.

[0325] In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include epithelial tumors, including ovarian tumors, uterine tumors, breast tumors, endometrial tumors, pancreatic tumors, kidney tumors, lung tumors, colorectal tumors, and brain tumors. In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include serous and endometrioid epithelial ovarian cancers, endometrial adenocarcinomas, adenocarcinoma subtypes of non-small cell lung cancer (NSCLC), squamous lung cancer, and triple-negative breast cancer (TNBC). In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include hematopoietic cancers, which include but are not limited to multiple myeloma (MM), including smoldering MM; monoclonal gammopathy (MGUS) of unknown significance (or unknown or unclear); plasma cell tumors (bone, extramedullary); lymphoplasmacytic lymphoma (LPL); Waldenstrom's macroglobulinemia; plasma cell leukemia and primary amyloidosis (AL). In embodiments, hematopoietic cancer is multiple myeloma.

[0326] In embodiments, the cancer is ovarian cancer. In embodiments, the cancer is breast cancer. In embodiments, the cancer is lung cancer. In embodiments, the cancer is triple negative breast cancer.

[0327] In embodiments, a therapeutically effective amount of an antibody-drug conjugate (ADC) provided herein is about 0.5 mg to about 3,000 mg per day, about 1 mg to about 2,000 mg per day, about 1 mg to about 1,500 mg per day, about 1 mg to about 1,000 mg per day, about 10 mg to about 1,000 mg per day, about 50 mg to about 1,000 mg per day, about 50 mg to about 800 mg per day, about 50 mg to about 700 mg per day, or about 100 mg to about 500 mg per day.

[0328] In embodiments, the therapeutically effective amount is about 1 mg, about 20 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, or about 1,000 mg per day.

[0329] In embodiments, the therapeutically effective amount is about 0.1 mg / kg / day to about 100 mg / kg / day, about 0.1 mg / kg / day to about 50 mg / kg / day, about 0.1 mg / kg / day to about 40 mg / kg / day, about 0.5 mg / kg / day to about 30 mg / kg / day, about 0.5 mg / kg / day to about 25 mg / kg / day, 1 mg / kg / day to about 25 mg / kg / day, about 1 mg / kg / day to about 20 mg / kg / day, about 1 mg / kg / day to about 15 mg / kg / day, or about 1 mg / kg / day to about 10 mg / kg / day.

[0330] Depending on the disease to be treated and the condition of the subject, the compound of formula (I) or a pharmaceutically acceptable salt thereof can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, CIV, intracisternal injection or infusion, subcutaneous injection or implantation), inhalation, nasal, vaginal, rectal, sublingual or topical (e.g., transdermal or topical) administration. The compound of formula (I) or a pharmaceutically acceptable salt thereof can be formulated alone or together with a pharmaceutically acceptable excipient, carrier, adjuvant and solvent in a suitable dosage unit suitable for each administration route.

[0331] The compound of formula (I) or a pharmaceutically acceptable salt thereof can be delivered as a single dose, such as, for example, a single bolus or oral tablet or pill; or varied over time, such as, for example, continuous infusion over time or single bolus doses divided over time. If necessary, the compound can be administered repeatedly, for example, until the patient experiences disease stabilization or regression, or until the patient experiences disease progression or unacceptable toxicity. For example, disease stability of a solid tumor generally means that the perpendicular diameter of a measurable lesion has not increased by 25% or more from the last measurement. Response Evaluation Criteria in Solid Tumors (RECIST) Guidelines, Journal of the National Cancer Institute, Vol. 92, No. 3: pp. 205-216 (2000). Disease stability or lack of disease stability is determined by methods known in the art, such as evaluation of patient symptoms; physical examination; visualization of tumors that have been imaged using X-rays, CAT, PET or MRI scans, and other generally accepted evaluation methods.

[0332] The compound of formula (I) or its pharmaceutically acceptable salt can be administered once a day (QD), or divided into multiple daily doses, such as twice a day (BID), three times a day (TID) and four times a day (QID). In addition, the administration can be continuous (i.e., for several consecutive days or every day), intermittent (e.g., periodic) (i.e., including taking a break for several days, weeks or months without taking the medicine). As used herein, the term "daily" is intended to indicate that a therapeutic compound such as a compound of formula (I) is administered once or more than once a day for a period of time. The term "continuously" is intended to indicate that a therapeutic compound such as a compound of formula (I) is administered every day for an uninterrupted period of at least 10 days to 52 weeks. As used herein, the term "intermittent" or "intermittently" is intended to indicate stopping and starting at regular or irregular intervals. For example, intermittent administration of a compound of formula (I) is administered one to six days a week, periodically (e.g., administered every day for two to eight consecutive weeks, followed by a rest period, not administered for up to a week) or administered every other day. As used herein, the term "cycle" is intended to mean daily administration of a therapeutic compound, such as a compound of Formula (I), or continuous administration of a therapeutic compound, but with rest periods.

[0333] In embodiments, the frequency of administration is in the range of about daily dose to about monthly dose. In embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once a week, once every two weeks, once every three weeks, or once every four weeks. In embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once a week. In another embodiment, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered twice a week. In embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered three times a week. In embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once every two weeks. In embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once every three weeks. In embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once every four weeks. In embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered once every eight weeks. Combination therapy utilizing a second active agent

[0334] The ADC of formula (I) or a pharmaceutically acceptable salt thereof may also be combined or used in combination with other therapeutic agents described herein that are useful for treating cancer.

[0335] In embodiments, provided herein are methods of treating cancer comprising administering to a patient an ADC of Formula (I) or a pharmaceutically acceptable salt thereof in combination with one or more second active agents, and optionally in combination with radiation therapy, transfusion, or surgery. It is believed that certain combinations act synergistically in the treatment of certain types of cancer and certain diseases and conditions associated with or characterized by undesirable angiogenesis.

[0336] One or more second active ingredients or agents can be used in the methods and compositions provided herein together with the compounds provided herein. In embodiments, the second active agent is an anticancer agent. The second active agent can be a macromolecule (e.g., protein) or a small molecule (e.g., a synthetic inorganic, organometallic or organic molecule).

[0337] Examples of macromolecular active agents include, but are not limited to, hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies. In certain embodiments, the macromolecular active agent is a biomolecule, such as a naturally occurring or artificially made protein.

[0338] Antibodies that can be used in combination with the compounds provided herein include monoclonal antibodies and polyclonal antibodies. In embodiments, antibodies include, but are not limited to, anti-VEGF antibodies. Examples of anti-VEGF antibodies include, but are not limited to, bevacizumab and ramucirumab.

[0339] VEGF inhibitors can be used in the methods and compositions provided herein. Inhibitors of VEGF receptors include anti-VEGF antibodies and small molecules (e.g., synthetic inorganic, organometallic or organic molecules). In embodiments, the VEGF inhibitor is an antibody. In embodiments, the VEGF inhibitor is a small molecule.

[0340] In embodiments, the VEGF inhibitor is an anti-VEGF antibody, including but not limited to bevacizumab and ramucirumab. In embodiments, the anti-VEGF antibody is bevacizumab or ramucirumab. In embodiments, the anti-VEGF antibody is bevacizumab. In embodiments, the anti-VEGF antibody is ramucirumab.

[0341] In embodiments, VEGF inhibitors are small molecules, including but not limited to sunitinib, sorafenib, axitinib, pazopanib, and regorafenib. In embodiments, the small molecule is sunitinib, sorafenib, axitinib, pazopanib, or regorafenib. In embodiments, the small molecule is sunitinib. In embodiments, the small molecule is sorafenib. In embodiments, the small molecule is axitinib. In embodiments, the small molecule is pazopanib. In embodiments, the small molecule is regorafenib.

[0342] As used herein, the term "combination" includes the use of more than one therapy (e.g., one or more therapeutic agents). However, the use of the term "combination" does not limit the order in which therapies (e.g., therapeutic agents) are administered to a patient suffering from a disease or disorder. The first therapy (e.g., a therapeutic agent, such as an ADC provided herein, e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof) can be administered to the subject prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) or concomitantly with the second therapy (e.g., a therapeutic agent). Triple therapy is also contemplated herein.

[0343] The compound of formula (I) and one or more second active agents can be administered to the patient simultaneously or sequentially by the same or different routes of administration. The combination of medicaments or compositions can be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via a separate intravenous tube) or sequentially (e.g., first administering one medicament and then administering the second medicament). Therefore, the term "combination" is used to refer to the concomitant, simultaneous or sequential administration of two or more medicaments or compositions. Depending on the specific characteristics of the subject and the selected treatment type, it is best to determine the course of treatment based on the individual. Treatments such as those disclosed herein can be administered to the subject once a day, twice a day, once every two weeks, once a month, or on any applicable basis for treatment effectiveness. Treatment can be administered alone or in combination with any other treatment disclosed herein or known in the art. Additional treatments can be administered simultaneously with the first treatment, at different times, or in completely different treatment regimens (e.g., the first treatment can be administered once a day, while additional treatments can be administered once a week).

[0344] The route of administration of the compound of formula (I) is independent of the route of administration of the second therapy. In one embodiment, the compound of formula (I) is administered orally. In another embodiment, the compound of formula (I) is administered intravenously. In another embodiment, the compound of formula (I) is administered intraperitoneally. Therefore, according to these embodiments, the compound of formula (I) is administered orally or intravenously, and the second therapy can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectal, transbuccally, intranasally, liposomes, by inhalation, vaginally, intraocularly, by catheter or stent local delivery, subcutaneously, intrafatally, intraarticularly, intrathecally or in a sustained release dosage form. In an embodiment, the compound of formula (I) and the second therapy are administered orally or by IV by the same mode of administration. In another embodiment, the compound of formula (I) is administered by a mode of administration, for example, by IV, and the second agent (anticancer agent) is administered by another mode of administration, for example, intraperitoneally.

[0345] In an embodiment, the second active agent is administered intravenously or intraperitoneally in an amount of about 1 mg to about 1000 mg, about 5 mg to about 500 mg, about 10 mg to about 500 mg, or about 50 mg to about 500 mg, and is administered once a week or every two weeks. The specific amount of the second active agent depends on the specific agent used, the type of disease to be treated or controlled, the severity and stage of the disease, and the compound of formula (I) provided herein and the amount of any optional additional active agent applied to the patient at the same time. In certain embodiments, the second active agent is bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib or regorafenib.

[0346] In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered with bevacizumab to a patient suffering from a cancer expressing FOLR1. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered with ramucirumab to a patient suffering from a cancer expressing FOLR1. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered with sunitinib to a patient suffering from a cancer expressing FOLR1. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered with sorafenib to a patient suffering from a cancer expressing FOLR1. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered with axitinib to a patient suffering from a cancer expressing FOLR1. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered with pazopanib to a patient suffering from a cancer expressing FOLR1. In an embodiment, a compound provided herein, e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof, is administered with regorafenib to a patient having a cancer expressing FOLR1.

[0347] In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with multiple myeloma together with bevacizumab. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with multiple myeloma together with sunitinib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with multiple myeloma together with sorafenib.

[0348] In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with bevacizumab. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with ramucirumab. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with sunitinib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with sorafenib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with axitinib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with pazopanib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with lung cancer together with regorafenib.

[0349] In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with bevacizumab. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with ramucirumab. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with sunitinib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with sorafenib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with axitinib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with pazopanib. In an embodiment, a compound provided herein, such as a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient with ovarian cancer together with regorafenib. Reagent test kit

[0350] In an embodiment, the active ingredients provided herein are not administered to a patient at the same time or by the same route of administration. Thus, kits that, when used by a medical practitioner, can simplify the administration of appropriate amounts of active ingredients to a patient are contemplated herein.

[0351] In embodiments, kits provided herein include dosage forms of compounds provided herein, e.g., compounds of formula (I) or pharmaceutically acceptable salts thereof. In embodiments, kits provided herein also include additional active ingredients, such as VEGF inhibitors. In embodiments, kits provided herein also include additional active ingredients, such as anti-VEGF antibodies or small molecule VEGF inhibitors. In embodiments, kits provided herein also include additional active ingredients, such as bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib or regorafenib.

[0352] In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, e.g., compounds of Formula (I) or pharmaceutically acceptable salts thereof, together with VEGF inhibitors. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, e.g., compounds of Formula (I) or pharmaceutically acceptable salts thereof, together with anti-VEGF antibodies. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, e.g., compounds of Formula (I) or pharmaceutically acceptable salts thereof, together with small molecule VEGF inhibitors.

[0353] In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with bevacizumab. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with ramucirumab. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with sunitinib. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with sorafenib. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with axitinib. In embodiments, kits provided herein may include pharmaceutical compositions comprising dosage forms of compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with pazopanib. In embodiments, kits provided herein may include dosage forms of pharmaceutical compositions comprising compounds provided herein, such as compounds of formula (I) or pharmaceutically acceptable salts thereof, together with regorafenib. In embodiments, the compound of formula (I) is ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6. In embodiments, the compound of formula (I) is ADC-1. In embodiments, the compound of formula (I) is ADC-2. In embodiments, the compound of formula (I) is ADC-3. In embodiments, the compound of formula (I) is ADC-4. In embodiments, the compound of formula (I) is ADC-5. In embodiments, the compound of formula (I) is ADC-6.

[0354] In embodiments, the kit provided herein further comprises additional active ingredients, such as bevacizumab. In embodiments, the kit provided herein further comprises additional active ingredients, such as ramucirumab. In embodiments, the kit provided herein further comprises additional active ingredients, such as sunitinib. In embodiments, the kit provided herein further comprises additional active ingredients, such as sorafenib. In embodiments, the kit provided herein further comprises additional active ingredients, such as axitinib. In embodiments, the kit provided herein further comprises additional active ingredients, such as pazopanib. In embodiments, the kit provided herein further comprises additional active ingredients, such as regorafenib.

[0355] In an embodiment, the kits provided herein also include a device for administering the active ingredient. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers.

[0356] In embodiments, the kit provided herein also includes cells or blood for transplantation and a pharmaceutically acceptable solvent that can be used to administer one or more active ingredients. For example, if the active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit may include a sealed container of a suitable solvent in which the active ingredient can be dissolved to form a particle-free sterile solution suitable for parenteral administration. Examples of pharmaceutically acceptable solvents include, but are not limited to: USP water for injection; aqueous solvents, such as, but not limited to, sodium chloride injection, Ringer's injection, glucose injection, glucose and sodium chloride injection, and lactated Ringer's injection; water-miscible solvents, such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous solvents, such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Sequence Listing:

[0357] Human FOLR1 sequence SEQ ID NO: 9

[0358] MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPED KLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCK SNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS

[0359] Sequence Listing: Table 2: Table 3: Note: Anti-FOLR1 antibodies FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20 and FOLR1-Ab23 are disclosed in WO2020 / 016661, wherein AMT-151 and FOLR1-Ab1 have the same 6 CDRs, and FOLR1-Ab14, FOLR1-Ab20 and FOLR1-Ab23 have the same 6 CDRs. Table 4: Example

[0360] The following examples are intended to be illustrative and useful for further understanding of the embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way.

[0361] The chemical reactions described in the embodiments can be easily adapted to prepare many other compounds of the present disclosure, and it is believed that alternative methods for preparing compounds of the present disclosure are within the scope of the present disclosure. For example, according to the present disclosure, the synthesis of non-illustrative compounds can be successfully carried out by modifications that are obvious to those skilled in the art, such as by utilizing other suitable reagents known in the art except those described, or by conventional modifications to reaction conditions, reagents and starting materials. Alternatively, other reactions disclosed herein or known in the art will be considered to be applicable to the preparation of other compounds of the present disclosure. The synthesis of compound I-1 and related compounds is disclosed in U.S. Patents 10,590165 and 9,981,046, which are incorporated herein in their entirety. Synthesis Example Example S1: Synthesis of compound 1-5.

[0362] To a 2 mL DMF solution of compound I-1 (TFA salt, 250 mg, 0.25 mmol) was added HATU (103 mg, 0.27 mmol), DIEA (188 μL, 1.08 mmol) and acid I-2 (142 mg, 0.27 mmol) in 2 mL DMF. The mixture was stirred for 30 minutes, then 160 μL DBU was added and stirred for 10 minutes. The mixture was purified by HPLC to give compound I-3 (214 mg). MS m / z 1057.6 (M+H).

[0363] To a 0.5 mL DMF solution of compound I-3 (TFA salt, 10 mg, 7.8 μmol) was added anhydride I-4 (16.5 mg, 23.5 μmol) and DIEA (5.4 μL, 31 μmol) in 0.5 mL DMF. The mixture was stirred for 10 minutes and then purified by HPLC to give compound I-5 (8.5 mg). MS m / z 1397.5 (M+H). Example S2: Synthesis of compound 1-6.

[0364] The synthesis of compound 1-6 is described in Example 3 of US Patent 10,590,165, which is incorporated herein in its entirety.

[0365] Antibody-drug conjugates (ADCs) were prepared by conjugating Compound 1-5 or Compound 1-6 to the anti-FOLR1 antibody AMT-151 clone. AMT-151 is a human IgG1 antibody. Example S3: Preparation of antibody-drug conjugate (ADC) anti-FOLR1 compound I-5 (151-C-LOCK-D5; ADC-1).

[0366] The six CDRs of the anti-FOLR1 antibody used in this example have the same antibody CDR sequences as the FOLR1 antibody anti-FLOR1-Ab1 described in WIPO Publication WO 2020 / 016661, which is incorporated herein in its entirety. The heavy chain sequence and light chain sequence of the anti-FOLR1 antibody used in this example are shown in SEQ ID NO: 7 and SEQ ID NO: 8 in Table 2 and SEQ ID NO: 10 and SEQ ID NO: 11 in Table 4, respectively. The affinity-purified anti-FOLR1 antibody was buffer exchanged into a coupling buffer (50 mM sodium phosphate buffer, pH 7.0-7.2, 4 mM EDTA) at a concentration of 5 mg / mL. A 20-fold molar excess of freshly prepared 10 mM tris(2-carboxyethyl)phosphine) (TCEP) in aqueous solution was added to a portion of the antibody stock solution. The resulting mixture was incubated overnight at 4°C-8°C. Excess TCEP was then removed by several rounds of centrifugal filtration with fresh coupling buffer. UV-Vis quantification of the recovered reduced antibody material was then performed to confirm adequate free thiol-antibody ratio (SH / Ab). Briefly, an aliquot of 1 mM freshly prepared Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid)) in coupling buffer was mixed with an equal volume of purified antibody solution. The resulting absorbance at 412 nm was measured and the 14,150 M -1 cm -1The reduced content of cysteine ​​was determined by the extinction coefficient of . Under these conditions, a SH / Ab ratio of about 6 was measured.

[0367] To effect conjugation of the toxin-linker material to the anti-FOLR1 antibody, compound I-5 was freshly dissolved in a 3:2 acetonitrile / water mixture to a concentration of 5 mM. Propylene glycol (PG) was then added to a portion of the reduced, purified (TCEP-removed) anti-FOLR1 antibody to give a final concentration of 30% (volume / volume) PG, followed immediately by the addition of a 4.5-fold molar excess of I-5. After thorough mixing and incubation for 2 hours at ambient temperature, the crude conjugation reaction was analyzed by HIC-HPLC chromatography to confirm that the reaction was complete (disappearance of the starting antibody peak) at a wavelength of 280 nm. The resulting ADC-1 conjugate was then purified by gel filtration chromatography using an AKTA system equipped with a Superdex 200 pg column (GE Healthcare) equilibrated with PBS. Based on the comparative peak area integration of the HIC-HPLC chromatogram, the average drug-antibody ratio (DAR) was calculated to be 4. Confirmation of the low percentage (<5%) of high molecular weight (HMW) aggregates of the resulting ADC-1 was determined using analytical SEC-HPLC. Example S4: Preparation of antibody-drug conjugate (ADC) anti-FOLR1 compound I-6 (151-K-LOCK-D5; ADC-5).

[0368] ADC anti-FOLR1 compound I-6 is prepared as described in U.S. Pat. No. 10,590,165, which is incorporated herein in its entirety. 0.1 equivalents to 10 equivalents of activated drug-linker conjugate (I-6) are added in batch or continuous flow to a 0.5 mg / mL to 50 mg / mL antibody solution in a buffer containing 0-30% organic solvent at pH 6.0-9.0. The reaction is carried out at 0-40°C with gentle stirring or shaking for 0.5 hours to 50 hours and monitored by HIC-HPLC. Using prior art procedures, the resulting crude ADC product is subjected to the necessary downstream steps of desalting, buffer exchange / formulation and optional purification. The ADC product is characterized by HIC-HPLC, SEC, RP-HPLC and optional LC-MS. According to HIC-HPLC analysis, the average DAR obtained for ADC-5 is 2.

[0369] Example S5: Preparation of antibody-drug conjugate (ADC) anti-FOLR1-GGFG-Dxd (151-GGFG-Dxd).

[0370] Antibody reduction: 151 antibody was exchanged with PBS 7.0 / EDTA. 7 equivalents of TCEP were added to the 151 antibody solution, and the reaction mixture was reacted at 37°C for 2 hours.

[0371] Coupling of antibody and linker-payload: The reduced antibody mixture was incubated at 4°C for 10 minutes. The linker-payload GGFG-DXd (developed by Daiichi Sankyo, purchased from DC Chemicals, DC50025) was dissolved in dimethylacetamide, and 12 equivalents of the GGFG-DXd solution were added to the reduced antibody mixture. The reaction was allowed to proceed at 22°C for 60 minutes.

[0372] The antibody-drug conjugate was desalted, buffer exchanged / formulated, and purified using state of the art procedures.

[0373] Example S6: Preparation of antibody-drug conjugate (ADC) anti-FOLR1-VC-MMAE (151-VC-MMAE).

[0374] Antibody reduction: 151 antibody was exchanged with PBS 7.0 / EDTA. 2.5 equivalents of TCEP were added to the 151 antibody solution, and the reaction mixture was reacted at 37°C for 2 hours.

[0375] Coupling of antibody and linker-payload: The reduced antibody mixture was incubated at 4°C for 10 minutes. The linker-payload VC-MMAE (purchased from DC Chemicals, DC7556) was dissolved in dimethylacetamide, and 7 equivalents of VC-MMAE solution were added to the reduced antibody mixture. The reaction was allowed to proceed at 22°C for 30 minutes.

[0376] The antibody-drug conjugate was desalted, buffer exchanged / formulated, and purified using state of the art procedures. Biological Examples The AMT-151 clone of anti-FOLR1 antibody was used to evaluate the in vitro and in vivo efficacy of the antibody-drug conjugate (ADC). Example B1: In vitro therapeutic efficacy of antibody-drug conjugates (ADC) 151-C-Lock-D5 (ADC-1) and 151-K-Lock-D5 (ADC-5).

[0377] The in vitro efficacy of ADC 151-C-Lock-D5 and 151-K-Lock-D5 was evaluated using the following human cancer cell lines: IGROV1, SKOV-3, and A549 (which are ovarian and lung cancer cell lines). Cells were cultured in RPMI-1640 medium (Gibco ThermoFisher; Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning; Corning, NY, USA) and maintained at 37°C in a humidified atmosphere of 5% CO2.

[0378] The in vitro assay was performed as follows: tumor cells were harvested by centrifugation at 300 g for 5 minutes, seeded in 96-well plates with white walls and transparent bottoms (2,000 cells / well to 3,000 cells / well in 50 μL complete medium) and maintained at 37°C. Cells were then treated in duplicate with 50 μL of test articles prepared at 2X final concentration, serially diluted in complete medium and incubated at 37°C for up to 120 hours. After treatment, inhibition of cancer cell growth was determined using a CCK-8 (cell counting kit-8) viability assay as described in the manufacturer's protocol.

[0379] Data were normalized to untreated controls using Microsoft Excel (Redmond, WA, USA) and analyzed using GraphPad Prism software (version 8; La Jolla, CA, USA). The half-maximal inhibitory concentration (IC 50 ) was obtained from the dose-response curve.

[0380] The cell viability of 151-C-Lock-D5 and 151-K-Lock-D5 is shown in FIG1 .

[0381] The in vitro cytotoxic activity and targeting specificity of the ADCs described herein were evaluated against high FOLR1 expressing IGROV1, low FOLR1 expressing SKOV-3, and FOLR1 negative A549 (negative control) cancer cell lines using a standard cell viability assay. As shown in FIG1 , anti-FOLR1 151-C-LOCK-D5 and 151-K-LOCK-D5 (where 151 is the AMT-151 clone of the FOLR1 antibody) reduced IGROV1 and SKOV-3 cell viability in a dose-dependent manner and showed no activity against A549 cells in a 5-day assay. Potency against FOLR1 expressing cell lines ranging from about 0.35 nM to 99 nM as determined by IC50 was observed in ADCs with different conjugation chemistries to the anti-FOLR1 antibody and in cell lines with different amounts of FOLR1 expression (Table 5).

[0382] The IC of anti-FOLR1 151-C-LOCK-D5 and 151-K-LOCK-D5 in human tumor cells is presented in Table 5 50 Summary of values ​​(nM). Table 5: IC of anti-FOLR1 151-C-LOCK-D5, 151-K-LOCK-D5 and their corresponding controls in human tumor cells 50 Value (nM)

[0383] In the IGROV1 (high FOLR1 expression) cell line, 151-C-LOCK-D5 and 151-K-LOCK-D5 showed comparable cell killing activity. In the SKOV-3 (low FOLR1 expression) cell line, 151-C-LOCK-D5 showed stronger cell killing activity than 151-K-LOCK-D5.

[0384] The control ADCs (IgG-C-LOCK-D5 and IgG-K-LOCK-D5) did not cause a significant loss of cell viability, as shown in FIG1 .

[0385] Example B2: In vitro therapeutic efficacy of antibody-drug conjugates (ADCs) 151-C-Lock-D5 (ADC-1) and 151-GGFG-Dxd.

[0386] The in vitro efficacy of ADCs 151-C-Lock-D5 and 151-K-Lock-D5 was evaluated using the SKOV-3 cell line.

[0387] The in vitro assay was performed as described in Example B1 above. Cell viability was determined after a 5-day incubation period. 151-mAb was used as a negative control in the assay. 151-mAb is an antibody comprising SEQ ID NO: 7 as the light chain variable region and SEQ ID NO: 8 as the heavy chain variable region.

[0388] The results of the cell viability assay of 151-C-Lock-D5 and 151-GGFG-Dxd are shown in Figure 5 middle. Figure 5 It was shown that 151-C-Lock-D5 exhibited better cell killing activity than 151-GGFG-Dxd in the SKOV-3 cell line. 151-mAb alone did not show a loss of cell viability. Example B3: In vivo efficacy of antibody-drug conjugates (ADC) 151-C-Lock-D5 (ADC-1) and 151-K-Lock-D5 (ADC-5).

[0389] Female Balb / c mice aged 6 to 8 weeks were purchased from JSJ Laboratory (Shanghai, China).

[0390] Human ovarian cancer tumor cell lines IGROV1 and SKOV-3 were cultured and expanded in RPMI 1640 medium supplemented with 10% FBS at 37°C and 5% CO2 in a humidified atmosphere for 2-3 weeks before harvesting for implantation. Cell viability determined by trypan blue dye exclusion assay prior to implantation was >90%. 5-10 million IGROV1 or SKOV-3 cells in 100 μL of PBS were inoculated into the right upper flank of each mouse by sc injection.

[0391] Tumor volume measurement was started on day 4 after tumor cell inoculation and was performed twice a week. The longest longitudinal diameter as length and the widest transverse diameter as width were measured using a digital caliper. Tumor volume (TV) was then calculated by the following formula: TV = [length × (width) 2 ] / 2 and analyze it in Excel.

[0392] When the average tumor size of IGROV1 and SKOV-3 reached about 150 mm 3 Processing starts when

[0393] When the tumor size reaches 2000mm 3 When , the mice were euthanized.

[0394] After randomization of tumor-bearing mice, 151-C-LOCK-D5, 151-K-LOCK-D5 or isotype ADC IgG-C-LOCK-D5 and IgG-K-LOCK-D5 diluted in PBS were administered to mice by ip injection. In Experiment 1, the treatment regimen included a single administration of 1.5 mg / kg, 3 mg / kg or 6 mg / kg of 151-C-LOCK-D5 on day 8 after tumor inoculation (IGROV1), or a single administration of 3 mg / kg or 6 mg / kg of 151-K-LOCK-D5 on day 8 after tumor inoculation (IGROV1). Figure 2A The changes in IGROV1 tumor volume over time are shown; Figure 2B Shown are IGROV1 tumor volumes at day 46 after tumor inoculation.

[0395] The body weights of all mice were measured twice a week after the first ADC administration, as Figure 2C shown.

[0396] In Experiment II, the treatment regimen included two consecutive rounds of 1.5 mg / kg, 3 mg / kg or 6 mg / kg of 151-C-LOCK-D5 once every two weeks (Q2W*2), or two consecutive rounds of 3 mg / kg or 6 mg / kg of 151-K-LOCK-D5 once every two weeks (Q2W*2), and the ADC was administered on days 15 and 29 after tumor inoculation (SKOV-3). Figure 3A Shown are the changes in SKOV-3 tumor volume over time; Figure 3B Shown are SKOV-3 tumor volumes at day 49 after tumor inoculation.

[0397] The body weights of all mice were measured twice a week after the first ADC administration, as Figure 3C shown.

[0398] The raw data of tumor measurement were analyzed in Excel. Tumor growth curves were plotted using GraphPad Prism 8.0 software, and values ​​are expressed as mean ± SEM.

[0399] In the IGROV1 xenograft tumor model of Experiment I ( Figure 2A and Figure 2B All treatment regimens using anti-FOLR1-151-K-LOCK-D5 (151-K-LOCK-D5; ADC-5) and anti-FOLR1-151-C-LOCK-D5 (151-C-LOCK-D5; ADC-1) significantly inhibited tumor growth compared to the IgG-C-LOCK-D5 isotype control. All regimens induced significant tumor regression even at low doses, and all tumors were eliminated approximately five weeks after initial treatment. Tumor growth inhibition is shown in Table 6. Table 6: Tumor Growth Inhibition

[0400] No toxicity was observed in any of these treatment regimens, as evidenced by the absence of body weight loss ( Figure 2C ).

[0401] In the SKOV-3 xenograft tumor model of Experiment II ( Figure 3A and Figure 3B As shown), anti-FOLR1-151-C-LOCK-D5 (151-C-LOCK-D5; ADC-1) showed antitumor activity compared with the negative control (IgG-K-LOCK-D5).

[0402] No toxicity was observed in any of these treatment regimens, as evidenced by the absence of significant body weight loss ( Figure 3C ). Example B4.151-K-Lock-D5 (ADC-5) anti-tumor effect in vivo on NSCLC cancer PDX model LU-01-1618

[0403] The antitumor effect of a single dose of 151-K-Lock-D5 (ADC-5) at 1.5 mg / kg, 3 mg / kg, 6 mg / kg and 10 mg / kg was evaluated using the FR-positive NSCLC LU-01-1618 PDX model (Wuxi WuXi AppTec (Shanghai) Co., Ltd.) transplanted on female BALB / c nude mice. Balb / c nude mice were purchased from Shanghai Lingchang Laboratory Animal Technology Co., Ltd. The LU-01-1618 tumor was cut into approximately 30 mm3 fragments and implanted subcutaneously into the flank of the mice. On day 18 after transplantation (average tumor volume ~170 mm3), mice were intravenously injected with one of the following substances: 151-K-Lock-D5 (1.5 mg / kg), 151-K-Lock-D5 (3 mg / kg), 151-K-Lock-D5 (6 mg / kg), 151-K-Lock-D5 (10 mg / kg), isotype ADC (10 mg / kg) and vehicle. From the start of treatment, tumor volume was measured twice a week by Caliper, and the calculation formula was as follows: TV = (width * length)2 / 2.

[0404] Figure 4A Table 7 shows the in vivo antitumor efficacy of 151-K-Lock-D5 (1.5 mg / kg...

Claims

1. An antibody-drug conjugate (ADC) of formula (I): or a pharmaceutically acceptable salt thereof, wherein: Ab is anti-FOLR1 antibody; m is an integer from 1 to 20; L 1 is a linker that binds to the anti-FOLR1 antibody; L 2 Contains or is a bond, -C(O)-, -NH-, an amino acid unit, –(CH2CH2O) n –, –(CH2) n – -(4-aminobenzyloxycarbonyl)-, -(C(O)CH2CH2NH)-, or any combination thereof, wherein n is an integer from 1 to 24; and D is the drug moiety.

2. The ADC according to claim 1, wherein: L 1 A linker that binds to one or two sulfur or nitrogen atoms of the anti-FOLR1 antibody.

3. The ADC according to claim 1 or 2, wherein: -L 1 -L 2 -for:

4. The ADC according to any one of claims 1 to 3, wherein: m is 1, 2, 3, 4, 5, 6, 7 or 8.

5. The ADC according to claim 4, wherein: m is 2 to 4.

6. The ADC according to any one of claims 1 to 5, wherein: L 2 Contains or is a bond, -C(O)-, -NH-, Val, Phe, Lys, –(4-aminobenzyloxycarbonyl)–, Gly, Ser, Thr, Ala, β-Ala, Citrulline (Cit), –(CH2) n –, –(CH2CH2O) n – or any combination thereof.

7. The ADC according to claim 6, wherein: L 2 Contains or is a bond, -C(O)-, -NH-, Val, Gly, Lys, Cit, –(CH2) n –, –(CH2CH2O) n – or any combination thereof.

8. The ADC according to claim 7, wherein: L 2 Contains or is a bond, -C(O)-, Val, Gly, Cit, –(CH2) n – or any combination thereof.

9. The ADC according to claim 8, wherein: L 2 contains or is a key, 10. The ADC according to claim 9, wherein: L 2 for 11. The ADC according to claim 9, wherein: L 2 for 12. The ADC according to claim 9, wherein: L 2 for 13. The ADC according to claim 9, wherein: L 2 for 14. The ADC according to claim 9, wherein: L 2 for 15. The ADC according to any one of claims 1 to 14, wherein: D is 16. The ADC according to any one of claims 1 to 14, wherein: D is in: R 1 is H or –C1-C8 alkyl; R 3 H, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR 3A 、-NR 3A R 3B 、-(CH2) v OR 6 、-C(O)NHSO2R 7 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 4 H, halogen, -OR 4A 、-NR 4A R 4B , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; Z 1 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl; Z 2 is substituted aryl, substituted heteroaryl, substituted cycloalkyl or substituted heterocycloalkyl; R 6 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO(CH2CH2O) w CH2CH2Y, -CONH(CH2CH2O) w CH2CH2Y, Charged groups or sugar derivatives, wherein: v is an integer from 1 to 24; w is an integer from 1 to 24; Y is -NH2, -OH, -COOH or -OCH3; R 7 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; R 10 is -OH, -OCH3 or -COOH; and Each R 3A , R 3B , R 4A and R 4B and R is independently H, or substituted or unsubstituted alkyl.

17. The ADC according to claim 16, wherein R 1 For H.

18. The ADC according to claim 16 or 17, wherein: R 3 H, -OR 3A 、-(CH2) v OR 6 、-C(O)NHSO2R 7 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

19. The ADC of claim 18, wherein: R 3 H, -OR 3A 、-(CH2) v OR 6 、-C(O)NHSO2R 7 , unsubstituted C1-C6 alkyl or substituted C1-C6 alkyl.

20. The ADC of claim 18, wherein: R 3 H, methyl, ethyl, propyl, butyl, –CH2OH, <h2 style=";text-align:left;direction:ltr">-CH2CH2OH、-CH2N3、-CH2CH2N3、-CH2OCH3、-CH2OCH2CH3、-CH2CH2OCH3、-CH2CH2OCH2CH3、<h2 style=";text-align:left;direction:ltr"> 21. The ADC of claim 20, wherein: R 3 is -CH2N3 or 22. The ADC according to any one of claims 16 to 21, wherein R 4 H, -OR 4A , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

23. The ADC of claim 22, wherein: R 4 It is H, -OH, methyl, ethyl, propyl or butyl.

24. The ADC of claim 23, wherein: R 4 It is H or -OH.

25. The ADC according to any one of claims 16 to 24, wherein: Z 1 is a substituted or unsubstituted aryl group.

26. The ADC according to any one of claims 16 to 24, wherein: Z 2 is a substituted aryl group.

27. The ADC of claim 25, wherein: Z 1 for in, Each X is independently Cl, Br, I or F; Each R' is independently -CH3, -CH2CH3 or -CH2CH2CH3; and q is an integer from 1 to 5.

28. The ADC of claim 27, wherein: Z 1 for 29. The ADC of claim 26, wherein: Z 2 for in, Each G is independently Cl, Br, I, F, -CH3, -CH2CH3, -CH2CH2CH3, -OCH3, -OCH2CH3, -OH or -NH2; and p is an integer from 0 to 4.

30. The ADC of claim 29, wherein: Z 2 for 31. The ADC according to any one of claims 1 to 30, wherein: D is:

32. The ADC of claim 31 , wherein: D is 33. The ADC according to any one of claims 1 to 32, wherein: The ADC is: or a pharmaceutically acceptable salt thereof, wherein m is an integer from 1 to 8.

34. The ADC of any one of claims 1 to 33, wherein the anti-FOLR1 antibody comprises (a) a VL CDR1 comprising the sequence of SEQ ID NO: 1; a VL CDR2 comprising the sequence of SEQ ID NO: 2; a VL CDR3 comprising the sequence of SEQ ID NO: 3; a VH CDR1 comprising the sequence of SEQ ID NO: 4; a VH CDR2 comprising the sequence of SEQ ID NO: 5; and a VH CDR3 comprising the sequence of SEQ ID NO: 6; (b) a VL CDR1 comprising the sequence of SEQ ID NO: 12; a VL CDR2 comprising the sequence of SEQ ID NO: 13; a VL CDR3 comprising the sequence of SEQ ID NO: 14; a VH CDR1 comprising the sequence of SEQ ID NO: 15; a VH CDR2 comprising the sequence of SEQ ID NO: 16; and a VH CDR3 comprising the sequence of SEQ ID NO: 17; or (c) a VL CDR1 comprising the sequence of SEQ ID NO: NO:12; VL CDR2, which comprises the sequence of SEQ ID NO:18; VL CDR3, which comprises the sequence of SEQ ID NO:19; VH CDR1, which comprises the sequence of SEQ ID NO:20; VH CDR2, which comprises the sequence of SEQ ID NO:21; and VH CDR3, which comprises the sequence of SEQ ID NO:

22.

35. The ADC of any one of claims 1 to 34, wherein the anti-FOLR1 antibody comprises: a VL having a sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:

30.

36. The ADC of any one of claims 1 to 35, wherein the anti-FOLR1 antibody comprises: a VH having a sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:

28.

37. The ADC of any one of claims 1 to 36, wherein the anti-FOLR1 antibody comprises: a VL having a sequence of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:

30.

38. The ADC of any one of claims 1 to 37, wherein the anti-FOLR1 antibody comprises: a VH having a sequence of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO:

28.

39. The ADC of any one of claims 1-38, wherein the anti-FOLR1 antibody is an IgG antibody, optionally wherein the anti-FOLR1 antibody is an IgG1 antibody.

40. The ADC of any one of claims 1 to 39, wherein the anti-FOLR1 antibody binds human FOLR1, optionally wherein the human FOLR1 has the amino acid sequence of SEQ ID NO:

9.

41. The ADC according to any one of claims 1 to 40 for use in therapy.

42. The ADC of claim 41 for use in treating a cancer expressing FOLR1, optionally wherein the cancer expressing FOLR1 is multiple myeloma, lung cancer or ovarian cancer.

43. The ADC according to claim 42, for use in treating epithelial-derived tumors.

44. A method of treating a FOLR1 -expressing cancer in a subject, the method comprising administering to a subject in need thereof an ADC according to any one of claims 1 to 40.

45. Use of the ADC according to any one of claims 1 to 40 in the manufacture of a medicament.

46. ​​Use of an ADC according to any one of claims 1 to 40 in the manufacture of a medicament for treating a cancer expressing FOLR1, optionally wherein the cancer expressing FOLR1 is multiple myeloma, lung cancer or ovarian cancer.

47. The ADC for use, use or method according to any one of claims 42, 44 or 46, wherein the cancer expressing FOLR1 is multiple myeloma.

48. The ADC for use according to claim 43, wherein the epithelial-derived tumor is a uterine, breast, endometrial, pancreatic, kidney, colorectal or brain tumor.

49. The ADC for use, use or method according to any one of claims 42, 44, 46 or 47, wherein the cancer expressing FOLR1 is in a mammal, optionally wherein the mammal is a human.

50. The ADC for use, use or method according to any one of claims 41 to 49, further comprising the administration of a therapeutically effective amount of one or more additional active agents.

51. The ADC for use, use or method according to claim 50, wherein the additional active agent is a VEGF inhibitor.

52. The ADC for use, use or method according to claim 51, wherein the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor.

53. The ADC for use, use or method according to claim 51 or 52, wherein the VEGF inhibitor is bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib or regorafenib.

54. A method of inhibiting proliferation of a cell expressing FOLR1, the method comprising contacting the cell expressing FOLR1 with an ADC according to any one of claims 1 to 40.

55. The use according to claim 45, wherein the medicament is used to inhibit the proliferation of cells expressing FOLR1.

56. The ADC according to any one of claims 1 to 40, for use in inhibiting the proliferation of cells expressing FOLR1.

57. The method, the use or the ADC for use according to any one of claims 54 to 56, wherein the cell expressing FOLR1 is a cancer cell expressing FOLR1, optionally wherein the cancer expressing FOLR1 is multiple myeloma, lung cancer or ovarian cancer.

58. The method, the use or the ADC for use according to any one of claims 54 to 56, wherein the cell expressing FOLR1 is a multiple myeloma cell expressing FOLR1.

59. The method of any one of claims 54, 57 or 58, which is in vitro.

60. The method of any one of claims 54, 57 or 58, wherein the method is in vivo.

61. A kit for treating a cancer expressing FOLR1, the kit comprising: (i) The ADC according to any one of claims 1 to 40; as well as (ii) VEGF inhibitors.

62. The kit of claim 61, wherein the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor.

63. The kit of claim 61 or 62, wherein the VEGF inhibitor is bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, or regorafenib.

64. A compound selected from: or a pharmaceutically acceptable salt thereof.

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