Antibody-drug conjugates including anti-FOLR1 antibodies

Abstract

Antibody drug conjugates (ADCs) that specifically bind to folate receptor 1 (FOLR1) are inter alia provided. Pharmaceutical compositions and methods for treating cancer are further disclosed. The present disclosure provides ADCs comprising an anti-FOLR1 antibody conjugated to a drug moiety by a linker moiety. In embodiments, the anti-FOLR1 antibody binds to FOLR1-expressing cancer cells, allowing selective uptake of the ADC into the cancer cells. In embodiments, the ADCs provided herein selectively deliver an effective amount of the drug moiety to tumor tissue, reducing non-specific toxicity associated with related ADCs. ADC compounds described herein include those that have anti-cancer activity.

Description

[Technical field]

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

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

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

[0004] Introduction and Overview Antibody-drug conjugates (ADCs) allow for targeted delivery of drug moieties to, and in some embodiments, intracellular accumulation in, tumors where systemic administration of unconjugated drugs would result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387). ADCs are targeted chemotherapeutic molecules that combine the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004), thus improving the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008) The Cancer Jour. 14(3):154-169; Chari, RV (2008) Acc. Chem. Res. 41:98-107). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Polakis P. (2005) Current Opinion in Pharmacology 5:382-387 [Non-Patent Document 2] Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004 [Non-Patent Document 3] Carter, PJ and Senter PD (2008) The Cancer Jour. 14(3):154-169 [Non-Patent Document 4] Chari, RV (2008) Acc. Chem. Res. 41:98-107 Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides an ADC comprising an anti-FOLR1 antibody conjugated to a drug moiety by a linker moiety. In embodiments, the anti-FOLR1 antibody binds to FOLR1-expressing cancer cells, allowing selective uptake of the ADC into the cancer cells. In embodiments, the ADC provided herein selectively delivers an effective amount of the drug moiety to tumor tissue, reducing non-specific toxicity associated with related ADCs. The ADC compounds described herein include those that have anti-cancer activity. Folate receptor 1 (FOLR1), also known as folate receptor alpha (FRα) or folate-binding protein (UniProt P15328), is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein with strong binding affinity to folate and reduced folate derivatives (Leung et al. (2013) Clin. Biochem. 46:1462- 1468). FOLR1 has important functions in cell proliferation and survival (Kelemen LE (2006) Int. J. Cancer 119(2):2430250) and mediates the delivery of 5-methyltetrahydrofolate, a physiological folate, to the cell interior. Expression of FOLR1 on normal tissues is restricted to the apical membrane of epithelial cells of the kidney proximal tubules, pneumocytes of the lung alveoli, bladder, testis, choroid plexus, and thyroid gland (Weitman SD et al. (1992) Cancer Res. 52:3396-3401; Antony AC (1996) Ann. Rev. Nutr. 16:501 -521; Kalli KR et al. (2008) Gynecol. Oncol. 108:619-626). FOLR1 is overexpressed in tumors of epithelial origin, including ovarian, uterine, breast, endometrial, pancreatic, renal, lung, colorectal, and brain tumors. This expression pattern of FOLR1 makes it a desirable target for FOLR1-directed cancer therapy.

[0007] There is a need for improved methods of modulating the immunoregulation of folate receptor alpha (FOLR1) and downstream signaling processes activated by folate receptor alpha (FOLR1). Furthermore, in light of the specific expression of folate receptor alpha (FOLR1) in cells transformed by cancer and carcinoma, and lower expression in non-cancerous tissues, there is a need for improved therapeutic agents that can specifically target cells and tissues that overexpress folate receptor alpha (FOLR1). Antibody conjugates with FOLR1 could be used to deliver therapeutic or diagnostic payload moieties to target cells expressing folate receptor alpha for the treatment of such diseases. Thus, antibody drug conjugates (ADCs) in which drugs are conjugated to anti-FOLR1 antibodies can provide highly targeted and potent anti-tumor activity.

[0008] In one aspect, provided herein is an antibody-drug conjugate (ADC) comprising a monoclonal antibody. In another aspect, provided herein is an antibody-drug conjugate (ADC) comprising an anti-FOLR1 antibody. In another aspect, provided herein is a method for treating cancer, such as FOLR1-expressing cancer, using the ADC disclosed herein.

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

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

[0011] In an aspect, provided herein is a method of treating a FOLR1-expressing cancer in a subject in need of such treatment, the method comprising administering to the subject an ADC described herein (including in the aspects, embodiments, tables, examples, or claims), or a pharma- ceutically acceptable salt thereof.

[0012] In an 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 (including in the aspects, embodiments, tables, examples, or claims), or a pharma- ceutically acceptable salt thereof, and further administering a therapeutically effective amount of one or more additional active agents. In an embodiment, 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 1AB]1A-C show 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: FIG. 1A IGROV1 (high expressing FOLR1) cells; FIG. 1B SKOV-3 (low expressing FOLR1) cells; and FIG. 1C A549 are FOLR1 negative (negative control). [Figure 1C] 1A-C show 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: FIG. 1A IGROV1 (high expressing FOLR1) cells; FIG. 1B SKOV-3 (low expressing FOLR1) cells; and FIG. 1C A549 are FOLR1 negative (negative control).

[0015] [Figure 2A] FIG. 2A shows the results of an in vivo efficacy study in IGROV1 xenografts in BALB / c 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 2BC] Figure 2B shows tumor volumes in mice 46 days after treatment as described in Figure 2A. Figure 2C shows the results of an in vivo toxicity study in mice (after treatment as described in Figure 2A). The figure shows the body weight change in mice treated with ADC.

[0016] [Figure 3AB]Figure 3A shows the results of an in vivo efficacy study in SKOV-3 xenografts in BALB / c nude mice treated with two doses of 151-K-Lock-D5 (at 3 mg / kg or 6 mg / kg each), 151-C-Lock-D5 (at 1.5 mg / kg, 3 mg / kg, or 6 mg / kg each) and control IgG-K-Lock-D5 (at 6 mg / kg each) on days 15 and 29. Figure 3B shows the tumor volume in mice 49 days after treatment described in Figure 3A. [Figure 3C] Figure 3C shows the results of an in vivo toxicity study in mice (following treatment as described in Figure 3A). The figure shows the body weight change in mice treated with the ADC.

[0017] [Figure 4A] FIG. 4A shows the results of an in vivo antitumor efficacy study in PDX model LU-01-1618 PDX in BALB / c nude mice treated 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. [Figure 4B] Figure 4B shows the results of an in vivo toxicity study in mice (after treatment as described in Figure 4A). The figure shows the weight change in the mice.

[0018] [Diagram 5] FIG. 5 shows the results of an in vitro efficacy study of 151-C-Lock-D5 and 151-GGFG-Dxd and negative control 151-mAb using SKOV-3 (low expressing FOLR1) cells.

[0019] [Figure 6]Figure 6A shows the results of an in vivo efficacy study in SKOV-3 xenografts in BALB / c nude mice treated with two doses of 151-C-Lock-D5 (at 3 mg / kg or 6 mg / kg each), 151-VC-MMAE (at 3 mg / kg or 6 mg / kg each) and control IgG-VC-MMAE (at 6 mg / kg each) on days 15 and 29. Figure 6B shows the results of an in vivo toxicity study in mice (after treatment as described in Figure 6A). The figure shows the weight change in mice treated with ADC.

[0020] [Figure 7AB] Figure 7A shows the results of an in vivo efficacy study in OVCAR-3 xenografts in BALB / c 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 was administered intravenously and bevacizumab was administered intraperitoneally) and control isotype ADC (10 mg / kg). Figure 7B shows the tumor volume in mice 48 days after treatment described in Figure 7A. [Figure 7C] Figure 7C shows the results of an in vivo toxicity study in mice (following treatment as described in Figure 7A). The figure shows the body weight change in mice treated with the ADC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention Definition: Unless otherwise defined, technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art.Generally, the terminology of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization techniques described herein are well known and commonly used in the art.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, unless otherwise indicated.See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992).Standard antibody production procedures are described in numerous basic textbooks, including Borrebaeck (ed) Antibody Engineering, 2nd Edition 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 Altos, Calif., and references cited therein;Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY, 1986, and Kohler and Milstein Nature 256: 495-497, 1975. All references cited herein are incorporated by reference in their entirety. Enzymatic reactions and concentration / purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein.The terminology used in connection with, and the laboratory and experimental techniques of, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical 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] As used herein, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as use of the singular form of any word, include plural referents unless expressly and unambiguously limited to one referent.

[0024] The use of the alternative (eg, "or") herein is to be understood to mean either one or both of the alternatives, or any combination thereof.

[0025] The term "and / or" as used herein should be understood to mean specific disclosure of each of the specified features or components with or without the others. For example, when the term "and / or" is used herein in a phrase such as "A and / or B", it is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, when the term "and / or" is used in a phrase such as "A, B and / or C", it is intended to include 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 foregoing elements, and further 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; and X, Y and Z, as well as further combinations in which one or more of X, Y, and Z occur more than once.

[0027] As used herein, the term "about" refers to a value or composition that is within an acceptable error range for the particular value and composition as determined by one of ordinary skill in the art, where the acceptable error range will depend in part on how the value or composition is measured or determined, i.e., on the constraints of the measurement system. For example, "about" or "approximately" may mean within 1 standard deviation or more than 1 standard deviation, according to practice in the art. Alternatively, "about" or "approximately" may mean a range of 10% (i.e., ±10%) or less, or a range of more than 10%, depending on the constraints of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms may mean an order of magnitude or 5-fold less than the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be considered to be within an acceptable error range for the particular value or composition. In an embodiment, about includes the value specified.

[0028] In this disclosure, "comprises," "comprising," "containing," "having," and the like may have the meaning ascribed to them in U.S. Patent Law and may mean "includes," "including," and the like. Similarly, "consisting essentially of" or "consists essentially of" has the meaning given to it in U.S. Patent Law, and the term is open-ended, thus permitting the presence of more than what is recited so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited, but excluding prior art embodiments.

[0029] As used herein, the terms "polypeptide", "peptide" and "protein" and other related terms are used interchangeably to refer to a polymer of amino acid residues, which may in embodiments be conjugated to a moiety that is not composed of amino acids. These terms apply to naturally occurring and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid. A "fusion protein" refers to a chimeric protein that includes sequences from two or more separate proteins that are, for example, recombinantly expressed as a single moiety, synthesized or conjugated to form a part thereof. Polypeptides include mature molecules that have undergone cleavage. These terms encompass native and artificial proteins, protein fragments, and polypeptide analogs of protein sequences (e.g., mutant proteins, variants, chimeric proteins and fusion proteins), as well as proteins that have been post-translationally modified or otherwise covalently or non-covalently modified. Two or more polypeptides (e.g., three polypeptide chains) may 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 the polypeptide chains may also include peptide folding. Thus, the polypeptide complex may be a dimer, trimer, tetramer, or higher order complex, depending on the number of polypeptide chains that form the complex.

[0030] As used herein, the terms "cancer", "neoplasm" and "tumor" are used interchangeably and refer to cells that have undergone malignant transformation, whether singular or plural, that render them pathogenic to the host organism. In an embodiment, 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 progenitor cells. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that usually manifest as solid tumors, a "clinically detectable" tumor is one that is detectable based on tumor burden, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during physical examination, and / or is detectable from the expression of one or more cancer-specific antigens in samples that can be taken from a patient. Tumors may also be hematopoietic cancers (or hematologic cancers or hematological cancers or blood-related cancers), such as cancers originating from blood cells or immune cells, which may be referred to as "liquid tumors". Specific examples of clinical conditions based on hematological tumors 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; etc.

[0031] Cancer can be any cancer that has abnormal number of blast cells or unwanted cell proliferation, or is diagnosed as blood cancer, including both lymphoid and myeloid malignancies.Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyelocytic (or promyelocytic or promyelocytic or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryocytic (megakaryoblastic) leukemia.These leukemias can be collectively referred to as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPDs), including, but not limited to, chronic myeloid (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), sometimes referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transition (RAEBT); and myelofibrosis with or without primary myelofibrosis (MFS).

[0032] Hematopoietic cancers also include lymphoid malignancies that may involve lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal sites. Cancers of the lymphatic system include B-cell malignancies, including but not limited to B-cell non-Hodgkin's lymphoma (B-NHL). B-NHL can be low-grade (or indolent), intermediate-grade (or aggressive), or high-grade (highly 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-NHL includes mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHL includes 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 Castleman's disease. NHL can also include T-cell non-Hodgkin's lymphoma (T-NHL), including but not limited to T-cell non-Hodgkin's lymphoma, not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome.

[0033] Hematopoietic cancers also include Hodgkin lymphoma (or Hodgkin's disease), including classical Hodgkin lymphoma, nodular sclerosing Hodgkin lymphoma, mixed cytology Hodgkin lymphoma, lymphocyte predominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphocytopenic Hodgkin lymphoma. Hematopoietic cancers also include plasma cell disorders 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 containing hematopoietic cells, referred to herein as "hematopoietic cell tissues," include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as the spleen, lymph nodes, lymphoid tissues associated with mucous membranes (e.g., gut-associated lymphoid tissue), tonsils, Peyer's patches, and appendix, and lymphoid tissues associated with other mucous membranes, such as the bronchial lining.

[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, endocrine system, brain, breast, cervical, colon, head and neck, liver, kidney, lung, ovarian, pancreatic, rectal, stomach, and uterine cancer. Additional examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, invasive breast cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, endocrine or exocrine pancreatic neoplasms, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer.

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

[0036] An "advanced" cancer is one that has spread outside of its site or organ of origin, either by local invasion or metastasis. The term "advanced" cancer includes both locally advanced and metastatic disease. A "metastatic" cancer refers to a cancer that has spread from one part of the body to another part of the body. A "refractory" cancer is one that progresses even if antitumor treatments such as chemotherapy are administered to the cancer patient. An example of a refractory cancer is one that is platinum-refractory. A "recurrent" cancer is one that has regrown either at the original site or at a distant site after responding to initial treatment.

[0037] As used herein, "antibody" and "antibodies" and related terms refer to an intact immunoglobulin or to an antigen-binding portion thereof that specifically binds to an antigen. Antigen-binding portions may 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, single chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin, which portion is sufficient to confer specific antigen binding to the polypeptide.

[0038] Antibodies include recombinantly produced antibodies and antigen-binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies. Antibodies include monospecific, multispecific (e.g., bispecific, trispecific, higher specific). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, 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-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies. Antibodies include monoclonal and polyclonal populations. Anti-FOLR1 antibodies are described herein.

[0039] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or arise during the production of a monoclonal antibody preparation, which typically are present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is an antibody against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals carrying all or portions of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0040] "Epitope" and related terms, as used herein, refer to a portion of an antigen that is bound by an antigen-binding protein (e.g., by an antibody or an antigen-binding portion thereof). An epitope may include portions of two or more antigens that are bound by an antigen-binding protein. An epitope may include non-contiguous portions of an antigen (e.g., amino acid residues that are not contiguous in the primary sequence of the antigen, but are in sufficient proximity to each other in the context of the antigen's tertiary and quaternary structure for binding by the antigen-binding protein) of the antigen. Generally, the variable regions of the antibody, particularly the CDRs, interact with the epitope. Anti-FOLR1 antibodies and their antigen-binding proteins that bind to epitopes of FOLR1 polypeptides 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 a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the 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); polypeptides that contain at least a portion of an antibody, the portion being sufficient to confer specific antigen binding to the polypeptide. Antigen-binding portions of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, 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 that is sufficient to confer antigen-binding properties to the antibody fragment. Antigen-binding fragments of anti-FOLR1 antibodies are described herein.

[0042] The antigen-binding protein may have the structure of, for example, an immunoglobulin. In one embodiment, "immunoglobulin" refers to a tetrameric molecule. Each tetrameric molecule is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. In an embodiment, the light chain is kappa. In an embodiment, the light chain is lambda. Heavy chains are classified as mu, delta, gamma, alpha or epsilon, defining the antibody's isotype as IgM, IgD, IgG, IgA and IgE, respectively. The variable and constant regions in the light and heavy chains are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), which is incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site, and thus an intact immunoglobulin has two antigen-binding sites. In one embodiment, the antigen-binding protein can be a synthetic molecule that has 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 include an antibody fragment, one to six 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 terms "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 a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH and VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). Antigen binding proteins with immunoglobulin-like properties that specifically bind to FOLR1 are described herein.

[0043] Examples of functional fragments of antibodies include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2', and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen (see, for example, FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed. 2001). As will be appreciated by those skilled in the art, various antibody fragments can be obtained in a variety of ways, for example, by digestion of intact antibodies with enzymes such as pepsin, or by de novo synthesis. Antibody fragments are often synthesized de novo, either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, includes either antibody fragments produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodology (e.g., single chain Fv), or those identified using phage display libraries (see, e.g., McCafferty et al., (1990) Nature 348:552). The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described, for example, in Kostelny et al. (1992) J. Immunol. 148:1547, Pack and Pluckthun (1992) Biochemistry 348:1547, and others. 31:1579, Hollinger et al. (1993), PNAS. USA 90:6444, Gruber et al. (1994) J Immunol. 152:5368, Zhu et al. (1997) Protein Sci. 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[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 that includes a portion that binds to an antigen, and optionally a 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. Antigen-binding proteins may include alternative protein scaffolds or artificial scaffolds, e.g., with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds that include mutations introduced, e.g., to stabilize the three-dimensional structure of the antigen-binding protein, as well as fully synthetic scaffolds, e.g., including biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimics ("PAMs") can be used, as can antibody mimic-based scaffolds that utilize fibronectin components as scaffolds. Antigen binding proteins that bind to FOLR1 are described herein.

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

[0046] "Specifically binds" as used throughout this specification with respect to anti-FOLR1 antigen binding proteins means that the antigen binding protein binds to human FOLR1 (hFOLR1) and has no or little binding to other human proteins. However, this term does not exclude that the antigen binding proteins of the present invention may be cross-reactive with other forms of FOLR1, such as primate FOLR1. In embodiments, the antibody binds to an antigen and is capable of binding to 10 -5 M or less, or 10 -6 M or less, or 10 -7 M or less, or 10 -8 M or less, or 10 -9 M or less, or 10 -10 M or a dissociation constant K D the antibody specifically binds to the target antigen.

[0047] The term "FOLR1", as used herein, refers to any native FOLR1 from any vertebrate source, including mammals, e.g., primates (e.g., humans, cynos) and rodents (e.g., mice and rats), unless otherwise indicated. FOLR1 is also referred to as "human folate receptor 1", "folate receptor alpha (FR-α)", and "FRα". FOLR1 is a single-chain membrane protein that can bind folate and its derivatives. The term encompasses "full-length" unprocessed FOLR1 as well as any form of FOLR1 that results from processing in cells. The term also encompasses naturally occurring variants of FOLR1, e.g., splice variants, allelic variants, and isoforms. Human FOLR1 sequences are known and include, for example, the sequence (including isoforms) published in UniProtKB Accession No. P 15328. An exemplary amino acid sequence of a human FOLR1 protein is set forth in SEQ ID NO:9.

[0048] The term "FOLR1-expressing cancer" refers to cancers that contain cells that express FOLR on their surface.

[0049] The term "increased expression" or "overexpression" of FOLR1 in a particular tumor, tissue or cell sample refers to FOLR1 (FOLR1 polypeptide, or a nucleic acid encoding such a polypeptide) being present at a higher level than that present in a healthy or non-diseased (native, wild-type) tissue or cell of the same type or origin. Such increased expression or overexpression can be caused, for example, by 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 can bind to FOLR1 with sufficient affinity such that the antibody is useful in targeting FOLR1 as a therapeutic agent. In one embodiment, the extent of binding of an 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 radioimmunoassay (RIA). In certain embodiments, an antibody that binds to FOLR1 has an affinity of ≦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 less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) of FOLR1. In certain embodiments, the anti-FOLR1 antibody binds to an epitope of FOLR1 that is conserved among FOLR1 from different species.

[0051] The term "chimeric antibody" and related terms as used herein refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a human antibody. In another embodiment, all of the CDRs are derived from a human antibody. In another embodiment, the CDRs from more than one human antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody contains CDR1 from a light chain of a first human antibody, CDR2 and CDR3 from a light chain of a second human antibody, and CDRs from a heavy chain of a third antibody. In another example, the CDRs originate from different species, for example, human and mouse, or human and rabbit, or human and goat. It will be understood by those skilled in the art that other combinations are possible.

[0052] Furthermore, the framework regions may be from the same antibody, from one or more different antibodies, such as a human antibody, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and / or light chain is identical to, homologous to, 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 to, homologous to, 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 function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on 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 receptor); and B cell activation.

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

[0055] "Humanized antibody" refers to an antibody having a sequence that differs from that of an antibody derived from a non-human species in terms of one or more amino acid substitutions, deletions and / or additions, such that the humanized antibody is less likely to induce an immune response and / or induces a less vigorous immune response when it is administered to a human subject, as compared to the non-human species antibody. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain from a human antibody is fused to a variable domain of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of the non-human antibody are altered to reduce the possible immunogenicity of the non-human antibody when it is administered to a human subject, and the altered amino acid residues are not important for the immunospecific binding of the antibody to its antigen, or the amino acid sequence changes made are not conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the 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 and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). These antibodies can be prepared in a variety of ways, examples of which are described below, including by recombinant methodology or by immunization with the antigen of interest of mice that have been genetically modified to express antibodies derived from human heavy and / or light chain encoding genes. Fully human anti-FOLR1 antibodies and their antigen-binding proteins are described herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0057] The term "isolated" means altered "by the hand of man" from its natural state, changed or removed from its original environment, or both. When applied to a nucleic acid or protein, the term "isolated" indicates that the nucleic acid or protein is essentially free of other cellular components with which it is naturally associated. It can, for example, be in a homogeneous state, or be in either a dry or aqueous solution. Purity and homogeneity are usually determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis, high performance liquid chromatography, or mass spectrometry. A protein that is the predominant species present in a preparation is substantially purified. For example, a polynucleotide or polypeptide that is naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide that is separated from coexisting materials in its natural state is "isolated," even if the cell is of the same species or type as the cell from which the polynucleotide or polypeptide was isolated, such as, but not limited to, when such polynucleotide or polypeptide is introduced back into a cell.

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

[0059] CDRs provide the majority of contact residues for the binding of an antibody to an antigen or epitope. CDRs of interest in 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 neutralizing ability as the donor antibody from which they were derived.

[0060] The CDR sequences of antibodies can be determined by the Kabat numbering system (Kabat et al; (Sequences of proteins of Immunological Interest NIH, 1987)) or they can be determined using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273, 927-948), the contact definition method (MacCallum RM, and Martin ACR and Thornton J. M, (1996), Journal of Molecular Biology, 262 (5), 732-745) or any other established method for numbering residues in an antibody and determining CDRs known to those skilled in the art.

[0061] Other numbering conventions for CDR sequences that can be used by those skilled in the art include the "AbM" (University of Bath) and "Contact" (University College London) methods. At least two of Kabat, Chothia, AbM and Contact methods can be used to determine the minimum overlapping area to obtain a "minimum binding unit". The minimum binding unit can be a subpart 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, which represents 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 expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific, illustrative and exemplary embodiments for measuring binding affinity are described below.

[0063] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved 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 in which one or more amino acid residues are inserted, deleted, and / or substituted into the amino acid sequence compared to a reference polypeptide sequence. Polypeptide variants include fusion proteins. Similarly, variant polynucleotides include nucleotide sequences in which one or more nucleotides are inserted, deleted, and / or substituted into the nucleotide sequence compared 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. In general, a domain is responsible for the distinct functional properties of a protein and can often be added, removed or transferred to other proteins without loss of function of the remainder of the protein and / or the domain. An "antibody single variable domain" is a folded polypeptide domain that contains a sequence that is typical of an antibody variable domain. It therefore includes complete antibody variable domains; and modified variable domains, for example, modified variable domains in which one or more loops are replaced by sequences that are not typical of antibody variable domains; or antibody variable domains that are truncated or contain N- or C-terminal extensions; as well as folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain.

[0066] The term "cytotoxic agent" or "payload" as used herein refers to a substance that inhibits or prevents the function of a cell and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Pb, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, e.g., nucleases; antibiotics; toxins, e.g., 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 chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine); acetogenins (particularly bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, and the like). camptothecin, scopoletin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its synthetic analogs adzelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; auristatins; duostatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); ereutherobiin; punk latistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine;Antibiotics, such as enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 11 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins (including dynemicin A); esperamicins; and neocarzinostatin chromophore and related enediyne antibiotic chromophores), aclacinomycins, actinomycins, anthramycins, azaserine, bleomycins, cactinomycins, carabicins, carminomycins, carzinophilins, chromomycins, dactinomycins, daunorubicins, detorubicins, 6-diazo-5-oxo-L-norleucine, doxorubicins, tetracyclines ... doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, diaminobutyricin, nostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens, e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; corticosteroid synthesis inhibitors, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone;elfornithine; elliptinium acetate; epothilones; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, e.g., maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A) A), Roridin A and Anguidine; urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and docetaxel (TAXOTERE®; Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate;topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine (XELODA®); pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone), CVP (abbreviation for cyclophosphamide, vincristine, and prednisolone), and FOLFOX (abbreviation for a treatment regimen of oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin);

[0068] An "antibody-drug conjugate" or "ADC" is an antibody conjugated 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 linked to the cytotoxic agent by a covalent bond, or the antibody can be indirectly linked to the cytotoxic agent via a linker. Typically, the linker is covalently attached to the antibody and also covalently attached to the cytotoxic agent. Such a linker can be a cleavable linker, e.g., 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 includes a protease cleavage site, which can contain 2-5 amino acid residues that are recognizable and / or cleavable by a suitable protease. Such peptides can include naturally occurring amino acid residues, non-naturally occurring amino acid residues, or a combination thereof. In one example, the peptide linker can be a dipeptide linker. Examples include valine-citrulline (val-cit) linker, phenylalanine-lysine (phe-lys) linker, or maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker. Alternatively, the linker can be non-cleavable, for example, a linker comprising an optionally substituted alkane or thioether. In some examples, the linker can include a functional group capable of forming a covalent bond with an antibody. Exemplary functional groups include, but are not limited to, a maleimide group, an iodoacetamide group, a vinylsulfone group, an acrylate group, an acrylamide group, an acrylonitrile group, or a methacrylate group. 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 "conjugated" when referring to two moieties means that the two moieties are linked together, and the bond or bonds connecting the two moieties may be covalent or non-covalent. In embodiments, the two moieties are covalently linked to each other (e.g., directly or through an intermediate step of covalent linkage). In embodiments, the two moieties are non-covalently linked (e.g., by 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, adolescent, child, or infant. In some embodiments, the terms "individual" or "patient" are used and intended to be synonymous with "subject."

[0071] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, by utilizing the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by utilizing the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by utilizing the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or by utilizing a computer program that uses said algorithm (for example, EMBOSS Needle or EMBOSS Water, available at www.ebi.ac.uk / Tools / psa / ). Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. "Percentage of sequence identity" or "percent (%) [sequence] identity" as used herein is determined by comparing two optimally locally aligned sequences over a comparison window defined by the length of the local alignment between these two sequences. (This may also be considered as percentage of homology, or "percent (%) homology.") The amino acid sequence within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence for optimal alignment between the two sequences. The local alignment between the two sequences includes only those segments of each sequence that appear to be sufficiently similar according to criteria that depend on the algorithm (e.g., EMBOSS Water) used to perform the alignment."Identical" or percent "identity" refers to two or more sequences or subsequences that are the same 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 higher identity when compared over a comparison window or designated region and aligned for maximum correspondence). The identity percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain 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 optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (Add. APL. Math. 2:482, 1981), the global homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), or by inspection.As a further example, GAP and BESTFIT can be used to determine the optimal alignment of two sequences identified for comparison.Normally, the default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used.

[0072] Comparison of sequences and determination of percent identity between two polypeptide sequences or 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 Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. A phrase such as "comprises a sequence having at least X% identity to Y" with respect to a subject sequence means that when aligned with sequence Y as described above, the subject sequence comprises residues that are identical to at least X% of the residues of Y.

[0073] In one embodiment, the amino acid sequence of the subject antibody may be similar, but not identical, to any of the amino acid sequences of the polypeptides constituting the multispecific antigen-binding protein complexes described herein. The similarity between the subject antibody and the polypeptide may be at least 95% identical, 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 constituting the multispecific antigen-binding protein complexes described herein. In one embodiment, the similar polypeptide may contain amino acid substitutions in the heavy and / or light chains. In one embodiment, the amino acid substitutions include one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of the protein. When two or more amino acid sequences differ from each other in terms of conservative substitutions, the percent sequence identity or similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 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: aspartate and glutamate, and (7) sulfur-containing side chains: cysteine ​​and methionine.

[0074] Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins with diverse antigen specificities. Such antibodies can be subjected to affinity purification to enrich them for a particular antigen specificity. Such enriched preparations of antibodies are usually made up of less than about 10% of antibodies that have specific binding activity for a particular antigen. By subjecting these preparations to several rounds of affinity purification, the proportion of antibodies that have specific binding activity for the antigen can be increased. Antibodies prepared in this manner are often referred to as "monospecific". Monospecific antibody preparations can be composed of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% of antibodies that have specific binding activity for a particular antigen. Recombinant nucleic acid techniques, as described below, can be used to produce antibodies.

[0075] The term "vector," as used herein, refers to a nucleic acid molecule capable of transmitting another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively 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 to 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, regardless of the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0077] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein.When a compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts.When a compound of the present disclosure contains a relatively basic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and the like, as well as salts derived from relatively non-toxic 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-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Salts of amino acids such as alginates, as well as salts of organic acids such as glucuronic acid or galacturonic acid, and the like are also included (see, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0078] Therefore, the compounds of the present disclosure can exist as salts, for example, salts with pharma- ceutically acceptable acids. The present disclosure includes such salts. Non-limiting examples of such salts include hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate (e.g., (+)-tartrate, (-)-tartrate, mixtures thereof, including racemic mixtures), succinate, benzoate, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, 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 form, the present disclosure provides compounds in prodrug form.Prodrugs of the compounds described herein are compounds that easily undergo chemical change under physiological conditions to provide the compounds of the present disclosure.Prodrugs of the compounds described herein can be converted in vivo after administration.In addition, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in ex vivo environments, for example, when contacted with suitable enzymes or chemical reagents.

[0081] Certain compounds of the present disclosure can exist in unsolvated form as well as in solvated form, including hydrated form.In general, solvated form is equivalent to unsolvated form and is included within the scope of the present disclosure.Certain compounds of the present disclosure can exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent in terms of the use contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0082] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of active agents to and by subjects, and can be included in the compositions of the present disclosure without causing significant adverse toxic effects to patients. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations can be sterilized and, if desired, can be mixed with auxiliary agents that do not adversely react with the compounds of the present disclosure, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring agents and / or flavoring agents. Those of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0083] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which it is to be administered.

[0084] The terms "administering", "administered" and grammatical variations refer to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration" as used herein means a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered by a parenteral route, for example, orally. Other parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administration can be, for example, once, multiple times, and / or over one or more extended periods of time.

[0085] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) administration or sequential administration in any order. Combination therapy can result in "synergism" and can be found to be "synergistic", i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects obtained as a result of using the compounds separately. Synergistic effects can be achieved when the active ingredients are (1) formulated together and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered sequentially, alternatingly, or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternating therapy, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Synergistic combinations produce effects that are greater than the additive effects of the individual components of the combination.

[0086] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at the dosages required, for the period of time required, to achieve the desired therapeutic or prophylactic result.

[0087] The abbreviations used herein have their conventional meaning within the chemical and biochemical arts. The chemical structures and formulas depicted herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0088] The description of the compounds of the present disclosure is restricted by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted by one or more of several substituents, such substitutions are selected to comply with the principles of chemical bonding and to obtain compounds that are not inherently unstable, and / or compounds that would be known to those skilled in the art to be likely to be unstable under ambient conditions, such as aqueous conditions, neutral conditions and some known physiological conditions.For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule through ring heteroatoms, complying with the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0089] Where substituents are specified by a conventional chemical formula written from left to right, they also encompass the chemically identical substituents that would result if the structure were written from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0090] The term saccharide refers to carbohydrates (or sugars). In embodiments, saccharides are monosaccharides. In embodiments, saccharides are polysaccharides. The majority of the building blocks of saccharides are carbohydrate monomers. The general formula is: n H 2n O n The term saccharide derivative refers to sugar molecules modified with substituents other than hydroxyl groups. Examples include glycosylamines, sugar phosphates, and sugar esters. Other saccharide derivatives include, for example, beta-D-glucuronyl, D-galactosyl, and D-glucosyl.

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

[0092] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight-chain (i.e., unbranched) or branched carbon chain (or carbons), or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and may include monovalent, divalent, and polyvalent radicals. An alkyl can contain a specified number of carbons (e.g., C1-C 10 means 1-10 carbons). An alkyl is a non-cyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those 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 homologs and isomers. An alkoxy is an alkyl that is attached to the remainder of the molecule via an oxygen linker (-O-). The alkyl moiety may be an alkenyl moiety. The alkyl moiety may be an alkynyl moiety. The alkyl moiety may be fully saturated. The alkenyl may contain more than one double bond and / or one or more triple bonds in addition to one or more double bonds. The alkynyl may contain more than one triple bond and / or one or more double bonds in addition to one or more triple bonds.

[0093] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited to, by -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred herein. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having 8 or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0094] The term "heteroalkyl," alone or in combination with another term, means, unless otherwise stated, a stable linear or branched chain, or any combination thereof, containing at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, or S), in which 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 placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is a non-cyclized 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). A heteroalkyl moiety may contain two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may contain three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may contain four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may contain five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may contain up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P). The term "heteroalkenyl," alone or in combination with another term, means a heteroalkyl containing at least one double bond, unless otherwise stated.Heteroalkenyl may optionally contain more than one double bond, and / or one or more triple bonds in addition to one or more double bonds. The term "heteroalkynyl," alone or in combination with another term, means a heteroalkyl containing at least one triple bond, unless otherwise stated. Heteroalkynyl may optionally contain more than one triple bond, and / or one or more double bonds in addition to one or more triple bonds.

[0095] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from a heteroalkyl, as exemplified by, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As explained above, heteroalkyl groups, as used herein, include groups that are attached to the remainder of the molecule by a heteroatom, e.g., -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'. When "heteroalkyl" is mentioned followed by a specific heteroalkyl group, e.g., -NR'R'', etc., it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is mentioned to add clarity. Thus, the term "heteroalkyl" should not be construed herein to exclude specific heteroalkyl groups, e.g., -NR'R'', etc.

[0096] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, mean cyclic versions of "alkyl" and "heteroalkyl", respectively, unless otherwise stated. Cycloalkyls and heterocycloalkyls are not aromatic. Additionally, for heterocycloalkyls, a heteroatom may occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyls include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0097] In embodiments, the term "cycloalkyl" refers to a monocyclic, bicyclic, or polycyclic cycloalkyl ring system. In embodiments, a monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, which may be saturated or unsaturated, but is not aromatic. In embodiments, a cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. A bicyclic heteroalkyl ring system is a bridged monocyclic ring or a fused bicyclic ring. In embodiments, a bridged monocyclic ring is one in which two non-adjacent carbon atoms of a monocyclic ring are joined by an alkylene bridge of between 1 and 3 additional carbon atoms (i.e., of the form (CH2)). wwhere w is 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, the fused bicyclic cycloalkyl ring system contains a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety by any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, the cycloalkyl group is optionally substituted with one or two groups that are independently oxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5- or 6-membered monocyclic cycloalkyl ring fused to either a phenyl 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, where the fused bicyclic cycloalkyl is optionally substituted with one or two groups, each independently being oxo or thia. In embodiments, the polycyclic cycloalkyl ring system is a monocyclic cycloalkyl ring (base ring) fused to either (i) a ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two other ring systems, independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. In embodiments, a polycyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.In embodiments, the polycyclic cycloalkyl ring system is a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two other ring systems independently selected from the group consisting of phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl, and perhydrophenoxazin-1-yl.

[0098] In embodiments, cycloalkyl is cycloalkenyl. The term "cycloalkenyl" is used according to its plain and ordinary meaning. In embodiments, cycloalkenyl is a monocyclic, bicyclic, or polycyclic cycloalkenyl ring system. In embodiments, a monocyclic cycloalkenyl ring system is a cyclic hydrocarbon group containing 3-8 carbon atoms, said group being unsaturated (i.e., containing at least one cyclic carbon-carbon double bond) but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, a bicyclic cycloalkenyl ring is a bridged monocyclic ring or a fused bicyclic ring. In embodiments, a bridged monocyclic ring is a ring in which two non-adjacent carbon atoms of a monocyclic ring are joined by an alkylene bridge of between 1-3 additional carbon atoms (i.e., of the form (CH2) wwhere w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct2enyl. In embodiments, the fused bicyclic cycloalkenyl ring system contains a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety by any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, the cycloalkenyl group is optionally substituted with one or two groups that are independently oxo or thia. In embodiments, the polycyclic cycloalkenyl ring contains a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two ring systems independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. In embodiments, the polycyclic cycloalkenyl is attached to the parent molecular moiety by any carbon atom contained within the base ring. In embodiments, the polycyclic cycloalkenyl ring contains a monocyclic cycloalkenyl ring (base ring) fused to either: (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of phenyl, monocyclic heteroaryl, monocyclic cycloalkyl, monocyclic cycloalkenyl, and monocyclic heterocyclyl.

[0099] In an embodiment, heterocycloalkyl is heterocyclyl. The term "heterocyclyl" as used herein means a monocyclic, bicyclic, or polycyclic heterocycle. A heterocyclyl monocyclic heterocycle is a 3-, 4-, 5-, 6-, or 7-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S, which is saturated or unsaturated, but not aromatic. A 3- or 4-membered ring contains one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring can contain zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6- or 7-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety by any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include 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, and oxazolinyl. Heterocyclyl bicyclic heterocycles include, but are not limited to, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. Heterocyclyl bicyclic heterocycles are monocyclic heterocycles fused with either phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocycle, or monocyclic heteroaryl. Heterocyclyl bicyclic heterocycles are connected to the parent molecular moiety by any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments, the heterocyclyl group is optionally substituted with one or two groups that are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5- or 6-membered monocyclic heterocyclyl ring fused to a phenyl 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 bicyclic heterocyclyl is optionally substituted with one or two groups that are independently oxo or thia. A polycyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two other ring systems independently selected from the group consisting of phenyl, bicyclic aryl, monocyclic or bicyclic heteroaryl, monocyclic or bicyclic cycloalkyl, monocyclic or bicyclic cycloalkenyl, and monocyclic or bicyclic heterocyclyl. The polycyclic heterocyclyl is attached to the parent molecular moiety by any carbon or nitrogen atom contained within the base ring. In embodiments, the polycyclic heterocyclyl ring system is a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of bicyclic aryl, bicyclic heteroaryl, bicyclic cycloalkyl, bicyclic cycloalkenyl, and bicyclic heterocyclyl, or (ii) two other ring systems independently selected from the group consisting of 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] The terms "halo" or "halogen," alone or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, 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, and the like.

[0101] The term "acyl," unless otherwise stated, means -C(O)R, in which 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] The term "aryl", unless otherwise stated, refers to a polyunsaturated, aromatic, hydrocarbon substituent that may be a single ring or multiple rings (preferably 1-3 rings) that are fused together (i.e., fused-ring aryl) or covalently linked together. Fused-ring aryl refers to multiple rings fused together, where at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) that contains at least one heteroatom, e.g., N, O, or S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., multiple rings fused together, where at least one of the fused rings is a heteroaromatic ring). 5,6-fused-ring heteroarylene refers to two rings fused together, where one ring has 5 members and the other ring has 6 members, and where at least one ring is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, one having 6 members and the other having 6 members, with at least one ring being a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, one having 6 members and the other having 5 members, with at least one ring being a heteroaryl ring. The heteroaryl group may be attached to the remainder of the molecule by a carbon or a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5 ... Examples of the aryl and heteroaryl ring systems include isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene," alone or as part of another substituent, refer to a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent can be -O- attached to a nitrogen ring heteroatom.

[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 can each independently be unsubstituted or substituted with one or more of the substituents described herein.

[0104] A spirocyclic ring is two or more rings in which adjacent rings are connected by a single atom. The individual rings in a spirocyclic ring can be the same or different. The individual rings in a spirocyclic ring can be substituted or unsubstituted and can have different substituents than the other individual rings in a set of spirocyclic rings. The possible substituents for the individual rings in a spirocyclic ring are the possible substituents for the same ring when not part of a spirocyclic ring (e.g., the substituents for a cycloalkyl or heterocycloalkyl ring). A spirocyclic ring can be a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene, and the individual rings in a spirocyclic ring group can be any of the immediately preceding lists, including those with all rings of one type (e.g., all rings that are substituted heterocycloalkylene, where each ring can be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, a heterocyclic spirocyclic ring means a spirocyclic ring in which at least one ring is a heterocyclic ring and each ring can be a different ring. When referring to a spirocyclic ring system, a substituted spirocyclic ring means that at least one ring is substituted and that each substituent can be optionally different.

[0105] symbol [ka] (wavy line) indicates the point of attachment of the chemical moiety to the remainder of the molecule or chemical formula.

[0106] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0107] The term "alkylsulfonyl" as used herein means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' can have a specified number of carbons (e.g., "C1-C4 alkylsulfonyl").

[0108] The term "alkylarylene" refers to an arylene moiety that is covalently linked to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, an alkylarylene group has the formula [ka] has.

[0109] The alkylarylene moiety may be substituted (e.g., at carbons 2, 3, 4, or 6) at the alkylene portion or the arylene linker with a substituent (e.g., halogen, oxo, -N3, -CF3, -CCl3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3-SO3H, -OSO3H, -SON2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, substituted or unsubstituted C1-C5 alkyl, or substituted or unsubstituted 2-5 membered heteroalkyl). In an embodiment, the alkylarylene is unsubstituted.

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

[0111] Substituents for alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are selected from zero to (2m'+1) in number: -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR'' R may be one or more of a variety of groups selected from, but not limited to, R''', -NR''C(O)R', -NR-C(NR'R''R'')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)R', -S(O)NR'R'', -NRSO2R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO2, -NR'SOR'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', where m' is the total number of carbon atoms in such radical. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When the compounds described herein include more than one R group, for example, each of the R groups is independently selected, and each R', R'', R''', and R'''' group is also independently selected when more than one of these groups is present. When R' and R'' are attached to the same nitrogen atom, they can combine 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, it will be understood by those of skill in the art that the term "alkyl" is intended to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF and -CHCF) and acyl (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0112] Similar to the substituents described for the alkyl radicals, the substituents for the aryl and heteroaryl groups are varied and include, for example, -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -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' in a number ranging from zero to the total number of open valences in the aromatic ring system. , -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'SOR'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', where 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 include more than one R group, for example, each of the R groups is independently selected, and each R', R'', R''', and R'''' group is also independently selected when more than one of these groups is present.

[0113] Substituents for a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as a substituent on the ring rather than on a specific atom of the ring (commonly referred to as a floating substituent). In such cases, the substituent may be attached (according to the rules of chemical valence) to any of the ring atoms, and in the case of a fused or spirocyclic ring, a substituent depicted as associated with one member of the fused or spirocyclic ring (a floating substituent on a single ring) may also be a substituent on any of the fused or spirocyclic rings (floating substituents on multiple rings). When a substituent is attached to a ring rather than to a specific atom (floating substituent), and the substituent subscript is an integer greater than 1, multiple substituents may be present on the same atom, the same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may be different, as appropriate. If the point of attachment of the ring to the rest of the molecule is not limited to a single atom (in the case of a floating substituent), the point of attachment may be any atom of the ring, or, in the case of a fused or spirocyclic ring, any atom of either the fused or spirocyclic ring, subject to the rules of chemical valence. If a ring, fused or spirocyclic ring contains one or more ring heteroatoms and the ring, fused or spirocyclic ring is shown with another floating substituent (including, but not limited to, the point of attachment to the rest of the molecule), the floating substituent may be attached to the heteroatom. If a ring heteroatom is shown attached to one or more hydrogens in a structure or formula with a floating substituent (e.g., a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen), it will be understood that when the heteroatom is attached to the floating substituent, the substituent is replaced with a hydrogen, subject to the rules of chemical valence.

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

[0115] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring optionally have the formula -TC(O)-(CRR') p -U-, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and p is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a ring of the formula -A-(CH2) r A and B can be optionally replaced with a substituent of the formula -B-, where 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 so formed can be optionally replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring can be optionally replaced with a substituent of the formula -(CRR') s -X'-(C''R''R'') d-, where 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 meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si).

[0117] "Substituent," as used herein, means a group selected from the following moieties: (A) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -S O4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI 2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl), and (B) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl, aryl, or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), which are substituted with at least one substituent selected from (i) and (ii) below: (i) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -S O4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI 2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl), and (ii) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10 aryl, aryl, or phenyl), heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), which are substituted with at least one substituent selected from (a) and (b) below: (a) Oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -S O4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI 2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl), and (b) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), aryl (e.g., C6-C 10 Aryl, C 10aryl, or phenyl), heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl) substituted with at least one substituent selected from the following: oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NH OH, -OCCl3, -OCF3, -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., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6 ... 10 Aryl, C 10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl).

[0118] A "size-limited substituent" or "size-limited substituent group" as used herein refers to a group in which each substituted or unsubstituted alkyl is substituted or unsubstituted. C1~C 20each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-20 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 3-8 membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C8 10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5-10 membered heteroaryl, meaning a group selected from all of the substituents listed above for "substituents."

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

[0120] In some embodiments, each substituted group described herein for compounds is substituted with at least one substituent. 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 herein for compounds is substituted with at least one substituent. In other embodiments, at least one or all of these groups are substituted with at least one size-limiting substituent. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent.

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

[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, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6 ... 10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5-9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2-8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-7 membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6 ...aryl is a substituted or unsubstituted 5-9 membered heteroaryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5-9 membered heteroaryl. 10 and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 9-membered heteroarylene. In some embodiments, the compound is a species shown in the Examples section below, in a figure, or in a table.

[0123] In embodiments, a 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, a 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 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 substituent, and when a substituted moiety is substituted with multiple substituents, each substituent can be optionally different. In embodiments, when a substituted moiety is substituted with multiple substituents, 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-limiting substituent, and when a substituted moiety is substituted with multiple size-limiting substituents, each size-limiting substituent can optionally be different. In embodiments, when a substituted moiety is substituted with multiple size-limiting substituents, each size-limiting substituent is different.

[0126] 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 lower substituent, and when a substituted moiety is substituted with multiple lower substituents, each lower substituent can optionally be different. In embodiments, when a substituted moiety is substituted with multiple lower substituents, each lower substituent is different.

[0127] In embodiments, a substituted moiety (e.g., a 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, size-limiting substituent, or lower-class substituent; when a substituted moiety is substituted with multiple groups selected from a substituent, a size-limiting substituent, and a lower-class substituent, each of the substituents, size-limiting substituents, and / or lower-class substituents can be different, if desired. In embodiments, when a substituted moiety is substituted with multiple groups selected from a substituent, a size-limiting substituent, and a lower-class substituent, each of the substituents, size-limiting substituents, and / or lower-class substituents 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 in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids as (D)- or (L)-), and individual isomers are encompassed 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 synthesize and / or isolate. The present disclosure is intended to include compounds in racemic and optically pure form. Optically active (R)- and (S)-isomers, or (D)- and (L)-isomers, can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. Where the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

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

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

[0131] It will be apparent to one of ordinary skill in the art that certain compounds of the present disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the present disclosure.

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

[0133] It should be noted that throughout this application, alternatives, e.g., each amino acid position that contains more than one possible amino acid, are described in terms of a Markush group. It is specifically contemplated that each member of a Markush group should be considered separately and thus constitutes a separate embodiment, and that a Markush group should not be read as a single unit.

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

[0135] An "amino acid unit" has the formula [ka] wherein R 0is 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 may also be referred to by its standard three letter abbreviation for an amino acid (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 moiety." The association can be direct or indirect. For example, conjugates of a first bioconjugate reactive group (e.g., -NH2, -C(O)OH, -N-hydroxysuccinimide, or -maleimide) with a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine side chain-containing amino acid, or carboxylate) provided herein can be direct, e.g., via a covalent bond or linker (e.g., a first linker or a second linker), or indirect, e.g., via a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions, etc.). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e., the association of two bioconjugate reactive groups), including, but not limited to, nucleophilic substitution (e.g., reaction of an amine with an alcohol bearing an acyl halide that is an active ester), electrophilic substitution (e.g., enamine reaction), and addition to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).These and other useful reactions are discussed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., 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, a first bioconjugate reactive group (e.g., a maleimide moiety) is covalently attached to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a haloacetyl moiety) is covalently attached to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a pyridyl moiety) is covalently attached to a second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, a first bioconjugate reactive group (e.g., a -N-hydroxysuccinimide moiety) is covalently attached to a second bioconjugate reactive group (e.g., an amine). In embodiments, a first bioconjugate reactive group (e.g., a fluorophenyl ester moiety) reacts with a second bioconjugate reactive group (e.g., an amine) to form a covalent bond. In embodiments, a first bioconjugate reactive group (e.g., a -sulfo-N-hydroxysuccinimide moiety) reacts with a second bioconjugate reactive group (e.g., an amine) to form a covalent bond.

[0137] Useful bioconjugate reactive moieties for use in the bioconjugate chemistry herein include, for example, the following: (a) the carboxyl group and its various derivatives (including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters); (b) a hydroxyl group, which can be converted to an ester, ether, aldehyde, etc. (c) haloalkyl groups, which can subsequently displace the halide with a nucleophilic group, such as an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the position of the halogen atom; (d) dienophile groups capable of participating in a Diels-Alder reaction, such as maleimido or maleimide groups; (e) aldehyde or ketone groups which can be subsequently derivatized by formation of carbonyl derivatives, such as imines, hydrazones, semicarbazones or oximes, or by mechanisms such as Grignard 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 conjugated to metals such as gold, or reacted with maleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized; (i) alkenes, which can undergo, for example, cycloaddition, acylation, Michael addition, etc.; (j) epoxides, which can react, for example, with amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (l) metal silicon oxide bond; and (m) The attachment of a metal to a reactive phosphorous group (e.g., a phosphine) to form, for example, a phosphodiester bond. (n) Azides coupled with alkynes using copper-catalyzed cycloaddition click chemistry. (o) A biotin conjugate that can react with avidin or streptavidin to form an avidin-biotin complex or a streptavidin-biotin complex.

[0138] Bioconjugate reactive groups can be selected such that they do not participate in or interfere with the chemical stability of the conjugates described herein. Alternatively, reactive functional groups can be protected from participating in crosslinking reactions by the presence of protecting groups. In embodiments, bioconjugates include molecular entities derived from the reaction of unsaturated bonds, such as maleimide and sulfhydryl groups.

[0139] "Analog" or "analogous" is used according to its plain and ordinary meaning in chemistry and biology to refer to a chemical compound that is structurally similar to another compound (i.e., a so-called "reference" compound) but differs 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 equivalent in function and appearance to the reference compound, but not in structure or origin.

[0140] As used herein, common organism and cell type abbreviations are defined as follows: Ac Acetyl Ala Alanine Asn Asparagine aq. Water-based β-Ala Beta-Alanine ℃ Temperature in degrees Celsius Cit 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 grams Gly Glycine h hour(singular) (hour(s)) 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 Lymphocyte Lys Lysine Me Methyl mg milligram MeOH Methanol mL Milliliters μL / μL microliter MONO Monocyte mol mmol μmol / umol micromol MS Mass Spectroscopy NHS N-Hydroxysuccinimide NEUT neutrophil PABC p-aminobenzyloxycarbonyl Phe phenylalanine PLT Platelets RP-HPLC Reversed Phase HPLC rt room temperature Ser Serin t-Bu tert-Butyl Tert, t Tertiary TFA Trifluoroacetic acid Thr Threonine Val Balin 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 that covalently attaches the monoclonal antibody to the drug moiety.

[0142] In another embodiment, an antibody drug conjugate (ADC) of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, provided herein, Ab is a monoclonal antibody; m is an integer from 1 to 8; L 1 is a linker attached to the monoclonal antibody; L 2 is the bond, -C(O)-, -NH-, and the amino acid unit, -(CH2CH2O) n -, -(CH2) n -, -(4-aminobenzyloxycarbonyl)-, -(C(O)CHCHNH)-, or any combination thereof, where n is an integer from 1 to 24; D is a drug moiety.

[0143] In another embodiment, an antibody drug conjugate (ADC) of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, provided herein, Ab is anti-FOLR1 antibody; m is an integer from 1 to 8; L 1 is a linker attached to the anti-FOLR1 antibody; L 2 is the bond, -C(O)-, -NH-, and the amino acid unit, -(CH2CH2O) n -, -(CH2) n-, -(4-aminobenzyloxycarbonyl)-, -(C(O)CHCHNH)-, or any combination thereof, where n is an integer from 1 to 24; D is a 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 an embodiment, n is an integer from 1 to 24. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6. In an embodiment, n is 7. In an embodiment, n is 8. In an embodiment, n is 9. In an embodiment, n is 10. In an embodiment, n is 11. In an embodiment, n is 12. In an embodiment, n is 13. In an embodiment, n is 14. In an embodiment, n is 15. In an embodiment, n is 16. In an embodiment, n is 17. In an embodiment, n is 18. In an embodiment, n is 19. In an embodiment, n is 20. In an embodiment, n is 21. In an embodiment, n is 22. In an embodiment, n is 23. In an embodiment, n is 24.

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

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

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

[0149] In an embodiment, L 1 teeth, [ka] It is.

[0150] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] In an embodiment, L 1 teeth, [ka] It is.

[0151] L 1 but, [ka] In the case where L is a 2-CH2 moiety shown on the right side of the structure, each of the two CH2 moieties may be attached to a separate sulfur of the anti-FOLR1 antibody. 1 but, [ka] In the example, the two alkene carbons shown at the bottom of the structure may each be attached to a separate sulfur in the anti-FOLR1 antibody.

[0152] L 1 but, [ka] where the carbonyl is attached to an amine of a lysine of the anti-FOLR1 antibody.

[0153] In an embodiment, D is [ka] It is.

[0154] In an embodiment, D is [ka] and R 1 is H or -C1-C8 alkyl; R 3 is 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 , -(O)NHSO2R 7 , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R 4 is 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 a 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(CHCHO) w CH2CH2Y, -CONH(CH2CH2O) w CH2CH2Y, [ka] a charged group, or a polysaccharide derivative; v is an integer from 1 to 24; w is an integer from 1 to 24; Y is -NH2, -OH, -COOH, or -OCH3; R 10 is -OH, -OCH3 or -COOH; R 7 are independently H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; Each R 3A , R 3B , R 4A , and R 4B is independently H or substituted or unsubstituted alkyl.

[0155] In an embodiment, L 2 are the bonds -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 is the bond, -C(O)-, -NH-, -Val-, -Phe-, -Lys-, -(4-aminobenzyloxycarbonyl)-, -(CH2) n -, -(CH2CH2O) n -, or any combination thereof.

[0157] In an embodiment, L 2 is the 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 is a bond, -C(O)-, -NH-, Val, Gly, Lys, Cit, -(CH2) n -, -(CH2CH2O) n -, or any combination thereof.

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

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

[0161] In an embodiment, L 2 In embodiments, L comprises or is a bond. 2 In embodiments, L comprises or is -C(O)-. 2 In embodiments, L comprises or is -NH-. 2 In an embodiment, L comprises or is -Val-. 2 In embodiments, L comprises or is -Phe-.2 In an embodiment, L comprises or is -Lys-. 2 In embodiments, L comprises or is -(4-aminobenzyloxycarbonyl)-. 2 is -(CH2) n In an embodiment, L 2 is -(CH2CH2O) n In an embodiment, L 2 In an embodiment, L comprises or is -Gly-. 2 In embodiments, L comprises or is -Ser-. 2 In embodiments, L comprises or is -Thr-. 2 In embodiments, L comprises or is -Ala-. 2 In an embodiment, L comprises or is -β-Ala-. 2 contains or is -Cit-.

[0162] In an embodiment, -L 1 -L 2 -teeth, [ka] It is.

[0163] In an embodiment, -L 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] and each of the two CH2 moieties shown on the left side of the structure may be attached to a separate sulfur of the anti-FOLR1 antibody. 1 -L 2 -teeth, [ka] In embodiments, the two CH2 moieties shown on the left side of the structure may each be attached to a separate sulfur of the anti-FOLR1 antibody. 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] and the two alkene carbons shown at the bottom of the structure may each be attached to a separate sulfur in the anti-FOLR1 antibody. 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] and each of the two CH2 moieties shown on the left side of the structure may be attached to a separate sulfur of the anti-FOLR1 antibody. 1 -L 2 -teeth, [ka] In an embodiment, -L1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] In an embodiment, -L 1 -L 2 -teeth, [ka] It is.

[0164] In embodiments, R 1 is H. In embodiments, R 1 is -C1 to C8 alkyl.

[0165] In embodiments, R 1 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl. 1 is methyl. In embodiments, R 1 is ethyl. In embodiments, R 1 is propyl. In embodiments, R 1 is isopropyl. In embodiments, R 1 is butyl. In an embodiment, R 1 is isobutyl. In embodiments, R 1 is tert-butyl. In embodiments, R1 is pentyl. In embodiments, R 1 is hexyl.

[0166] In embodiments, R 3 is 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 , -(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., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl).

[0167] In embodiments, R 3 H, -OR 3A , -(CH2) v OR 6 , -(O)NHSO2R 7 , substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) 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, size-limiting substituent, or lower substituent) or unsubstituted heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl).

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

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

[0170] In embodiments, R 3 are methyl, ethyl, propyl, butyl, -CH2OH, -CH2CH2OH, -CH2N3, -CH2CH2N3, -CH2OCH3, -CH2OCH2CH3, -CH2CH2OCH3, -CH2CH2OCH2CH3, [ka] In an embodiment, R 3 is methyl. In embodiments, R 3 is ethyl. In embodiments, R 3 is propyl. In embodiments, R 3 is butyl. In an embodiment, R 3 is -CHOH. In embodiments, R 3 is -CHCHOH. In embodiments, R 3 is -CH2N3. In embodiments, R3 is -CHCHN. 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 is -OH. In embodiments, R 3 is H. In embodiments, R 3 teeth, [ka] In an embodiment, R 3 teeth, [ka] It is.

[0171] In embodiments, R 3 is methyl, -CH2OH, [ka] or -CH2N3. In embodiments, R 3 teeth, [ka] , or -CH2N3.

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

[0173] In embodiments, R 4 is 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., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl).

[0174] In embodiments, R 4 H, -OR 4A , substituted (e.g., substituted with at least one substituent, size-limiting substituent, or lower substituent) 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, size-limiting substituent, or lower substituent) or unsubstituted heteroalkyl (e.g., 2-8 membered heteroalkyl, 2-6 membered heteroalkyl, or 2-4 membered heteroalkyl).

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

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

[0177] In embodiments, R 4 is H or -OH.

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

[0179] In an embodiment, Z 1 teeth, [ka] 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 embodiments, q is 1. In embodiments, q is 2. In embodiments, q is 3. In embodiments, q is 4. In embodiments, q is 5.

[0181] In embodiments, X is Cl. In embodiments, X is Br. In embodiments, X is I. In embodiments, 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 teeth, [ka] In an embodiment, Z 1 teeth, [ka] In an embodiment, Z 1 teeth, [ka] In an embodiment, Z 1 teeth, [ka] It is.

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

[0185] In an embodiment, Z 2 teeth, [ka] wherein 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 embodiments, p is 0. In embodiments, p is 1. In embodiments, p is 2. In embodiments, p is 3. In embodiments, 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 teeth, [ka] In an embodiment, Z 2 teeth, [ka] In an embodiment, Z 2 teeth, [ka] In an embodiment, Z 2 teeth, [ka] It is.

[0189] In embodiments, 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(CHCHO) w CH2CH2Y, -CONH(CH2CH2O) w CH2CH2Y, [ka] , a charged group, or a polysaccharide derivative; w is an integer from 1 to 24; Y is -NH2, -OH, -COOH, or -OCH3; R 10 is -OH, -OCH3 or -COOH.

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

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

[0192] In embodiments, R 6 is H or substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heterocycloalkyl (e.g., a 3-8 membered heterocycloalkyl, a 3-6 membered heterocycloalkyl, or a 5-6 membered heterocycloalkyl).

[0193] In embodiments, R 6 is H or [ka] It is.

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

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

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

[0197] In embodiments, R 6 teeth, [ka] In an embodiment, R 6 teeth, [ka] In an embodiment, R 6 teeth, [ka] In an embodiment, R 6 teeth, [ka] It is.

[0198] In embodiments, R 6 is a saccharide derivative. In embodiments, R 6 teeth, [ka] In an embodiment, R 6 teeth, [ka] In an embodiment, R 6 teeth, [ka] It is.

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

[0200] In embodiments, R 7 is H or substituted (e.g., substituted with at least one substituent, size-limited substituent, or lower substituent) 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, size-limited substituent, or lower substituent) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl).

[0201] In embodiments, R7 is H, substituted (e.g., substituted with a substituent, a size-limited substituent, or a lower substituent) or unsubstituted cycloalkyl (e.g., 3-8 membered cycloalkyl, 3-6 membered cycloalkyl, or 5-6 membered cycloalkyl). 7 is a substituted (e.g., substituted, size-limited, or lower substituted) cycloalkyl (e.g., 3-8 membered cycloalkyl, 3-6 membered cycloalkyl, or 5-6 membered cycloalkyl). 7 is an unsubstituted cycloalkyl (eg, a 3- to 8-membered cycloalkyl, a 3- to 6-membered cycloalkyl, or a 5- to 6-membered cycloalkyl).

[0202] In embodiments, R 7 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. 7 is cyclopropyl. In embodiments, R 7 is cyclobutyl. In embodiments, R 7 is cyclopentyl. In embodiments, R 7 is cyclohexyl. In embodiments, R 7 is cycloheptyl.

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

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

[0205] In an embodiment, each R 3A , R 3B , R 4A , and R 4B 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 embodiments, each R 3A , R 3B , R 4A , and R 4B is independently ethyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently propyl. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently isopropyl. In embodiments, each R 3A , R 3B , R4A , and R 4B is independently butyl. 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. In embodiments, each R 3A , R 3B , R 4A , and R 4B is independently pentyl.

[0206] In an embodiment, D is [ka] It is.

[0207] In an embodiment, D is [ka] It is.

[0208] In embodiments, an ADC (e.g., an anti-FOLR1 ADC) comprises: [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein m is an integer of 1 to 8. Drug burden

[0209] Drug loading is represented by m, the average number of drug moieties (i.e., D) per monoclonal antibody in an antibody drug conjugate (ADC) of formula (I) or variants thereof. Drug loading can range from 1 to 20 drug moieties per antibody. An ADC of formula (I), and any embodiment, variant, or aspect thereof, includes a collection of antibodies conjugated with drug moieties ranging from 1 to 20, such as 1 to 8. The average number of drug moieties per antibody in a preparation of ADCs from a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution in the ADC in terms of m can also be determined. In some cases, separation, purification, and characterization of a homogeneous ADC with a certain value of m 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 embodiments, the average number of drug moieties (ie, D) per anti-FOLR1 antibody can range from 1 to 20, 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, if the linkage is a cysteine ​​thiol, as in some 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 to which a linker can be attached. In embodiments, the average drug loading of the ADC ranges from 1 to about 8, or from about 2 to about 8. In embodiments, L 1 can form a covalent bond with the thiol group of a free cysteine ​​in an IgG antibody. 1 can form covalent bonds with the amine groups of lysines in IgG antibodies.

[0211] In embodiments, less than the theoretical maximum amount of drug moiety is conjugated to antibody during conjugation reaction.Generally, antibody does not contain many free and reactive cysteine ​​thiol groups that can be linked to drug moiety, and in fact, most cysteine ​​thiol residues in antibody are present as disulfide bridges.In embodiments, antibody is reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups.In embodiments, antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

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

[0213] In embodiments, antibody drug conjugates are provided herein, in which the linker moiety reacts in a controlled manner with either cysteine ​​or lysine on the antibody.In embodiments, the drug antibody ratio (DAR) for conjugation with antibody lysine is 2 for the majority of conjugates.In embodiments, the drug antibody ratio (DAR) for conjugation with antibody cysteine ​​is 4 for the majority of conjugates.

[0214] It is understood that if 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 that is both antibody-specific and drug-specific. Individual ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; 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, Volume 45, March 2004; Alley, SC, et al. "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, (See, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004.) In embodiments, homogenous ADCs having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.In embodiments, enhanced selectivity for lysine may result in a less heterogeneous mixture (US Pat. No. 9,981,046).

[0215] Anti-FOLR1 antibody The disclosure of WO2020 / 016661 is incorporated by reference in its entirety into this specification. i. Exemplary Antibodies and Antibody Sequences In embodiments, the ADC comprises an antibody that binds to FOLR1, which has been reported to be overexpressed in tumors of epithelial origin, including ovarian, uterine, breast, endometrial, pancreatic, renal, lung, colorectal and brain tumors.

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

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

[0218] In embodiments, the ADCs provided herein include anti-FOLR1 antibodies 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 embodiments, the ADCs provided herein comprise 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: 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 embodiments, the ADCs comprise an anti-FOLR1 antibody comprising at least one CDR selected from (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 embodiments, 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: 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least three CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least four CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least five CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least six CDRs selected from (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 embodiments, the ADCs provided herein comprise 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 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 embodiments, the ADCs comprise 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 embodiments, 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least three 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least four 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least five 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least six 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.

[0221] In embodiments, the ADCs provided herein comprise 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 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 embodiments, the ADCs comprise 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 embodiments, 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least three 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least four 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least five 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising at least six 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.

[0222] In embodiments, the ADC comprises an anti-FOLR1 antibody comprising one CDR selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising two CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising three CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising four CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising five CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising six CDRs selected from (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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising three 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising four 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising five 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising six 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.

[0224] In embodiments, the ADC comprises an anti-FOLR1 antibody comprising 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising three 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising four 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising five 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 embodiments, the ADC comprises an anti-FOLR1 antibody comprising six 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.

[0225] In an embodiment, the 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, the anti-FOLR1 antibody comprises a VL CDR1 comprising the sequence of SEQ ID NO:1. In an embodiment, the anti-FOLR1 antibody comprises a VL CDR2 comprising the sequence of SEQ ID NO:2. In an embodiment, the anti-FOLR1 antibody comprises a VL CDR3 comprising the sequence of SEQ ID NO:3. In an embodiment, the anti-FOLR1 antibody comprises a VH CDR1 comprising the sequence of SEQ ID NO:4. In an embodiment, the 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, the anti-FOLR1 antibody comprises 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. In an embodiment, the anti-FOLR1 antibody comprises a VL CDR1 comprising the sequence of SEQ ID NO: 12. In an embodiment, the anti-FOLR1 antibody comprises a VL CDR2 comprising the sequence of SEQ ID NO: 13. In an embodiment, the anti-FOLR1 antibody comprises a VL CDR3 comprising the sequence of SEQ ID NO: 14. In an embodiment, the anti-FOLR1 antibody comprises a VH CDR1 comprising the sequence of SEQ ID NO: 15. In an embodiment, the anti-FOLR1 antibody comprises a VH CDR2 comprising the sequence of SEQ ID NO: 16. In an embodiment, the anti-FOLR1 antibody comprises a VH CDR3 comprising the sequence of SEQ ID NO: 17.

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

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

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

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

[0231] In an embodiment, the anti-FOLR1 antibody comprises a VL having a sequence with 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. In an embodiment, the anti-FOLR1 antibody comprises a VL having a sequence of SEQ ID NO:7. In an embodiment, the VL sequence with 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 compared 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 1-10 amino acids are 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 embodiments, a total of 1-5 amino acids are 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 embodiments, the substitutions, insertions or deletions are made in regions outside the CDRs (i.e., in the FRs). In embodiments, the anti-FOLR1 antibody comprises a 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, including post-translational modifications of that sequence.

[0232] In an embodiment, the anti-FOLR1 antibody comprises a VH having a sequence with at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28. In an embodiment, the anti-FOLR1 antibody comprises a VH having a sequence with SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28. In an embodiment, the VH sequence with at least 95%, 96%, 97%, 98% or 99% identity 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 compared 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 1-10 amino acids are 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, a total of 1-5 amino acids are substituted, inserted and / or deleted in SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28. In embodiments, the substitutions, insertions or deletions are made in regions outside the CDRs (i.e., in the FRs). In embodiments, the anti-FOLR1 antibody comprises a VH sequence of SEQ ID NO:8, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28, including post-translational modifications 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, the 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 CDRs as in any of the above embodiments, and further comprises a human acceptor framework, for example, 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 CDRs as in any of the above embodiments, and further comprises a human acceptor framework, for example, 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 CDRs as in any of the above embodiments, and further comprises a human acceptor framework, for example, 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 embodiments, 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 a chimeric, humanized or human antibody. In one embodiment, the anti-FOLR1 antibody is an antibody fragment, such as an Fv, Fab, 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 antibody provided herein binds 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 antibody binds 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 skilled in the art. For example, whether an anti-FOLR1 antibody "binds with an affinity of" ≦10 nM, or ≦5 nM, or ≦4 nM, or ≦3 nM, or ≦2 nM can be determined using standard Scatchard analysis utilizing a nonlinear curve fitting program (see, for example, Munson et al., Anal Biochem, 107: 220-239, 1980).

[0241] In embodiments, the anti-FOLR1 antibodies provided herein have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM, and optionally a dissociation constant of ≧10 -13 M. (For example, 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10-13 M).

[0242] In embodiments, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen, as illustrated by the following assay: The solution binding affinity of the Fab for the antigen is measured by binding the Fab to a minimal concentration of ( 125 I) Equilibration with labeled antigen and then measurement by capturing bound antigen on anti-Fab antibody-coated plates (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23° C.). In non-absorbent plates (Nunc #269620), 100 pM or 26 pM of [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., according to the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight. However, this incubation may continue for a longer period (e.g., up to about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20™; Packard) is added and the plate is counted in a TOPCOUNT™ gamma counter (Packard) for 10 minutes. Concentrations of each Fab that result in binding less than or equal to 20% of maximal binding are selected for use in competitive binding assays.

[0243] According to another embodiment, Kd is measured using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25° C. using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. with a flow rate of approximately 25 μl / min. Association rates (kon) and dissociation rates (koff) are calculated by simultaneous fitting of association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). Equilibrium dissociation constants (Kd) are calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). The on-rate of 106 M by the surface plasmon resonance assay method described above was 1.0 μg / min. -1 s -1Above 100 nm, the on-rate can be measured by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band pass 16 nm) of 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 with a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer with stirred cuvette (ThermoSpectronic). iii.Antibody fragment

[0244] In embodiments, the anti-FOLR1 antibody provided herein is an antibody fragment. 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. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), and also see WO93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see US Pat. No. 5,869,046.

[0245] Diabodies are antibody fragments that have two antigen-binding sites, and can be bivalent or bispecific.See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0246] A single domain antibody is an antibody fragment that contains 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, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see also, e.g., U.S. Patent No. 6,248,516 B1).

[0247] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells (e.g., E. coli or phages), as described herein. iv. Chimeric and Humanized Antibodies

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

[0249] In an embodiment, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, in which the HVR, e.g., CDR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody will also optionally comprise at least a portion of a human constant region. In an embodiment, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0250] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0251] Human framework regions that may 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. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996). v. Human antibodies

[0252] In embodiments, the anti-FOLR1 antibody provided herein is a human antibody.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. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0253] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals usually have all or part of the human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are extrachromosomal or randomly integrated into the animal's chromosomes. The endogenous immunoglobulin loci in such transgenic mice are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with a different human constant region.

[0254] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 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., 147: 86 (1991).) Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods are described, for example, in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0255] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below. vi. Library-derived antibodies

[0256] In embodiments, the anti-FOLR1 antibody provided herein is derived from an antibody library. The antibody can be isolated by screening a combinatorial library for an antibody with a desired activity or activities. For example, various methods are known in the art for generating a phage display library and screening such a library for an antibody with a desired binding property. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0257] In the phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without any immunization to provide a single source of antibodies against a wide range of non-self and also self antigens, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode hypervariable CDR3 regions, and performing rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication 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 a human antibody library are considered herein to be human antibodies or human antibody fragments. vii. Multispecific antibodies

[0259] In embodiments, the anti-FOLR1 antibody provided herein is a multispecific antibody, e.g., a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In embodiments, one of the binding specificities is for FOLR1, and the other is for any other antigen. In embodiments, a bispecific antibody 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 making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). engineering electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999). 152:5368 (1994)); as well as multispecific antibodies can be made by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0261] Engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies," are also included herein (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576A1).

[0262] The antibodies or fragments herein also include "dual acting FAbs" or "DAFs" that contain antigen binding sites that bind to FOLR1 as well as another distinct antigen. viii. Antibody variants

[0263] In embodiments, amino acid sequence variants of the 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 the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at 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 of "preferred substitutions." More significant changes are provided in Table 1 under the heading of "exemplary substitutions," as further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] Amino acids can be classified according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0265] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have altered (e.g., improved) biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0266] HVRs can be altered (e.g., substituted) to, for example, improve antibody affinity. Such alterations can be made in HVR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).). In an embodiment of affinity maturation, diversity is introduced into the variable genes 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 generated. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-specific approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomly assigned. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0267] In embodiments, substitutions, insertions or deletions can be made within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such changes can also be outside of HVR "hot spots" or SDRs. In the embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or contains at most one, two or three amino acid substitutions.

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

[0269] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the N- or C-terminal fusion of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases 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 to which the antibody is glycosylated. Adding glycosylation sites to an antibody or deleting glycosylation sites can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0271] If an antibody comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells usually contain branched biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In embodiments, the oligosaccharides in the antibody can be modified to generate antibody variants with certain improved properties.

[0272] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 in the glycan relative to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region, although Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294-300, due to small sequence variations within the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108; WO2000 / 61739; WO2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 200 4 / 0132140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include: , Lec13 CHO cells which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0273] Further provided are antibody variants with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.). c) Fc domain variants

[0274] In embodiments, an Fc region variant can be generated by introducing one or more amino acid modifications into the Fc region of an anti-FOLR1 antibody provided herein. The Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0275] In embodiments, antibody variants are contemplated that have some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack 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 thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas 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. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be utilized (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® 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 additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. For example, see the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

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

[0277] Certain antibody variants with improved or diminished binding to FcR have been described. (See, e.g., U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0278] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976), and Kim et al., J. Immunol. 24:249 (1994)), have been described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of 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., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0279] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 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 can be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may 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] For recombinant production of anti-FOLR1 antibodies, nucleic acids encoding the antibodies, such as the antibodies described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

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

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

[0285] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. A large number of baculovirus strains have been identified that can be used for the transfection of insect cells, and in particular Spodoptera frugiperda cells.

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

[0287] Vertebrate cells can also be used as hosts, for example, mammalian cell lines that have been adapted to growth in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 lines (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (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, e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC Other useful mammalian host cell lines include DHFR 5 cells; and FS4 cells. -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (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 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003); Dhara, VG et al., BioDrugs 32: 571-584 (2018); Kunert, R. and Reinhart, D. Applied microbiology and biotechnology, 100(8): 3451-3461 (2016). xi. Assay

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

[0289] In embodiments, the antibody is tested for its antigen binding activity by known methods such as, for example, ELISA, BIACore®, FACS or Western blot.

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

[0291] In an exemplary competitive assay, immobilized FOLR1 is incubated in a solution containing a first labeled antibody that binds to FOLR1 and a second unlabeled antibody that will be tested for its ability to compete with the first antibody for binding to FOLR1. The second antibody may be present in a hybridoma supernatant. As a control, immobilized FOLR1 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody 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 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to FOLR1. In an embodiment, immobilized FOLR1 is present on the surface of a cell or in a membrane preparation obtained from a cell expressing FOLR1 on its surface. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Methods for preparing antibody-drug conjugates

[0292] The ADC of formula (I) is prepared by (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'' by covalent bond, followed by reacting with a drug moiety D or a drug-linker molecule D-L 2 ; and (2) reacting a nucleophilic group of the drug moiety D with a nucleophilic group of a bivalent linker reagent (L 2 and / or B, B' or B'') to form D-L 2 , D-L 2 -B, D-L 2 -B' or D-L 2 -B'' by covalent bond, followed by reacting with a nucleophilic group of an antibody or a reduced antibody, and can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art. Some such methods are described by Agarwal et al., (2015), Bioconjugate Chem., 26: 176-192.

[0293] Nucleophilic groups on the antibody include, but are not limited to, (i) the N-terminal amine group, (ii) side-chain amine groups such as lysine, (iii) side-chain thiol groups such as cysteine, and (iv) sugar hydroxyl or amino groups if the antibody is glycosylated. Amine, thiol and hydroxyl groups are nucleophilic and can react with electrophilic groups of the linker moiety and linker reagent: (i) active esters such as NHS ester, HOBt ester, haloformate, and acid halide; (ii) alkyl and benzyl halides such as haloacetamide; and (iii) aldehyde, ketone, carboxyl, and maleimide groups to form covalent bonds. In addition to NHS ester, functional groups used for conjugation with antibody lysine include, as non-limiting examples, pentafluorophenyl, tetrafluorophenyl, tetrafluorobenzenesulfonate, nitrophenyl, isocyanate, isothiocyanate, and sulfonyl chloride.

[0294] In yet another embodiment, the antibodies may be conjugated to a "receptor" (e.g., streptavidin) for use in pre-targeting of tumors, in which the antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) that has been conjugated to a cytotoxic agent (e.g., a drug or radionuclide).

[0295] Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive by treatment with reducing agents, such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), to fully or partially reduce the antibody for conjugation with a linker reagent. As a result, each cysteine ​​bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by modification of lysine residues, for example, by reacting the lysine residues with 2-iminothiolane (Traut's reagent), thereby converting the amine to a thiol. Reactive thiol groups can also be introduced into the antibody by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a variant antibody containing one or more non-native 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 embodiments, the antibody can be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups. The interchain cysteine ​​residues can then be alkylated, for example, using maleimide. Alternatively, the interchain cysteine ​​residues can be subjected to cross-linking alkylation, for example, using a bissulfone linker or propargyldibromomaleimide, followed by Cu-click ligation. In embodiments, the antibody can be conjugated through a lysine amino acid. Such conjugation can be a one-step conjugation or a two-step conjugation. In embodiments, one-step conjugation involves the coupling of a drug-linker molecule (DL) containing an amine-reactive group with the ε-amino group of the lysine residue. 2 , D.L. 2 -B, DL 2 -B' or DL 2 -B″) via an amide bond. In embodiments, the amine reactive group is an activated ester. In embodiments, the antibody can be conjugated by a two-step conjugation. Two-step conjugation involves, in a first step, reacting a bifunctional reagent containing both an amine-reactive functional group and a thiol-reactive functional group with the lysine ε-amino group. In a second step, a drug-linker molecule (DL 2 , D.L. 2 -B, DL 2 -B' or DL 2 -B″) is conjugated to the thiol-reactive group of the bifunctional reagent. Some examples are provided by Jain et al., (2015), Pharm. Res., 32:3526-3540. In an embodiment, the first step is functionalization of the antibody with an azide, followed by the addition of an alkyne-modified linker or drug-linker molecule (DL 2 , D.L. 2 -B, DL 2 -B' or DL 2-B″)。 In embodiments, the first step may involve a click chemistry reaction with an alkyne, followed by the functionalization of the antibody with an azide-modified linker or drug-linker molecule (DL 2 , D.L. 2 -B, DL 2 -B' or DL 2 -B″)。 In embodiments, the first step may involve a click chemistry reaction with an aldehyde functionalization of the antibody, followed by the addition of an alkoxyamine or hydrazine modified linker or drug-linker molecule (DL 2 , D.L. 2 -B, DL 2 -B' or DL 2 -B″). In embodiments, the first step involves functionalization of the antibody with a tetrazine, followed by a click chemistry reaction with a trans-cyclooctene or cyclopropene modified linker or drug-linker molecule (DL 2 , D.L. 2 -B, DL 2 -B' or DL 2 -B″). In embodiments, the first step involves functionalization of the antibody with trans-cyclooctene or cyclopropene, followed by the addition of a tetrazine modified linker or a drug-linker molecule (DL 2 , D.L. 2 -B, DL 2 -B' or DL 2 -B″). Some examples are described by Pickens et al., (2018), Bioconjug. Chem., 29:686-701; Li et al., (2018), MAbs, 10:712-719; and Chio et al., (2020), Methods Mol. Biol., 2078:83-97.

[0297] In embodiments, the ADC of Formula (I) comprises an anti-FOLR1 antibody (Ab) coupled to a molecule of formula (PI): [ka] or a pharma- ceutically acceptable salt thereof, wherein B is a reactive moiety capable of forming a bond with an anti-FOLR1 antibody.

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

[0299] In embodiments, the ADC of formula (I) comprises an anti-FOLR1 antibody (Ab) coupled to a molecule of formula (P-II): [ka] or a pharma- ceutically acceptable salt thereof, wherein B' is a reactive moiety capable of forming a bond with an anti-FOLR1 antibody.

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

[0301] In embodiments, the ADC of formula (I) comprises an anti-FOLR1 antibody (Ab) coupled to a molecule of formula (P-III): [ka] or a pharma- ceutically acceptable salt thereof, wherein B″ is a reactive moiety capable of forming a bond with an anti-FOLR1 antibody (the reaction is described in Example 3 of US Pat. No. 10,590,165).

[0302] In embodiments, B″ is a reactive moiety capable of forming a bond with an amine group of an anti-FOLR1 antibody. In embodiments, B″ is [ka] It is.

[0303] In embodiments, PI, P-II, or P-III is [ka]

[0304] [ka]

[0305] [ka]

[0306] [ka]

[0307] [ka] and

[0308] [ka]

[0309] A compound selected from

[0310] or a pharma- ceutically acceptable salt thereof. Pharmaceutical Compositions

[0311] In aspects, provided herein is a pharmaceutical composition comprising an ADC as described herein, including embodiments, and a pharma- ceutically acceptable carrier. In embodiments, the ADC as described herein is included in a therapeutically effective amount.

[0312] In embodiments, the pharmaceutical composition is formulated as a tablet, powder, capsule, pill, cachet or lozenge as described herein. The pharmaceutical composition can be formulated as a tablet, capsule, pill, cachet or lozenge for oral administration. The pharmaceutical composition can be formulated for dissolution in a solution for administration by such techniques as intravenous administration. The pharmaceutical composition can be formulated as described herein 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.

[0313] The ADCs and pharmaceutical compositions thereof are particularly useful for parenteral administration, i.e., subcutaneously (sc), intrathecally, intraperitoneally, intramuscularly (im) or intravenously (iv). In embodiments, the ADCs and pharmaceutical compositions thereof are administered intravenously or subcutaneously.

[0314] The compositions may contain pharma- ceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, etc. The concentration of the antigen binding protein of the invention in such pharmaceutical formulations can vary greatly, i.e., from less than about 0.5% by weight, usually about or at least about 1% by weight, to as high as about 15 or 20% by weight, and will be selected primarily based on fluid volumes, viscosities, etc. in accordance with the particular method of administration selected.

[0315] Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Sciences, vol. 15, No. 1, pp. 111-115, 1997. thed., Mack Publishing Company, Easton, Pa. For the preparation of intravenously administrable antigen-binding protein formulations of the present invention, see Lasmar U and Parkins D "The formulation of Biopharmaceutical products", Pharma. Sci. Tech. today, pages 129-137, Vol. 3 (3 Apr. 2000); Wang, W "Instability, stabilisation and formulation of liquid protein pharmaceuticals", Int. J. Pharm 185 (1999) 129-188; Stability of Protein Pharmaceuticals Part A and Bed Ahern TJ, Manning MC, New York, NY: Plenum Press (1992); Akers, MJ "Excipient-Drug interactions in Parenteral Formulations", J. Pharm Sci 91 (2002) 2283-2300; Imamura, K et al "Effects of types of sugar on stabilization of Protein in the dried state", J Pharm Sci 92 (2003) 266-274;Izutsu, Kkojima, S. "Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying", J. Pharm. Pharmacol, 54 (2002) 1033-1039;Johnson, R, "Mannitol-sucrose mixtures-versatile formulations for protein peroxidise19g19n", J. Pharm. Sci, 91 (2002) 914-922; and Ha, E Wang W, Wang Y. j.See "Peroxide formation in polysorbate 80 and protein stability", J. Pharm Sci, 91, 2252-2264, (2002), the entire contents of which are incorporated herein by reference and the reader is specifically referred to these references.

[0316] In embodiments, the pharmaceutical composition may comprise an optical isomer, diastereomer, enantiomer, isoform, polymorph, hydrate, solvate or product, or a pharmaceutically acceptable salt of the compound described herein. The compound described herein (including its pharmaceutically acceptable salt) contained in the pharmaceutical composition may be covalently bound to a carrier moiety as described above. In embodiments, the compound described herein (including its pharmaceutically acceptable salt) contained in the pharmaceutical composition is not covalently linked to a carrier moiety. A combination of the covalently linked and non-covalently linked compounds described herein may be present in the pharmaceutical composition herein. How to use

[0317] In an embodiment, a method of treating a disease in a subject in need thereof comprises administering to a subject an IgG antibody and a conjugation linker moiety (L) attached to a thiol of a cysteine ​​residue or to an amine of a lysine residue of the IgG antibody. 1 ) and either L 1 , or optionally another linker L 2 and a drug moiety covalently attached to said antibody. In embodiments, the IgG antibody binds to FOLR1.

[0318] In one aspect, the ADCs provided herein are used in a method of inhibiting proliferation of a FOLR1 expressing cell, comprising contacting the cell with the ADC, e.g., exposing the cell to the ADC under conditions that allow binding of the anti-FOLR1 antibody of the ADC at the cell surface, thereby inhibiting proliferation of the cell. In an embodiment, the method is an in vitro or in vivo method. In an embodiment, the cell is a B cell. In an embodiment, the cell is a cancer cell. In an embodiment, the cell is a multiple myeloma cell. In any of these embodiments, the cell can be a mammalian cell, e.g., a human cell.

[0319] In vitro cell proliferation inhibition can be assayed using the CellTiter-Glo™ luminescent cell viability assay available from Promega (Madison, WI). The assay determines the number of viable cells in culture based on quantification of ATP present, an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay can be performed in 96- or 384-well format, making it amenable to automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding a single reagent (CellTiter-Glo® Reagent) directly to cultured cells. This results in cell lysis and the generation of a luminescent signal by a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imaging device. Luminescence output data is expressed as relative light units (RLU).

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

[0321] In an embodiment, the disease is cancer. In an embodiment, the cancer is associated with overexpression of FOLR1. In an embodiment, an ADC is provided herein for use in a method of treating an individual with a FOLR1-expressing cancer, the method comprising administering to the individual an effective amount of the ADC. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. In an embodiment, the additional therapeutic agent is a VEGF inhibitor. In an embodiment, the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor. In an embodiment, the VEGF inhibitor is an anti-VEGF antibody. In an embodiment, the VEGF inhibitor is a small molecule VEGF inhibitor. In an embodiment, the anti-VEGF antibody is bevacizumab or ramucirumab. In an embodiment, the anti-VEGF antibody is bevacizumab. In an embodiment, the anti-VEGF antibody is ramucirumab. In an embodiment, the small molecule VEGF inhibitor is sunitinib, sorafenib, axitinib, pazopanib, or regorafenib. In an embodiment, the small molecule VEGF inhibitor is sunitinib. In an embodiment, the small molecule VEGF inhibitor is sorafenib. In an embodiment, 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 a further aspect, the present disclosure provides the use of the ADC in the manufacture or preparation of a medicament. In an embodiment, the medicament is for the treatment of FOLR1-expressing cancer. In a further embodiment, the medicament is for use in a method for treating FOLR1-expressing cancer, the method comprising administering an effective amount of the medicament to an individual having FOLR1-expressing cancer. 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 disclosure provides for the use of an ADC described herein, including embodiments, in combination with an additional therapeutic agent in the manufacture or preparation of a medicament for the treatment of a FOLR1-expressing cancer. In embodiments, the disclosure provides for the use of an ADC described herein, including embodiments, in combination with a VEGF inhibitor in the manufacture or preparation of a medicament for the treatment of a FOLR1-expressing cancer. 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 an embodiment, the small molecule VEGF inhibitor is sorafenib. In an embodiment, 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. In an embodiment, the ADC described herein is ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6. In an embodiment, the ADC is ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6. In an embodiment, the ADC is ADC-1. In an embodiment, the ADC is ADC-2. In an embodiment, the ADC is ADC-3. In an embodiment, the ADC is ADC-4. In an embodiment, the ADC is ADC-5. In an embodiment, the ADC is ADC-6.

[0324] In embodiments, the methods provided herein are for treating cancer in a mammal. In embodiments, the methods provided herein are for treating cancer in a human.

[0325] In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include tumors of epithelial origin, including ovarian, uterine, breast, endometrial, pancreatic, renal, lung, colorectal and brain tumors. In embodiments, cancers that can be treated with the immunoconjugates or methods provided herein include serous and endometrioid epithelial ovarian cancer, endometrioid adenocarcinoma, adenocarcinoma subtypes of non-small cell lung cancer (NSCLC), squamous cell 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, including, but not limited to, multiple myeloma (MM), including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's hypergammaglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). In embodiments, the 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 to about 3,000 mg per day, about 1 to about 2,000 mg per day, about 1 to about 1,500 mg per day, about 1 to about 1,000 mg per day, about 10 to about 1,000 mg per day, about 50 to about 1,000 mg per day, about 50 to about 800 mg per day, about 50 to about 700 mg per day, or about 100 to about 500 mg per day.

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

[0329] In embodiments, the therapeutically effective amount is about 0.1 to about 100 mg / kg / day, about 0.1 to about 50 mg / kg / day, about 0.1 to about 40 mg / kg / day, about 0.5 to about 30 mg / kg / day, about 0.5 to about 25 mg / kg / day, 1 to about 25 mg / kg / day, about 1 to about 20 mg / kg / day, about 1 to about 15 mg / kg / day, or about 1 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 implant), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal or topical) administration route. The compound of formula (I) or a pharmaceutically acceptable salt thereof can be formulated into a suitable dosage unit, either alone or together, using pharmaceutically acceptable excipients, carriers, adjuvants and vehicles suitable for each administration route.

[0331] The compound of formula (I) or its pharma- ceutically acceptable salt can be delivered as a single dose, for example, a single bolus injection, or an oral tablet or pill, or can be delivered over time, for example, by continuous infusion over time or divided bolus doses over time. The compound can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. For example, stable disease for solid tumors generally means that the perpendicular diameter of measurable lesions has not increased by 25% or more since the last measurement. Response Evaluation Criteria in Solid Tumors (RECIST) Guidelines, Journal of the National Cancer Institute 92(3): 205-216 (2000). Stable disease or lack thereof is determined by methods known in the art, for example, evaluation of the patient's symptoms, physical examination, visualization of tumors imaged using X-ray, CAT, PET or MRI scan, and other commonly accepted evaluation modalities.

[0332] The compound of formula (I), or a pharma- ceutically acceptable salt thereof, 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, administration can be continuous (i.e., daily, or every day for consecutive days) or intermittent, e.g., cyclic (i.e., with days, weeks, or months off). As used herein, the term "daily" is intended to mean that the therapeutic compound, such as the compound of formula (I), is administered once or more each day, e.g., for a period of time. The term "continuous" is intended to mean that the therapeutic compound, such as the compound of formula (I), is administered daily for an uninterrupted period of at least 10 days to 52 weeks. The term "intermittent" or "intermittently" as used herein is intended to mean that the administration is stopped or started at either regular or irregular intervals. For example, intermittent administration of a compound of formula (I) can be administration for 1-6 days per week, administration in cycles (e.g., daily administration for 2-8 consecutive weeks followed by a drug-free period of 1 week or less), or administration every other day. The term "cycling," as used herein, is intended to mean that a therapeutic compound, such as a compound of formula (I), is administered daily or continuously, but with a drug-free period.

[0333] In an embodiment, the frequency of administration ranges from about a daily dose to about a monthly dose. In an embodiment, 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 an embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered once a week. In another embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered twice a week. In an embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered three times a week. In an embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered once every two weeks. In an embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered once every three weeks. In an embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered once every four weeks. In an embodiment, the compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered once every eight weeks.

[0334] Combination therapy with a second active agent The ADC of Formula (I), or a pharma- ceutically acceptable salt thereof, may also be combined or used in combination with other therapeutic agents useful in the treatment of cancer as described herein.

[0335] In embodiments, provided herein are methods of treating cancer comprising administering to a patient an ADC of Formula (I), or a pharma- ceutically acceptable salt thereof, in combination with one or more second active agents, and optionally in combination with radiation therapy, blood transfusion, or surgery. Certain combinations are believed to act synergistically in the treatment of certain types of cancer, as well as in the treatment of 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 large molecule (e.g., protein) or a small molecule (e.g., synthetic inorganic molecule, organometallic molecule or organic molecule).

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

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

[0339] VEGF inhibitors can be used in the methods and compositions provided herein. VEGF receptor inhibitors include anti-VEGF antibodies and small molecules (e.g., synthetic inorganic, organometallic or organic molecules). In an embodiment, the VEGF inhibitor is an antibody. In an embodiment, 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 an embodiment, the VEGF inhibitor is a small molecule, including but not limited to sunitinib, sorafenib, axitinib, pazopanib and regorafenib. In an embodiment, the small molecule is sunitinib, sorafenib, axitinib, pazopanib or regorafenib. In an embodiment, the small molecule is sunitinib. In an embodiment, the small molecule is sorafenib. In an embodiment, the small molecule is axitinib. In an embodiment, the small molecule is pazopanib. In an embodiment, the small molecule is regorafenib.

[0342] As used herein, the term "in combination" includes the use of more than one therapies (e.g., one or more therapeutic agents). However, the use of the term "in combination" does not restrict the order in which therapies (e.g., therapeutic agents) are administered to a patient with a disease or disorder. A first therapy (e.g., a therapeutic agent, e.g., an ADC provided herein, e.g., a compound of Formula (I), or a pharma- ceutically acceptable salt thereof) can be administered 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), concomitantly with, or following (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) administration of a second therapy (e.g., a therapeutic agent) to a subject. Triple therapy is also contemplated herein.

[0343] The administration of the compound of formula (I) and one or more second active agents to a patient can be performed simultaneously or sequentially by the same or different routes of administration. The combination of agents or compositions can be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., by separate intravenous lines), or sequentially (e.g., one agent is administered first, followed by the second agent). Thus, the term combination is used to refer to the concomitant, simultaneous, or sequential administration of two or more agents or compositions. The course of treatment is best determined on an individual basis, depending on the specific characteristics of the subject and the type of treatment selected. Treatments such as those disclosed herein can be administered to a subject daily, twice daily, biweekly, monthly, or any applicable frequency that is therapeutically effective. Treatments can be administered alone or in combination with any other treatments disclosed herein or known in the art. The additional treatments can be administered at the same time as the first treatment, can be administered at different times, or can be administered on entirely different therapeutic schedules (e.g., the first treatment can be daily, while the additional treatments are weekly).

[0344] The route of administration of the compound of formula (I) is independent of the route of administration of the second treatment. 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. Thus, according to these embodiments, the compound of formula (I) is administered orally or intravenously, and the second treatment can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, by liposomes, via inhalation, vaginally, intraocularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form. In an embodiment, the compound of formula (I) and the second treatment are administered by the same mode of administration, orally or by IV. In another embodiment, the compound of formula (I) is administered by one mode of administration, e.g., by IV, while the second agent (the anti-cancer agent) is administered by another mode of administration, e.g., intraperitoneally.

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

[0346] In an embodiment, a compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with bevacizumab. In an embodiment, a compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with ramucirumab. In an embodiment, a compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with sunitinib. In an embodiment, a compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with sorafenib. In an embodiment, a compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with axitinib. In an embodiment, the compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with pazopanib. In an embodiment, the compound provided herein, for example, a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with a FOLR1-expressing cancer with regorafenib.

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

[0348] In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered to a patient with lung cancer with bevacizumab. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered to a patient with lung cancer with ramucirumab. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered to a patient with lung cancer with sunitinib. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered to a patient with lung cancer with sorafenib. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered to a patient with lung cancer with axitinib. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is administered to a patient with lung cancer with pazopanib. In embodiments, a compound provided herein, e.g., a compound of Formula (I), or a pharma- ceutically acceptable salt thereof, is administered with regorafenib to a patient with lung cancer.

[0349] In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with ovarian cancer with bevacizumab. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with ovarian cancer with ramucirumab. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with ovarian cancer with sunitinib. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with ovarian cancer with sorafenib. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with ovarian cancer with axitinib. In an embodiment, a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered to a patient with ovarian cancer with pazopanib. In embodiments, a compound provided herein, e.g., a compound of Formula (I), or a pharma- ceutically acceptable salt thereof, is administered with regorafenib to a patient with ovarian cancer.

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

[0351] In an embodiment, the kit provided herein comprises a dosage form of a compound provided herein, for example, a compound of formula (I), or a pharma- ceutically acceptable salt thereof. In an embodiment, the kit provided herein further comprises an additional active ingredient, for example, a VEGF inhibitor. In an embodiment, the kit provided herein further comprises an additional active ingredient, for example, an anti-VEGF antibody or a small molecule VEGF inhibitor. In an embodiment, the kit provided herein further comprises an additional active ingredient, for example, bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, or regorafenib.

[0352] In embodiments, the kits provided herein may include a pharmaceutical composition comprising a dosage form of a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, together with a VEGF inhibitor. In embodiments, the kits provided herein may include a pharmaceutical composition comprising a dosage form of a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, together with an anti-VEGF antibody. In embodiments, the kits provided herein may include a pharmaceutical composition comprising a dosage form of a compound provided herein, e.g., a compound of formula (I), or a pharma- ceutically acceptable salt thereof, together with a small molecule VEGF inhibitor.

[0353] In embodiments, the kits provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with bevacizumab. In embodiments, the kits provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with ramucirumab. In embodiments, the kits provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with sunitinib. In embodiments, the kits provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with sorafenib. In embodiments, the kits provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with axitinib. In an embodiment, the kit provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with pazopanib. In an embodiment, the kit provided herein may include a pharmaceutical composition comprising a compound provided herein, e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with regorafenib. In an embodiment, the compound of formula (I) is ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, or ADC-6. In an embodiment, the compound of formula (I) is ADC-1. In an embodiment, the compound of formula (I) is ADC-2. In an embodiment, the compound of formula (I) is ADC-3. In an embodiment, the compound of formula (I) is ADC-4. In an embodiment, the compound of formula (I) is ADC-5. In an embodiment, the compound of formula (I) is ADC-6.

[0354] In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., bevacizumab. In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., ramucirumab. In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., sunitinib. In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., sorafenib. In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., axitinib. In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., pazopanib. In embodiments, the kits provided herein further comprise an additional active ingredient, e.g., regorafenib.

[0355] In embodiments, the kits provided herein further comprise a device used to administer the active ingredient. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers.

[0356] In an embodiment, the kit provided herein further comprises a pharma- ceutically acceptable vehicle that can be used to administer cells or blood for transplantation and 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 can comprise a sealed container of a suitable vehicle that can dissolve the active ingredient to form a particulate-free sterile solution that is suitable for parenteral administration.Examples of pharma-ceutically acceptable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles, such as, but are not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles, such as, but are not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles, such as, but are not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0357] Sequence Listing: Human FOLR1 sequence SEQ ID NO:9

[0358] MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKER VLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS

[0359] Array table: [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] NOTE: Anti-FOLR1 antibodies FOLR1-Ab1, FOLR1-Ab4, FOLR1-Ab14, FOLR1-Ab20 and FOLR1-Ab23 were disclosed in WO2020 / 016661, among which AMT-151 and FOLR1-Ab1 share the same six CDRs, and FOLR1-Ab14, FOLR1-Ab20 and FOLR1-Ab23 share the same six CDRs. [Table 4] EXAMPLES

[0360] Working Example The following examples are intended to be illustrative and can be used to further understand 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 this example can be easily adapted to the preparation of many other compounds of the present disclosure, and alternative methods of preparing the compounds of the present disclosure are considered to be within the scope of the present disclosure.For example, the synthesis of compounds not exemplified according to the present disclosure 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 other than those described, or by making changes in the routing of reaction conditions, reagents and starting materials. Alternatively, it will be recognized that other reactions disclosed herein or known in the art can be applied to the preparation of other compounds of the present disclosure.The synthesis of compound I-1 and related compounds is disclosed in U.S. Pat. Nos. 10,590,165 and 9,981,046, and these references are incorporated herein in their entirety. Synthesis Examples (Example S1) Synthesis of compound I-5. [ka]

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

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

[0364] (Example S2) Synthesis of compound I-6. The synthesis of compound I-6 is described in Example 3 of US Pat. No. 10,590,165, which is incorporated herein in its entirety. [ka]

[0365] Antibody-drug conjugates (ADCs) were prepared by conjugating compound I-5 or compound I-6 with the AMT-151 clone of anti-FOLR1 antibody. AMT-151 is a human IgG1 antibody.

[0366] (Example S3) Preparation of antibody-drug conjugate (ADC), anti-FOLR1-compound I-5 (151-C-LOCK-D5; ADC-1). The six CDRs of the anti-FOLR1 antibody used in this example have the same antibody CDR sequences as the FORL1 antibody anti-FLOR1-Ab1 described in WIPO Publication No. WO2020 / 016661, which is incorporated herein in its entirety. The heavy and light chain sequences of the anti-FOLR1 antibody used in this example are shown in Table 2 as SEQ ID NO:7 and SEQ ID NO:8 and in Table 4 as SEQ ID NO:10 and SEQ ID NO:11, respectively. The affinity purified anti-FOLR1 antibody was buffer exchanged into conjugation buffer (50 mM sodium phosphate buffer, pH 7.0-7.2, 4 mM EDTA) at a concentration of 5 mg / mL. To a portion of this antibody stock, a freshly prepared 10 mM aqueous solution of tris(2-carboxyethyl)phosphine) (TCEP) was added at a 20-fold molar excess. The resulting mixture was incubated overnight at 4-8 °C. Excess TCEP was then removed by several rounds of centrifugal filtration with fresh conjugation buffer. Following UV-Vis quantification of the recovered reduced antibody material, sufficient free thiol-to-antibody ratio (SH / Ab) was confirmed. Briefly, a 1 mM aliquot of freshly prepared Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) in conjugation buffer was mixed with an equal volume of purified antibody solution. The resulting absorbance at 412 nm was measured, and the reduced cysteine ​​content was determined to be 14,150 mM. -1 cm -1 The extinction coefficient was determined using 0.05%. Under these conditions, the SH / Ab ratio was approximately 6.

[0367] To initiate the 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 and purified (TCEP-removed) anti-FOLR1 antibody to obtain a final concentration of 30% (v / v) PG, immediately followed by the addition of I-5 in a 4.5-fold molar excess. After thorough mixing and incubation at ambient temperature for 2 hours, the crude conjugation reaction was analyzed by HIC-HPLC chromatography to confirm reaction completion (disappearance of the starting antibody peak) with 280 nm wavelength detection. The resulting ADC-1 conjugate was then purified by gel filtration chromatography using an AKTA system equipped with a Superdex 200pg column (GE Healthcare) equilibrated with PBS. The average drug-antibody ratio (DAR) was calculated to be 4 based on the comparative peak area integration of the HIC-HPLC chromatograms. Evidence of a low percentage (<5%) of high molecular weight (HMW) aggregates for the resulting ADC-1 was determined using analytical SEC-HPLC.

[0368] (Example S4) Preparation of antibody-drug conjugate (ADC), anti-FOLR1-compound I-6 (151-K-LOCK-D5; ADC-5). The ADC, anti-FOLR1-compound I-6, was prepared as described in U.S. Pat. No. 10,590,165, which is incorporated herein in its entirety. To a solution of 0.5-50 mg / mL of antibody in a buffer of pH 6.0-9.0 containing 0-30% organic solvent, 0.1-10 equivalents of activated drug linker conjugate (I-6) was added in portions or in a continuous flow. The reaction was carried out at 0-40° C. for 0.5-50 h with gentle stirring or shaking and monitored by HIC-HPLC. The resulting crude ADC product was subjected to the necessary downstream steps of desalting, buffer exchange / formulation and purification as required using state-of-the-art procedures. The ADC products were characterized by HIC-HPLC, SEC, RP-HPLC, and LC-MS as required. The average DAR obtained for ADC-5 was 2 according to HIC-HPLC analysis.

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

[0370] Reduction of antibody: 151 antibody was exchanged into PBS 7.0 / EDTA. 7 equivalents of TCEP were added to the 151 antibody solution and the reaction mixture was allowed to react for 2 hours at 37°C.

[0371] Conjugation of antibody and linker-payload: The reduced antibody mixture was incubated for 10 minutes at 4° C. Linker-payload GGFG-DXd (developed by Daiichi Sankyo Co., Ltd. and 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 for 60 minutes at 22° C.

[0372] Antibody-drug conjugates were desalted, buffer exchanged / formulated and purified using state-of-the-art procedures. [ka]

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

[0374] Reduction of antibody: 151 antibody was exchanged into PBS 7.0 / EDTA. 2.5 equivalents of TCEP were added to the 151 antibody solution and the reaction mixture was allowed to react for 2 hours at 37°C.

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

[0376] Antibody-drug conjugates were desalted, buffer exchanged / formulated and purified using state-of-the-art procedures. [ka] Biological Examples The in vitro and in vivo efficacy of antibody-drug conjugates (ADCs) was evaluated using the AMT-151 clone of anti-FOLR1 antibodies.

[0377] Example B1 In vitro efficacy of antibody-drug conjugates (ADCs), 151-C-Lock-D5 (ADC-1) and 151-K-Lock-D5 (ADC-5). The in vitro efficacy of ADCs 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 5% CO2 humidified environment.

[0378] In vitro assays were performed as follows: tumor cells were harvested by centrifugation at 300 g for 5 min, plated in 96-well clear-bottom white-walled plates (2,000-3,000 cells / well in 50 μL complete medium) and maintained at 37°C. Cells were then treated in duplicate with 50 μL of test substances prepared at 2× final concentration, serially diluted in complete medium, and incubated at 37°C for up to 120 h. After treatment, inhibition of cancer cell growth was determined using a CCK-8 (Cell Counting Kit-8) viability assay as described by 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). 50 ) was derived from dose-response curves.

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

[0381] Standard cell viability assays were used to evaluate the in vitro cytotoxic activity and targeting specificity of the ADCs described herein against high FOLR1 expressing IGROV1, low FOLR1 expressing SKOV-3 and FOLR1 negative A549 (negative control) cancer cell lines. As shown in Figure 1, anti-FOLR1 151-C-LOCK-D5 and 151-K-LOCK-D5 (where 151 is the AMT-151 clone of the FOLR1 antibody) dose-dependently reduced IGROV1 and SKOV-3 cell viability in a 5-day assay and showed no activity against A549 cells. A range of potency, as determined by IC50s of approximately 0.35-99 nM against FOLR1 expressing cell lines, was observed among ADCs with different conjugation chemistries with the anti-FOLR1 antibody and in cell lines with different amounts of FOLR1 expression (Table 5).

[0382] IC of anti-FOLR1 151-C-LOCK-D5 and 151-K-LOCK-D5 in human tumor cells 50 A summary of the values ​​(nM) is presented in Table 5. [Table 5]

[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 result in a significant decrease in cell viability, as shown in FIG. 1.

[0385] (Example B2) In vitro 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] In vitro assay was carried out as described in Example B1 above. Cell viability was determined after 5 days of incubation. 151-mAb was used as a negative control in the assay. 151-mAb is an antibody that comprises 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 cell viability assay for 151-C-Lock-D5 and 151-GGFG-Dxd are shown in Figure 5. Figure 5 shows that 151-C-Lock-D5 showed better cell killing activity than 151-GGFG-Dxd in SKOV-3 cell line. 151-mAb alone did not show any decrease in cell viability.

[0389] (Example B3) In vivo efficacy of antibody-drug conjugates (ADCs) 151-C-Lock-D5 (ADC-1) and 151-K-Lock-D5 (ADC-5). Six- to eight-week-old female Balb / c mice 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 in a 5% CO2 humidified environment for 2-3 weeks before being harvested for implantation. Cell viability, as determined by trypan blue dye exclusion assay, was >90% prior to implantation. Five to ten million IGROV1 or SKOV-3 cells in 100 μl PBS were inoculated into the right upper flank of each mouse by sc injection.

[0391] Tumor volume measurements were performed twice weekly starting on day 4 after tumor cell inoculation. The longest longitudinal diameter was measured as length and the widest traverse diameter was measured as width by using digital calipers. Tumor volume (TV) was then calculated according to the formula: TV = [length x (width) 2 ] / 2 and analyzed using Excel.

[0392] The mean tumor size of IGROV1 and SKOV-3 was approximately 150 mm 3 Treatment was initiated when

[0393] Tumor size is 2000mm 3 When the endothelial cell count reached 100 mg / kg, the mice were euthanized.

[0394] After randomization of tumor-bearing mice, 151-C-LOCK-D5, 151-K-LOCK-D5, or isotype ADCs IgG-C-LOCK-D5 and IgG-K-LOCK-D5 diluted in PBS were administered to the mice by ip injection. In experiment I, the treatment regimen included 1.5, 3, or 6 mg / kg of 151-C-LOCK-D5 administered once on day 8 after tumor (IGROV1) inoculation, or 3 or 6 mg / kg of 151-K-LOCK-D5 administered once on day 8 after tumor (IGROV1) inoculation. Figure 2A shows IGROV1 tumor volume over time, and Figure 2B shows IGROV1 tumor volume 46 days after tumor inoculation.

[0395] The body weights of all mice were measured twice weekly after a single ADC dose as shown in Figure 2C.

[0396] In experiment II, the treatment regimen included 1.5, 3 or 6 mg / kg 151-C-LOCK-D5 administered once every 2 weeks for 2 doses (Q2W x 2), or 3 or 6 mg / kg 151-K-LOCK-D5 administered once every 2 weeks for 2 doses (Q2W x 2), and ADC was administered on days 15 and 29 after inoculation of the tumor (SKOV-3). Figure 3A shows SKOV-3 tumor volume over time, and Figure 3B shows SKOV-3 tumor volume 49 days after inoculation of the tumor.

[0397] The body weights of all mice were measured twice weekly after administration of the first dose of ADC as shown in Figure 3C.

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

[0399] In the IGROV1 tumor xenograft model of experiment I (as shown in Figure 2A and Figure 2B), all treatment regimens with anti-FOLR1-151-K-LOCK-D5 (151-K-LOCK-D5; ADC-5) and with 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 dramatic tumor regression even at low doses, with all tumors disappearing approximately 5 weeks after the first treatment. Tumor growth inhibition is shown in Table 6. [Table 6]

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

[0401] In the SKOV-3 tumor xenograft model of experiment II (as shown in Figures 3A and 3B), anti-FOLR1-151-C-LOCK-D5 (151-C-LOCK-D5; ADC-1) demonstrated antitumor activity compared to the negative control (IgG-K-LOCK-D5).

[0402] No toxicity was observed with any of the treatment regimens, as evidenced by the absence of significant weight loss (Figure 3C).

[0403] (Example B4) In vivo antitumor effect of 151-K-Lock-D5 (ADC-5) on NSCLC cancer PDX model LU-01-1618 The antitumor efficacy of single doses of 1.5 mg / kg, 3 mg / kg, 6 mg / kg and 10 mg / kg of 151-K-Lock-D5 (ADC-5) was evaluated on FRα-positive NSCLC LU-01-1618 PDX tumors (WuXi AppTec (Shanghai) Co., Ltd.) implanted in female BALB / c nude mice. BALB / c nude mice were purchased from Shanghai Lingchang Laboratory Animal Technology Co., LTD. LU-01-1618 tumors were sliced ​​into approximately 30 mm 3 The tumors were cut into pieces and implanted subcutaneously into the flanks of mice. On the 18th day after implantation (average tumor size was approximately 170 mm 3 ), mice were injected intravenously with o...

Claims

1. Antibody-drug conjugate (ADC) of formula (I): 【Chemical 94】 or a pharmaceutically acceptable salt thereof, in the formula, Ab is an anti-FOLR1 antibody; m is an integer between 1 and 20; L １ This is a linker bound to the anti-FOLR1 antibody; L ２ is a combination, -C(O)-, -NH-, an amino acid unit, -(CH ２ CH ２ O) ｎ -, -(CH ２ ) ｎ -, -(4-aminobenzyloxycarbonyl)-, -(C(O)CH ２ CH ２ NH)-, or any combination thereof, or is the same, and n is an integer from 1 to 24; D is the drug part. ADC or a pharmaceutically acceptable salt thereof.

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

3. -L １ -L ２ -but, 【Chemical 95】 The ADC according to claim 1.

4. The ADC according to claim 1, 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. L ２ However, the bond is -C(O)-, -NH-, Val, Phe, Lys, -(4-aminobenzyloxycarbonyl)-, Gly, Ser, Thr, Ala, β-Ala, citrulline (Cit), -(CH ２ ) ｎ -, - (CH ２ CH ２ O) ｎ - or any combination thereof, the ADC according to claim 1.

7. L ２ is a bond, -C(O)-, -NH-, Val, Gly, Lys, Cit, -(CH ２ ) ｎ -, - (CH ２ CH ２ O) ｎ - or any combination thereof, the ADC according to claim 6.

8. L ２ However, 【Chemistry 96】 The ADC according to claim 7, which includes or is the ADC.

9. D is 【Chemical Engineering 102】 The ADC according to claim 1.

10. D is 【Chemistry 103】 And in the formula, R １ is H or -C １ ~C ８ It is alkyl; R ３ H, halogen, -CCl ３ , - CBr ３ , -CF ３ , -CI ３ -CHCl ２ , -CHBr ２ ,-CHF ２ , -CHI ２ ien-CH ２ Cl, -CH ２ Br, -CH ２ F, -CH ２ I, -CN, -OR ３Ａ , -NR ３Ａ R ３Ｂ ,-(CH ２ ) ｖ OR ６ , -C(O)NHSO ２ R 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 a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl; Z ２ is a substituted aryl, substituted heteroaryl, substituted cycloalkyl, or substituted heterocycloalkyl; R ６ H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -CO(CH ２ CH ２ O) ｗ CH ２ CH ２ Y, -CONH(CH ２ CH ２ O) ｗ CH ２ CH ２ Y, 【Chemical 104】 , a charged group, or a sugar derivative, where v is an integer between 1 and 24; w is an integer between 1 and 24; Y is -NH ２ -OH, -COOH, or -OCH ３ And; R ７ These are independently H, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; R １０ is -OH, -OCH ３ or -COOH; Each R ３Ａ , R ３Ｂ , R ４Ａ , and R ４Ｂ These are independently H or substituted or unsubstituted alkyl groups. The ADC according to claim 1.

11. R １ However, the ADC according to claim 10 is H.

12. R ３ However, H, -OR ３Ａ ,-(CH ２ ) ｖ OR ６ , -C(O)NHSO ２ R ７ , substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl, R3 is H, -OR3A, -(CH2)vOR6, -C(O)NHSO2R7, unsubstituted C1-C6 alkyl, or substituted C1-C6 alkyl, R3 is H, methyl, ethyl, propyl, butyl, -CH2OH, -CH2CH2OH, -CH2N3, -CH2CH2N3, -CH2OCH3, -CH2OCH2CH3, -CH2CH2OCH3, -CH2CH2OCH3, 【Chemistry 105】 The ADC according to claim 10.

13. R ３ However, -CH ２ N ３ or 【Chemistry 106】 The ADC according to claim 12.

14. R ４ However, H, -OR ４Ａ The ADC according to claim 10, which is a substituted or unsubstituted alkyl, or a substituted or unsubstituted heteroalkyl.

15. R ４ The ADC according to claim 14, wherein the ADC is H, -OH, methyl, ethyl, propyl, or butyl.

16. Z １ The ADC according to claim 10, wherein Z is a substituted or unsubstituted aryl.

17. Z ２ The ADC according to claim 10, wherein the substitution aryl is.

18. Z １ but, 【Chemistry 107】 And in the formula, Each X is independently Cl, Br, I, or F; Each R' is independently, -CH ３ , -CH ２ CH ３ or -CH ２ CH ２ CH ３ ; q is an integer between 1 and 5. The ADC according to claim 16.

19. Z １ but, 【Chemistry 108】 The ADC according to claim 18.

20. Z ２ but, 【Chemistry 109】 In the formula, each G is independently Cl, Br, I, F, -CH ３ ien-CH ２ CH ３ ien-CH ２ CH ２ CH ３ , -OCH ３ , -OCH ２ CH ３ -OH, or -NH ２ And; p is an integer from 0 to 4. The ADC according to claim 17.

21. Z ２ but, 【Chemical 110】 The ADC according to claim 20.

22. D is 【Chemistry 111】 The ADC according to claim 1.

23. D is 【Chemistry 112】 The ADC according to claim 22.

24. The ADC, 【Chemistry 113-1】 【Chemistry 113-2】 The ADC according to claim 1, or a pharmaceutically acceptable salt thereof, wherein m in the formula is an integer from 1 to 8.

25. The anti-FOLR1 antibody is (a) VL CDR1 containing the sequence of SEQ ID NO: 1, VL CDR2 containing the sequence of SEQ ID NO: 2, VL CDR3 containing the sequence of SEQ ID NO: 3, VH CDR1 containing the sequence of SEQ ID NO: 4, VH CDR2 containing the sequence of SEQ ID NO: 5, and VH CDR3 containing the sequence of SEQ ID NO: 6; (b) VL CDR1 containing the sequence of SEQ ID NO: 12, VL CDR2 containing the sequence of SEQ ID NO: 13, VL CDR3 containing the sequence of SEQ ID NO: 14, VH CDR1 containing the sequence of SEQ ID NO: 15, VH CDR2 containing the sequence of SEQ ID NO: 16, and VH CDR3 containing the sequence of SEQ ID NO: 17; or (c) VL CDR1 containing the sequence of SEQ ID NO: 12, VL CDR2 containing the sequence of SEQ ID NO: 18, VL CDR3 containing the sequence of SEQ ID NO: 19, VH CDR1 containing the sequence of SEQ ID NO: 20, VH CDR2 containing the sequence of SEQ ID NO: 21, and VH CDR3 containing the sequence of SEQ ID NO: 22 Including CDR3, and / or The anti-FOLR1 antibody has a sequence that is 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, or contains a VL having the 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, or The anti-FOLR1 antibody has 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, or includes a VH having the sequence of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO:

28. The ADC according to claim 1.

26. The anti-FOLR1 antibody is an IgG antibody, and optionally the anti-FOLR1 antibody is an IgG1 antibody, and / or The anti-FOLR1 antibody binds to human FOLR1, and if necessary, the human FOLR1 has the amino acid sequence of SEQ ID NO:

9. The ADC according to claim 1.

27. A composition comprising the ADC according to any one of claims 1 to 26 for use in the treatment of medicine.

28. The composition according to claim 27, wherein the treatment is the treatment of a FOLR1-expressing cancer, and the FOLR1-expressing cancer is, if necessary, multiple myeloma, lung cancer, or ovarian cancer, or the treatment is the treatment of an epithelial tumor, and the epithelial tumor is, if necessary, a tumor of the uterus, breast, endometrium, pancreas, kidney, colorectal, or brain.

29. Use of the ADC according to any one of claims 1 to 26 for the manufacture of a pharmaceutical product.

30. Use of the ADC according to any one of claims 1 to 26 for the manufacture of a pharmacopoeia for treating FOLR1-expressing cancer, which is, if necessary, multiple myeloma, lung cancer, or ovarian cancer.

31. The composition according to claim 27, wherein the treatment comprises administering a therapeutically effective amount of one or more additional active agents.

32. The composition according to claim 31, wherein the additional active agent is a VEGF inhibitor, and optionally the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor.

33. The composition according to claim 32, wherein the VEGF inhibitor is bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, or regorafenib.

34. The composition according to claim 28, wherein the treatment further comprises administering a therapeutically effective amount of one or more additional active agents.

35. The composition according to claim 34, wherein the additional active agent is a VEGF inhibitor, and optionally the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor.

36. The composition according to claim 34, wherein the VEGF inhibitor is bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, or regorafenib.

37. A kit for treating FOLR1-expressing cancer, (i) The ADC according to any one of claims 1 to 26, and (ii) VEGF inhibitors A kit that includes this.

38. The kit according to claim 37, wherein the VEGF inhibitor is an anti-VEGF antibody or a small molecule VEGF inhibitor.

39. The kit according to claim 37, wherein the VEGF inhibitor is bevacizumab, ramucirumab, sunitinib, sorafenib, axitinib, pazopanib, or regorafenib.

40. below: 【Chemistry 114-1】 【Chemistry 114-2】 A compound selected from, or a pharmaceutically acceptable salt thereof.

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