Binding molecules for FRα

Abstract

The present disclosure provides binding molecules (e.g., antibodies or antigen-binding fragments thereof) and related antibody-drug conjugates to FRa, as well as pharmaceutical compositions and kits comprising the same. Methods of using the binding molecules and related antibody-drug conjugates in the treatment of cancer are also provided.

Description

[Technical field]

[0001] The present invention relates to binding molecules (eg, antibodies or binding fragments thereof) against FRα, and related antibody-drug conjugates, for the treatment of cancer. [Background technology]

[0002] Cancer remains one of the most prevalent diseases worldwide, despite years of research into the mechanisms of cancer development and the development of many potential anticancer drugs. In particular, lung and ovarian cancers are the third and fifth most common cancers in women, respectively. Chemotherapy and radiotherapy are the most common cancer treatments. Nevertheless, these treatments are linked to a variety of negative side effects, including fatigue, nausea, and hair loss. These problems are compounded by the fact that chemotherapy treatments are administered frequently and for extended periods of time. Over the past few decades, many antibody therapies for cancer have been developed and marketed, leading to a decrease in the need for traditional forms of chemotherapy for many cancer types. Although the availability of methods for making antibodies (e.g., monoclonal antibodies) has improved greatly throughout this period, there are relatively few clinically available anticancer antibodies, and even fewer that can be used to target a wide range of cancer types. Furthermore, there is a need to increase the efficacy of therapeutic antibodies, which is generally limited by the expression rate of the target antigen and the subsequent effects in cancer cells after antibody binding. Conjugating monoclonal antibodies to cytotoxic molecules to generate antibody-drug conjugates (ADCs) is a novel approach to deliver targeted therapy for malignant tumors. This approach has been successfully implemented with specific targets (HER2, CD30, and CD79b), leading to commercial ADCs such as fam-trastuzumab deruxtecan-nxki (breast and gastric cancer), brentuximab vedotin (Hodgkin's lymphoma), and polatuzumab vedotin-piiq (non-Hodgkin's lymphoma), respectively.

[0003] Folate receptors (FRs) are membrane-bound proteins present on the cell surface and therefore can be utilized to develop new ADCs. The FR family includes FRα, FRβ, FRγ, and FRδ. FRs bind folate molecules and transport them into cells, so that the folate molecules are delivered to the folate cycle and support the metabolism of nucleotides. In particular, folate is important for DNA synthesis, methylation, and repair (Non-Patent Document 1).

[0004] FRα is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high affinity for 5-methyltetrahydrofolate (5-MTF), the active form of folate. FRα is expressed by the FOLR1 gene. Previous studies have shown that FRα plays an important role in embryogenesis (Non-Patent Document 2). However, folate transport in adults is mainly driven by the ubiquitous expression of reduced folate carriers and proton-bound folate transporters (Non-Patent Document 3). The distribution of FRα expression in adults is usually restricted to the apical surface of polarized epithelia such as the choroid plexus, kidney, lung, and placenta.

[0005] Overexpression of FRα, also known as folate receptor 1 (FOLR1) or folate-binding protein (FBP), is frequently observed in tumor cells, such as ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), and breast carcinoma (Non-Patent Document 4). In particular, previous studies have found that the levels of soluble FRα are elevated in the blood of ovarian cancer patients, supporting the possibility of applying FRα as a biomarker for early ovarian cancer (Non-Patent Document 5). Preclinical ovarian models have also revealed that overexpression of FRα is associated with tumor progression, and that binding of folate to FRα can mediate activation of the oncogenic gene STAT3 (Non-Patent Document 6).

[0006] The antibodies against FRα in the art have drawbacks such as poor internalization, short half-life, and poor cytotoxicity. In addition, the FRα-targeted ADCs in the art use microtubule inhibitors that have been associated with certain toxicities in clinical trials, such as corneal inflammation (mirvetuximab soravtansine, consisting of anti-FRα antibody M9346A conjugated via a maytansinoid warhead DM4 and a sulfo-SPBD linker) (Non-Patent Document 7), interstitial lung disease (MORAb-202, consisting of humanized antibody farletuzumab derived from LK26 conjugated to an eribulin warhead) (Non-Patent Document 8), and neuropathy and neutropenia (STRO-002, consisting of anti-FRα antibody SP8166 conjugated to a hemiasterlin warhead) (Non-Patent Document 9). Recent studies have also found that cancer cells can acquire resistance to microtubule inhibitors (Non-Patent Document 10).

[0007] Moreover, recent FRα-targeting ADCs use antibodies with complex structures. For example, the ADC IMGN151 was developed from the ADC mirvetuximab soravtansine. The antibody of IMGN151 is an asymmetric, biparatopic, bispecific molecule that contains a knob-in-hole mutation that forces heterodimerization. Half of the antibody consists of IgG1 heavy and light chains, whose variable domains are shared with mirvetuximab. The other half consists of an scFv-Fc fusion protein that binds to a second, separate epitope on FRα. Modification of IgG may have negative consequences in terms of physiochemical properties (and therefore developability or manufacturability) and immunogenicity. Bispecific formats are known to have a tendency to reduce expression titers and increase aggregation, which may lead to more complex manufacturing processes and increased product costs. On the other hand, the antibody of STRO-002 contains the unnatural amino acid p-azidomethylphenylalanine (pAMF) at two designated sites on each heavy chain for site-specific conjugation of drugs. Introduction of unnatural amino acids, although specific, can require substantial cell line or cell-free engineering efforts and can result in lower mAb production titers.

[0008] Therefore, there is a need for the development of FRα-targeted ADCs that can deliver high concentrations of cytotoxic payloads to target cells and mediate highly effective tumor cell killing with less toxicity to patients. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Cheung,et al.,Oncotarget.2016;7(32):52553-52574 [Non-Patent Document 2] Kelemen, Int J Cancer.2006;119(2):243-250 [Non-Patent Document 3] Zhao,et al.,Annu Rev Nutr.2011;31:177-201 [Non-Patent Document 4] Shi,et al.,Drug Des Devel Ther.2015;9:4989-4996 [Non-Patent Document 5] Basal,et al.,PLoS One.2009;4(7):e6292 [Non-Patent Document 6] Hansen,et al.,Cell Signal.2015;27(7):1356-1368) [Non-Patent Document 7] Moore et al. (2017) Cancer 123:3080-7 [Non-Patent Document 8] Sato, et al. (2020) ESMO Abstract https: / / doi.org / 10.1016 / j.annonc.2020.01.026 [Non-Patent Document 9] Naumann, et al. (2021) J Clin Oncol 39(Suppl 15 / abstr 5550) https: / / doi10.1200 / JCO.2021.39.15_suppl.5550 [Non-Patent Document 10] Ganguly, et al., Biochim Biophys Acta.2011 Dec;1816(2):164-171 Summary of the Invention [Means for solving the problem]

[0010] The invention provides, inter alia, nucleic acid molecules, vectors, host cells, anti-FRα antibodies including pharmaceutical compositions, and kits including the pharmaceutical compositions, as well as uses of the pharmaceutical compositions including methods of treatment.

[0011] In one aspect, an anti-FRα antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment thereof comprising: (a) heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, light chain CDR2 (KASGLES) of SEQ ID NO: 5, and light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6; (b) heavy chain CDR1 (SYAMS) of SEQ ID NO: 7, heavy chain CDR2 (SISSGRSYIYYADSVKG) of SEQ ID NO: 8, heavy chain CDR3 (EMQQLALDY) of SEQ ID NO: 9, light chain CDR1 (RASQGISNFLA) of SEQ ID NO: 10, light chain CDR2 (AASSLQS) of SEQ ID NO: 11, and light chain CDR3 (QQYNSYPFT) of SEQ ID NO: 12; (c) heavy chain CDR1 (SNSAAWN) of SEQ ID NO: 13, heavy chain CDR2 (RTYYRSNWYNDYTLSVKS) of SEQ ID NO: 14, heavy chain CDR3 (GVGRFDS) of SEQ ID NO: 15, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 16, light chain CDR2 (KASSLES) of SEQ ID NO: 17, and light chain CDR3 (QEYKTYSIFT) of SEQ ID NO: 18; (d) heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, heavy chain CDR2 (SISSGSSYIYYADSMKG) of SEQ ID NO: 20, heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 21, light chain CDR1 (RASQGISTFLA) of SEQ ID NO: 22, light chain CDR2 (AASSLQS) of SEQ ID NO: 23, and light chain CDR3 (QQYISYPLT) of SEQ ID NO: 24; (e) a heavy chain CDR1 (SYSMN) of SEQ ID NO: 25, a heavy chain CDR2 (SISSRSSYVYYADSVKG) of SEQ ID NO: 26, a heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 27, a light chain CDR1 (RASQGISSFLA) of SEQ ID NO: 28, a light chain CDR2 (AASSLQS) of SEQ ID NO: 29, and a light chain CDR3 (QQYNSYPLT) of SEQ ID NO: 30; or (f) heavy chain CDR1 (SDSATWN) of SEQ ID NO: 31, heavy chain CDR2 (RTYYRSKWYSDYAVSVKS) of SEQ ID NO: 32, heavy chain CDR3 (GGAPFDY) of SEQ ID NO: 33, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 34, light chain CDR2 (KASSLES) of SEQ ID NO: 35, and light chain CDR3 (QQYNSYSMYT) of SEQ ID NO: 36. Includes.

[0012] In some embodiments of any aspect of the invention, the anti-FRα antibody, or antigen-binding fragment thereof, (a) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 37, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38; (b) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 39, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 40; (c) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 41, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 42; (d) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 43, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 44; (e) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 46; or (f) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48; Includes.

[0013] In some embodiments of any aspect of the invention, the anti-FRα antibody, or antigen-binding fragment thereof, (a) L at the N-terminus (e.g., position 1) of VH; (b) E at the N-terminus (e.g., position 1) of VH; or (c) Q at the N-terminus (e.g., position 1) of VH Includes.

[0014] In some embodiments of any aspect of the invention, the anti-FRα antibody, or antigen-binding fragment thereof, (a) a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (b) VH of SEQ ID NO: 39 and VL of SEQ ID NO: 40 (c) a VH of SEQ ID NO: 41 and a VL of SEQ ID NO: 42; (d) a VH of SEQ ID NO: 43 and a VL of SEQ ID NO: 44; (e) a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46; or (f) VH of SEQ ID NO: 47 and VL of SEQ ID NO: 48 Includes.

[0015] In some embodiments of any aspect of the invention, the anti-FRα antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 109 or 111, and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 110.

[0016] In some embodiments of any aspect of the invention, the anti-FRα antibody comprises the constant heavy chain amino acid sequence of SEQ ID NO:109 or 111 and the constant light chain amino acid sequence of SEQ ID NO:110.

[0017] In some embodiments of any aspect of the invention, the anti-FRα antibody is (a) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 49, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 50; (b) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 52; (c) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 54; (d) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 55, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 56; (e) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 57, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 58; or (f) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 59, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 60. Includes.

[0018] In some embodiments of any aspect of the invention, the anti-FRα antibody is (a) a heavy chain amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence of SEQ ID NO: 50; (b) a heavy chain amino acid sequence of SEQ ID NO:51 and a light chain amino acid sequence of SEQ ID NO:52; (c) a heavy chain amino acid sequence of SEQ ID NO: 53 and a light chain amino acid sequence of SEQ ID NO: 54; (d) a heavy chain amino acid sequence of SEQ ID NO: 55 and a light chain amino acid sequence of SEQ ID NO: 56; (e) a heavy chain amino acid sequence of SEQ ID NO: 57 and a light chain amino acid sequence of SEQ ID NO: 58; or (f) the heavy chain amino acid sequence of SEQ ID NO: 59 and the light chain amino acid sequence of SEQ ID NO: 60 Includes.

[0019] In some embodiments of any aspect of the invention, the antigen-binding fragment is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0020] In some embodiments of any of the aspects of the invention, the anti-FRα antibody or antigen-binding fragment thereof is humanized, chimeric or fully human, preferably the anti-FRα antibody or antigen-binding fragment thereof is fully human.

[0021] In some embodiments of any of the aspects of the invention, the anti-FRα antibody, or antigen-binding fragment thereof, is monoclonal, polyclonal, recombinant, or multispecific.

[0022] In some embodiments of any of the aspects of the invention, the anti-FRα antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 type, preferably of the IgG1 type.

[0023] In some embodiments of any of the aspects of the invention, the anti-FRα antibody, or antigen-binding fragment thereof, is conjugated to one or more heterologous agents.

[0024] In some embodiments of any aspect of the invention, the one or more heterologous agents are selected from the group consisting of a cytotoxin, an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a xenogenous antibody, a fragment of a xenogenous antibody, a detectable label, polyethylene glycol (PEG), a radioisotope, or a combination thereof.

[0025] In some embodiments of any of the aspects of the invention, the heterologous agent is a cytotoxin.

[0026] In another aspect, an antibody-drug conjugate (ADC) is provided comprising an anti-FRα antibody or antigen-binding fragment thereof of the present invention, wherein the anti-FRα antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0027] In some embodiments of any of the aspects of the invention, the cytotoxin is linked to a linker R selected from: L is linked to the anti-FRα antibody or antigen-binding fragment thereof via: [ka] [In the formula, Q is [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and; X is [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0)). and; G L is a linker for conjugating to an anti-FRα antibody or antigen-binding fragment thereof]; [ka] [In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1; or [ka] [In the formula, RL1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group.

[0028] In some embodiments of any aspect of the invention, G L teeth, [ka] It is.

[0029] In some embodiments of any aspect of the invention, R L teeth, [ka] It is.

[0030] In some embodiments of any aspect of the invention, the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof, hi preferred embodiments, the cytotoxin is a topoisomerase I inhibitor.

[0031] In another aspect, an ADC is provided that comprises an anti-FRα antibody, or antigen-binding fragment thereof, linked to a cytotoxin, where the cytotoxin is a topoisomerase I inhibitor.

[0032] In some embodiments of any of the aspects of the invention, the topoisomerase I inhibitor has formula (I): [ka] and salts and solvates thereof; (In the formula, R L is defined above).

[0033] In some embodiments of any of the aspects of the invention, the topoisomerase I inhibitor is: [ka] [ka] and preferably the topoisomerase I inhibitor is [ka] It is.

[0034] In some embodiments of any aspect of the invention, the drug-antibody ratio (DAR) is in the range of about 1-20, optionally with the DAR in a range selected from about 1-10, about 2-10, about 2-8, about 2-6, and about 4-10.

[0035] In some embodiments of any aspect of the invention, the DAR is about 8 or about 4, preferably the DAR is about 8.

[0036] In another aspect, an ADC comprising an anti-FRα antibody, or antigen-binding fragment thereof, linked to a cytotoxin, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2; a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3; a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4; a light chain CDR2 (KASGLES) of SEQ ID NO: 5; and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, optionally wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG3932. [ka] and (iii) the DAR is about 8; ADCs are provided.

[0037] In another aspect, an isolated polynucleotide encoding an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is provided.

[0038] In another aspect, (a) a polynucleotide operably linked to a promoter, or (b) a polynucleotide encoding a VH region as defined herein, and a polynucleotide encoding a VL region as defined herein, wherein the polynucleotide is operably linked to one or more promoters. A vector comprising the

[0039] In some embodiments, the vector further comprises a polynucleotide encoding a constant heavy chain region as defined in the present invention, and a polynucleotide encoding a constant light chain region as defined in the present invention.

[0040] In another aspect, there is provided a host cell comprising a polynucleotide of the invention or a vector of the invention.

[0041] In another aspect, there is provided a method for preparing an anti-FRα antibody or antigen-binding fragment thereof of the invention, comprising the steps of: (a) transfecting a host cell with a vector of the present invention; (b) culturing the host cell under conditions that allow synthesis of the antibody or antigen-binding fragment; and (c) recovering the antibody or antigen-binding fragment from the culture. A method is provided that includes:

[0042] In another aspect, a pharmaceutical composition is provided comprising an anti-FRα antibody of the invention or an antigen-binding fragment thereof, or an ADC of the invention, and a pharma- ceutically acceptable excipient.

[0043] In another aspect, there is provided an anti-FRα antibody or antigen-binding fragment thereof, an ADC of the invention, or a pharmaceutical composition of the invention for use in a method of depleting a population of FRa-positive cells in a subject, the method comprising administering the anti-FRα antibody or antigen-binding fragment thereof, the ADC, or the pharmaceutical composition to a subject. Typically, the FRa-positive cells are FRa-positive cancer cells.

[0044] In a related aspect, there is provided a method of depleting a population of FRa-positive cells in a subject, the method comprising administering to the subject an anti-FRα antibody or antigen-binding fragment thereof of the invention, an ADC of the invention, or a pharmaceutical composition of the invention. Typically, the FRa-positive cells are FRa-positive cancer cells.

[0045] In another aspect, there is provided an anti-FRα antibody or antigen-binding fragment thereof, an ADC of the invention, or a pharmaceutical composition of the invention for use in treating a cancer associated with FRa expression.

[0046] In a related aspect, a method for treating a cancer associated with FRa expression is provided, the method comprising administering to a subject an anti-FRα antibody or antigen-binding fragment thereof of the present invention, an ADC of the present invention, or a pharmaceutical composition of the present invention.

[0047] In some embodiments of any aspect of the invention, the cancer comprises cancer cells that have heterogeneous expression of FRα and / or low expression of FRα, and optionally the cancer cells have FRα expression similar to the Igrov-1 cell line.

[0048] In some embodiments of any aspect of the invention, the cancer is selected from ovarian cancer, lung cancer, endometrial cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinoma (HNSCC), breast cancer (e.g., TNBC), cervical cancer, and malignant pleural mesothelioma, preferably, the cancer is selected from ovarian cancer and lung cancer.

[0049] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), optionally wherein the NSCLC is selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof.

[0050] Aspects and embodiments of the invention are set out in the accompanying claims. These and other aspects and embodiments of the invention are also described herein.

[0051] The invention will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 shows the binding of (A) AB1370026, (B) AB1370035, (C) AB1370049, (D) AB1370083, (E) AB1370096 and (F) AB1370117 to human FRα, cynomolgus monkey FRα, mouse FRα, human FRβ or human FRγ by HTRF assay. [Diagram 2] Figure 2 shows a High Content Profiler multi-parameter analysis of antibody internalization into KB cells plotting intensity on the vertical axis against sample concentration on the horizontal axis. Antibodies with uptake above the cutoff determined by the positive control sample (circle) were shortlisted and shown as grey diamonds, and those below the cutoff as white diamonds. Data points for six exemplary antibodies run in duplicate in the assay are shown as black diamonds and labeled with the sample name. At least one sample per exemplary antibody was above the cutoff. [Diagram 3] FIG. 3 shows six exemplary IgGs in an HTRF epitope competition assay with (A) Comparator 1 IgG, (B) Comparator 2 IgG, and (C) Comparator 3 IgG. [Figure 4] Figure 4 shows the results of the AC-SINS self-interaction assay. The tendency of antibodies to self-associate in HSA (black column) buffer was examined. The negative control antibody showed a low level of self-interaction, while the positive control antibody shows a high level of self-interaction in HSA, as expected. The threshold for flagging at-risk antibodies (>5 nm) is indicated by the dashed horizontal line. [Diagram 5] FIG. 5 shows internalization of anti-FRα antibody into (A) Jeg-3 cells (moderately expressing FRα) and (B) KB cells (highly expressing FRα) over time, as measured by increase in fluorescence on a CX7 instrument. [Figure 6]FIG. 6 shows chromatograms of AB1370049-SG3932 DAR8 obtained by (A) UHPLC-RP (reduced) at 214 nm, (B) UHPLC-RP (reduced) at 330 nm, (C) UHPLC-SEC at 280 nm, and (D) UHPLC-HIC at 214 nm. [Figure 7] FIG. 7 shows chromatograms of AB1370049-SG3932 DAR4 obtained by (A) UHPLC-RP (reduced) at 214 nm, (B) UHPLC-RP (reduced) at 330 nm, (C) UHPLC-SEC at 280 nm, and (D) UHPLC-HIC at 214 nm. [Figure 8] 8 shows the progression of chemical conversion to a series of DAR8 ADCs upon exposure to various sera. The chemical processes observed are (A) deconjugation and (B) maleimide hydrolysis. [Figure 9] FIG. 9 shows a 6-day cytotoxicity assay with the lead DAR8 ADC against (A) KB cells (high expression of FRα), (B) Jeg-3 cells (medium to high expression of FRα), and (C) Igrov-1 cells (medium expression of FRα). [Figure 10] Figure 10 shows the bystander killing activity of AB1370049-SG3932 DAR8. FRα positive and FRα negative KB cells were treated with 1 nM AB1370049-SG3932 DAR8 ADC alone or at a 1:1 ratio for 6 days. After 6 days, cytotoxicity activity was measured by flow cytometry. [Figure 11] Figure 11 shows a KB xenograft study with the lead DAR8 ADC. A single dose of (A) 1.25 mg / kg or (B) 5 mg / kg was administered intravenously on day 6. Dose administration is indicated by a black asterisk. [Figure 12] Figure 12 shows the OVCAR-3 xenograft study with the lead DAR8 ADC. A single dose of (A) 1.25 mg / kg or (B) 5 mg / kg was administered intravenously on day 33. Dose administration is indicated by a black asterisk. [Figure 13]Figure 13 shows an IGROV-1 xenograft study with AB1370049-SG3932 DAR8. A single intravenous dose of 5 mg / kg was administered on day 33. [Figure 14] Figure 14 shows a KB xenograft study with the lead DAR4 ADC. A single intravenous dose of 5 mg / kg was administered on day 7. [Figure 15] Figure 15 shows (A) OVCAR-3 and (B) CaCo-2 transplant studies with single doses of 1.25, 2.5 and 5 mg / kg AB1370049-SG3932 DAR8 or FRα-DM4 ADC. Dose administration is indicated by black asterisks. [Figure 16] FIG. 16 shows median tumor growth rates following a single dose of (A) 5 mg / kg AB1370049-SG3932 DAR8 in 51 PDX models and (B) 2.5 mg / kg AB1370049-SG3932 DAR8 in 39 PDX models. [Figure 17] FIG. 17 shows PDX model studies of a single dose of 2.5 mg / kg or 5 mg / kg AB1370049-SG3932 DAR8 in (A) ovarian PDX model CTG-0711, (B) NSCLC PDX model CTG-2367, and (C) endometrial PDX model CTG-2268. [Figure 18] Figure 18 shows cell viability signals measured for (A) megakaryocytic progenitor cells, (B) myeloid progenitor cells, (C) erythroid progenitor cells, (D) megakaryocytic expanded and differentiated cells, (E) myeloid expanded and differentiated cells, and (F) erythroid expanded and differentiated cells. The X-axis represents drug concentration and the Y-axis represents percent viability (values ​​are mean- / +SD of triplicates). AB1370049-SG3932 DAR8 shows no worsening toxicity than non-targeted ADC control in primary CD34+ bone marrow derived hematopoietic stem progenitor cells induced to differentiate into erythroid, myeloid or megakaryocytic lineages. [Figure 19]Figure 19 shows the mean (±SD) concentration-time profiles of unconjugated mAb and AB1370049-SG3932 DAR8 in cynomolgus monkeys. PK profiles in 15 and 25 mg / kg plasma samples were collected and processed using an immunocapture LC-MS / MS assay and non-compartmental PK. Total mAb is a measure of intact antibody (in the case of ADC, this includes ADC or deconjugated mAb). Total ADC is a measure of intact ADC only. [Figure 20] FIG. 20 shows the mean tumor growth rate in OVO857 PDX models treated with a single dose of 1.25, 2.5, 5, and 10 mg / kg AB1370049-SG3932 DAR8, or 2.5 and 5 mg / kg FRα-DM4 ADC. [Figure 21] FIG. 21 shows the mean tumor growth rate in CTG3226 PDX models treated with a single dose of 1.25, 2.5, 5, and 10 mg / kg AB1370049-SG3932 DAR8, or 2.5 and 5 mg / kg FRα-DM4 ADC. [Figure 22] FIG. 22 shows the mean tumor growth rate in CTG-0956 PDX models treated with a single dose of 0.15, 0.3, 0.6, 1.25, 2.5, 5 and 10 mg / kg AB1370049-SG3932 DAR8. [Diagram 23] Figure 23 shows the average tumor growth rate in OVCAR3 xenograft models of four mice (designated 2_2718, 4_7C2A, 18_2146, and 27_2438, respectively) that received eight rounds of FRα-DM4ADC administered intravenously Q2W at 5 mg / kg, followed by two rounds of AB1370049-SG3932 DAR8 administered intravenously Q2W again at 5 mg / kg. Dashed vertical lines indicate the dose administered to the mice. [Figure 24]Figure 24 shows the mean tumor growth rate in an OVCAR3 xenograft model in which FRα-DM4-resistant tumors were isolated from mice and reimplanted into new host mice. After tumors reached an average size of 280-360 mm3, mice were dosed twice (Q2W) with 5 mg / kg of AB1370049-SG3932 DAR8 or FRα-DM4. The dashed vertical lines indicate the doses administered to the mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0054] Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; an amino acid sequence is written left to right in amino to carboxy orientation, respectively.

[0055] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, reference to "the agent" includes a reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth.

[0056] "About" may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurement method. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of the indicated value or range of values. Preferably, the term "about" is understood herein as plus or minus (±) 5% of the numerical value of the number used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%. An embodiment described herein as "comprising" one or more features may also be considered as a disclosure of a corresponding embodiment "consisting of" such features.

[0057] As used herein, amino acids are referred to using the amino acid name, three-letter abbreviation, or one-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter code for amino acids as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0058] Concentrations, amounts, volumes, ratios, and other numerical values ​​may be presented herein in a range format, with it being understood that such range formats are used merely for convenience and brevity and should be interpreted flexibly not only to include the numerical values ​​explicitly recited as the limits of the range, but also to include all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited.

[0059] Anti-FRα antibody The inventors developed an exemplary array of anti-FRα antibodies with high affinity and specific binding to FRα on cancer cells (e.g., not specifically binding to other FR family members such as FRβ and FRγ). The inventors performed a thorough evaluation of the antibody's developability, checking the tendency for reversible self-association, internalization, non-specific binding and hydrophobicity, as well as the stability of the mAbs against heat and light stress. In addition, the inventors used in vivo mouse PK studies for a focused panel of mAbs to remove those that showed short half-life and elevated clearance in vivo. To reduce the possibility of anti-drug antibody (ADA) reactions in humans, a computational immunogenicity assessment was performed to remove antibodies predicted to have an increased risk of ADA reactions. Through the above screening strategy, the inventors identified a panel of six antibodies with similar beneficial properties.

[0060] Antibody sequence The present invention encompasses antibodies or antigen-binding fragments as defined herein having the recited CDR sequences or variable heavy and light chain sequences (reference antibody) as well as functional variants thereof. The functional variants bind to the same target antigen as the reference antibody and preferably exhibit the same antigen cross-reactivity as the reference antibody. The functional variants may have different affinities for the target antigen when compared to the reference antibody, but preferably have substantially the same affinity.

[0061] In some embodiments, functional variants of a reference antibody show sequence variations in one or more CDRs when compared to the corresponding reference CDR sequence. Thus, functional antibody variants can include functional variants of CDRs. When the term "functional variant" is used in connection with a CDR sequence, it means that the CDR has at most two, preferably at most one amino acid difference compared to the corresponding reference CDR sequence, and when combined with the remaining five CDRs (or variants thereof), the variant antibody can bind to the same target antigen as the reference antibody and preferably exhibit the same antigen cross-reactivity as the reference antibody. A functional variant can be referred to as a "variant antibody".

[0062] Tables 1-5 show the CDR, VH and VL, heavy and light chain, FR and constant domain sequences of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117, respectively. In case of discrepancies, the sequences in the tables take precedence.

[0063] In one aspect, the anti-FRα antibody or antigen-binding fragment thereof comprises the six CDRs of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 in Table 2 (wherein the CDRs are determined by Kabat, Chothia or IMGT).

[0064] In another aspect of the invention, an anti-FRα antibody or antigen-binding fragment thereof is provided, the anti-FRα antibody or antigen-binding fragment thereof comprising: Heavy chain CDR1 of SEQ ID NO:1 (SDSATWN); Heavy chain CDR2 of SEQ ID NO:2 (RTYYRSKWYNDYAVSVKS); Heavy chain CDR3 of SEQ ID NO:3 (GVGSFDY); Light chain CDR1 of SEQ ID NO:4 (RASQSISSWLA); Light chain CDR2 of SEQ ID NO:5 (KASGLES); and Light chain CDR3 of SEQ ID NO:6 (QQYNSYSQLT) Including, Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0065] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, Heavy chain CDR1 of SEQ ID NO:1 (SDSATWN); Heavy chain CDR2 of SEQ ID NO:2 (RTYYRSKWYNDYAVSVKS); Heavy chain CDR3 of SEQ ID NO:3 (GVGSFDY); Light chain CDR1 of SEQ ID NO:4 (RASQSISSWLA); Light chain CDR2 of SEQ ID NO:5 (KASGLES); and Light chain CDR3 of SEQ ID NO:6 (QQYNSYSQLT) Includes.

[0066] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 37 and a VL of SEQ ID NO:38.

[0067] In some embodiments, the anti-FRα antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 49, and a light chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-FRα antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 49, and the light chain amino acid sequence of SEQ ID NO:50.

[0068] In another aspect of the invention, an anti-FRα antibody or antigen-binding fragment thereof is provided, the anti-FRα antibody or antigen-binding fragment thereof comprising: Heavy chain CDR1 of SEQ ID NO:7 (SYAMS); Heavy chain CDR2 of SEQ ID NO:8 (SISSGRSYIYYADSVKG); Heavy chain CDR3 of SEQ ID NO:9 (EMQQLALDY); Light chain CDR1 of SEQ ID NO:10 (RASQGISNFLA); Light chain CDR2 of SEQ ID NO: 11 (AASSLQS); and Light chain CDR3 of SEQ ID NO: 12 (QQYNSYPFT) Including, Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0069] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, Heavy chain CDR1 of SEQ ID NO:7 (SYAMS); Heavy chain CDR2 of SEQ ID NO:8 (SISSGRSYIYYADSVKG); Heavy chain CDR3 of SEQ ID NO:9 (EMQQLALDY); Light chain CDR1 of SEQ ID NO:10 (RASQGISNFLA); Light chain CDR2 of SEQ ID NO: 11 (AASSLQS); and Light chain CDR3 of SEQ ID NO: 12 (QQYNSYPFT) Includes.

[0070] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 39, and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 39 and a VL of SEQ ID NO: 40.

[0071] In some embodiments, the anti-FRα antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 51, and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-FRα antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 51, and the light chain amino acid sequence of SEQ ID NO:52.

[0072] In another aspect of the invention, an anti-FRα antibody or antigen-binding fragment thereof is provided, the anti-FRα antibody or antigen-binding fragment thereof comprising: Heavy chain CDR1 of SEQ ID NO: 13 (SNSAAWN); Heavy chain CDR2 of SEQ ID NO: 14 (RTYYRSNWYNDYTLSVKS); Heavy chain CDR3 of SEQ ID NO: 15 (GVGRFDS); Light chain CDR1 of SEQ ID NO: 16 (RASQSISSWLA); Light chain CDR2 of SEQ ID NO: 17 (KASSLES); and Light chain CDR3 of SEQ ID NO: 18 (QEYKTYSIFT) Including, Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0073] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, Heavy chain CDR1 of SEQ ID NO: 13 (SNSAAWN); Heavy chain CDR2 of SEQ ID NO: 14 (RTYYRSNWYNDYTLSVKS); Heavy chain CDR3 of SEQ ID NO: 15 (GVGRFDS); Light chain CDR1 of SEQ ID NO: 16 (RASQSISSWLA); Light chain CDR2 of SEQ ID NO: 17 (KASSLES); and Light chain CDR3 of SEQ ID NO: 18 (QEYKTYSIFT) Includes.

[0074] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 41, and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 41 and a VL of SEQ ID NO:42.

[0075] In some embodiments, the anti-FRα antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 53, and a light chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-FRα antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 53, and the light chain amino acid sequence of SEQ ID NO:54.

[0076] In another aspect of the invention, an anti-FRα antibody or antigen-binding fragment thereof is provided, the anti-FRα antibody or antigen-binding fragment thereof comprising: Heavy chain CDR1 of SEQ ID NO: 19 (SYNMN); Heavy chain CDR2 of SEQ ID NO:20 (SISSGSSYIYYADSMKG); Heavy chain CDR3 of SEQ ID NO:21 (GMTTLTFDY); Light chain CDR1 of SEQ ID NO:22 (RASQGISTFLA); Light chain CDR2 of SEQ ID NO: 23 (AASSLQS); and Light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT) Including, Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0077] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, Heavy chain CDR1 of SEQ ID NO: 19 (SYNMN); Heavy chain CDR2 of SEQ ID NO:20 (SISSGSSYIYYADSMKG); Heavy chain CDR3 of SEQ ID NO:21 (GMTTLTFDY); Light chain CDR1 of SEQ ID NO:22 (RASQGISTFLA); Light chain CDR2 of SEQ ID NO: 23 (AASSLQS); and Light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT) Includes.

[0078] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 43, and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 43 and a VL of SEQ ID NO:44.

[0079] In some embodiments, the anti-FRα antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 55, and a light chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the anti-FRα antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 55, and the light chain amino acid sequence of SEQ ID NO:56.

[0080] In another aspect of the invention, an anti-FRα antibody or antigen-binding fragment thereof is provided, the anti-FRα antibody or antigen-binding fragment thereof comprising: Heavy chain CDR1 of SEQ ID NO:25 (SYSMN); Heavy chain CDR2 of SEQ ID NO:26 (SISSRSSYVYYADSVKG); Heavy chain CDR3 of SEQ ID NO:27 (GMTTLTFDY); Light chain CDR1 of SEQ ID NO:28 (RASQGISSFLA); Light chain CDR2 of SEQ ID NO: 29 (AASSLQS); and Light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT) Including, Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0081] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, Heavy chain CDR1 of SEQ ID NO:25 (SYSMN); Heavy chain CDR2 of SEQ ID NO:26 (SISSRSSYVYYADSVKG); Heavy chain CDR3 of SEQ ID NO:27 (GMTTLTFDY); Light chain CDR1 of SEQ ID NO:28 (RASQGISSFLA); Light chain CDR2 of SEQ ID NO: 29 (AASSLQS); and Light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT) Includes.

[0082] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 45, and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 45 and a VL of SEQ ID NO:46.

[0083] In some embodiments, the anti-FRα antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 57, and a light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the anti-FRα antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 57, and the light chain amino acid sequence of SEQ ID NO:58.

[0084] In another aspect of the invention, an anti-FRα antibody or antigen-binding fragment thereof is provided, the anti-FRα antibody or antigen-binding fragment thereof comprising: Heavy chain CDR1 of SEQ ID NO:31 (SDSATWN); Heavy chain CDR2 of SEQ ID NO:32 (RTYYRSKWYSDYAVSVKS); Heavy chain CDR3 of SEQ ID NO:33 (GGAPFDY); Light chain CDR1 of SEQ ID NO:34 (RASQSISSWLA); Light chain CDR2 of SEQ ID NO: 35 (KASSLES); and Light chain CDR3 of SEQ ID NO: 36 (QQYNSYSMYT) Including, Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0085] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, Heavy chain CDR1 of SEQ ID NO:31 (SDSATWN); Heavy chain CDR2 of SEQ ID NO:32 (RTYYRSKWYSDYAVSVKS); Heavy chain CDR3 of SEQ ID NO:33 (GGAPFDY); Light chain CDR1 of SEQ ID NO:34 (RASQSISSWLA); Light chain CDR2 of SEQ ID NO: 35 (KASSLES); and Light chain CDR3 of SEQ ID NO: 36 (QQYNSYSMYT) Includes.

[0086] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof has a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 47, and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO: 47 and a VL of SEQ ID NO:48.

[0087] In some embodiments, the anti-FRα antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 59, and a light chain comprising an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 60. In some embodiments, the anti-FRα antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 59, and the light chain amino acid sequence of SEQ ID NO: 60.

[0088] In some embodiments of any of the aspects described herein, the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain VH FR1, VH FR2, VH FR3 and / or VH FR4 that is at least 80%, 85%, 90% or 95% identical or identical to the reference heavy chain VH FR1, VH FR2, VH FR3 and / or VH FR4, respectively, of any one of the constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 4, and the antibody or fragment alone (e.g., in the form of a single chain antibody fragment) is capable of binding to FRα.

[0089] In some embodiments of any of the aspects described herein, the anti-FRα antibody or antigen-binding fragment thereof comprises a light chain VL FR1, VL FR2, VL FR3 and / or VL FR4 that is at least 80%, 85%, 90% or 95% identical or identical to the reference light chain VL FR1, VL FR2, VL FR3 and / or VL FR4, respectively, of any one of the constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 4, and the antibody or fragment alone (e.g., in the form of a single chain antibody fragment) is capable of binding to FRα.

[0090] In some embodiments of any of the aspects described herein, the anti-FRα antibody or antigen-binding fragment thereof comprises: (a) a light chain VL FR1, VL FR2, VL FR3 and VL FR4 that are at least 80%, 85%, 90% or 95% identical or identical to the reference light chain VL FR1, VL FR2, VL FR3 and VL FR4, respectively, of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 4; and (b) a reference heavy chain VH FR1, VH FR2, VH FR3 and VH FR4, respectively, of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 4. The present invention comprises heavy chain VH FR1, VH FR2, VH FR3 and VH FR4 which are at least 80%, 85%, 90% or 95% identical or identical to VH FR1, VH FR2, VH FR3 and VH FR4, respectively.

[0091] In some embodiments of any of the aspects described herein, the anti-FRα antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 109, and a constant light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-FRα antibody comprises the constant heavy chain amino acid sequence of SEQ ID NO: 109, and the constant light chain amino acid sequence of SEQ ID NO:110.

[0092] In some embodiments of any of the antigen-binding fragment aspects described herein, the anti-FRα antigen-binding fragment comprises a constant heavy chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 111, and a constant light chain comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-FRα antigen-binding fragment comprises the constant heavy chain amino acid sequence of SEQ ID NO: 111, and the constant light chain amino acid sequence of SEQ ID NO:110.

[0093] [Table 1]

[0094] [Table 2]

[0095] [Table 3]

[0096] [Table 4]

[0097] [Table 5]

[0098] [Table 6]

[0099] [Table 7]

[0100] [Table 8]

[0101] [Table 9]

[0102] Minor variations in the amino acid sequence of the antibodies of the invention are considered to be encompassed by the present invention, provided that the variations in amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, and most preferably at least 99% sequence identity with the antibodies or antigen-binding fragments thereof of the invention as defined anywhere herein.

[0103] The antibodies of the present invention may include variants in which an amino acid residue from one species is substituted with the corresponding residue in another species at a conserved or non-conserved position. In some embodiments, an amino acid residue at a non-conserved position is substituted with a conserved or non-conserved residue. In particular, conservative amino acid substitutions are contemplated.

[0104] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include, for example, basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in the antibodies of the invention does not exclude other forms of variants, such as polymorphic variants, interspecies homologs, and alleles.

[0105] "Non-conservative amino acid substitutions" include (i) a residue having a positively charged side chain (e.g., Arg, His, or Lys) is replaced with or by a negatively charged residue (e.g., Glu or Asp); (ii) a hydrophilic residue (e.g., Ser or Thr) is replaced with or by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) a cysteine ​​or proline is replaced with or by any other residue; or (iv) a residue having a large hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is replaced with or by one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

[0106] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. The antibodies of the invention may also include unnatural amino acid residues.

[0107] Unnatural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing E. coli S30 extracts and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996). In a third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of a desired unnatural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine), and the unnatural amino acid is incorporated into the polypeptide in place of its natural counterpart.See Koide et al., Biochem. 33:7470-6, 1994. In vitro chemical modification can convert naturally occurring amino acid residues to non-naturally occurring species. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0108] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0109] Essential amino acids in the polypeptides of the invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structures as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of amino acids at putative contact sites. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the antibodies of the invention (e.g., translocation component or protease component).

[0110] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed in Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of permissible substitutions at each position. Other methods that may be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0111] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, and the length of each gap. The sequence comparison and percent identity determination between two or more sequences can be performed using a specific mathematical algorithm, such as BLAST, which is well known to those skilled in the art.

[0112] Percent identity can be determined using any of a variety of sequence alignment methods, including but not limited to global methods, local methods, and hybrid methods such as, for example, segment approach methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by applying gap penalties. Non-limiting methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994)), and iterative refinement methods (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J.MoI. Biol. 823-838(1996)). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509(1992)), Gibbs sampling (see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214(1993)), and Align-M (see, e.g., Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435(2004)).

[0113] Percent sequence identity can be determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by standard one-letter code):

[0114] In some embodiments, both the heavy and light chain variable domains of the antibody or antigen-binding fragment thereof are modified by at least partial replacement of one or more CDRs and / or by replacement and sequence alteration of partial framework regions. The CDRs may be derived from antibodies of the same class or subclass as the antibody from which the framework regions are derived, but it is envisaged that the CDRs are derived from antibodies of different classes, and in certain embodiments, from antibodies of different species. To transfer the antigen-binding capacity of one variable domain to another, it is not necessary to replace all CDRs with the complete CDRs of the donor variable region. Rather, it is sufficient to transfer only those residues necessary to maintain the activity of the antigen-binding site. Given the explanations in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it is well within the capabilities of the skilled artisan to carry out routine experimentation to obtain functional antibodies with reduced immunogenicity.

[0115] In some embodiments, an antibody or antigen-binding fragment thereof can include a heavy chain constant region or fragment thereof in addition to a VH and a VL, in some embodiments, the heavy chain constant region is a human heavy chain constant region, such as a human IgG constant region, such as a human IgG1 constant region.

[0116] In some embodiments (preferably where the antibody or antigen-binding fragment thereof is conjugated to an agent, such as a cytotoxic agent), residues are inserted into the heavy chain constant region for site-specific conjugation. For example, a cysteine ​​residue may be inserted between amino acids S239 and V240 in the CH2 region of IgG1, which may be referred to as a "239 insertion" or "239i".

[0117] In some embodiments, the antibodies disclosed herein may include modifications or alterations to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some embodiments, modified constant regions in which one or more domains are partially or completely deleted are contemplated. In some embodiments, the modified antibodies will include domain deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the deleted constant region domain may be replaced by a short amino acid spacer (e.g., 10 residues) that provides some molecular flexibility normally conferred by the missing constant region. Deleting or inactivating a constant region domain (by point mutation or other means) may reduce Fc receptor binding of the modified antibody in circulation. In other cases, modifying the constant region may modulate complement binding, thus reducing serum half-life and non-specific association of the complexed cytotoxin, consistent with the present disclosure. Yet other modifications of the constant region can be used to remove disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility, hi some embodiments, the antibody or antigen-binding fragment thereof has no antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity.

[0118] In some embodiments, antibodies or antigen-binding fragments thereof may be engineered to fuse the CH3 domain directly to the hinge region of the individual modified antibodies or fragments thereof. In other constructs, a peptide spacer may be inserted between the hinge region and the modified CH2 and / or CH3 domain. For example, a compatible construct may be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a spacer of 5-20 amino acids. Adding such a spacer may ensure, for example, that the regulatory elements of the constant domain remain free and available or that the hinge region remains mobile. In some cases, amino acid spacers are found to be immunogenic and induce undesirable immune responses against the construct. In some embodiments, any spacers added to the construct may be relatively non-immunogenic or omitted entirely to maintain the desired biochemical qualities of the modified antibodies.

[0119] In addition to the deletion of the entire constant region domain, the antibodies or antigen-binding fragments thereof provided herein may be modified by partial deletion or substitution of several or single amino acids in the constant region. For example, the mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase tumor localization. Similarly, one or more constant region domains that control effector functions (e.g., complement C1Q binding) may be deleted in whole or in part. Such partial deletion of the constant region may improve selected properties of the antibody or antigen-binding fragment thereof (e.g., serum half-life) while leaving other desirable functions associated with the constant region domain of interest intact. Furthermore, the constant regions of the antibodies and antigen-binding fragments thereof may be modified by the mutation or substitution of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenicity profile of the modified antibody or antigen-binding fragment thereof. In some embodiments, there may be one or more amino acid additions to the constant region to enhance a desired property, such as reduced or increased effector function, or to provide more cytotoxin or carbohydrate binding. In some embodiments, it may be desirable to insert or duplicate a particular sequence from a selected constant region domain. In some embodiments, the heavy chain constant region or fragment thereof, such as a human IgG constant region or fragment thereof, may contain one or more amino acid substitutions compared to a wild-type IgG constant domain, where the modified IgG has an extended half-life compared to the half-life of an IgG having a wild-type IgG constant domain. For example, the IgG constant domain may contain one or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, where the numbering of the amino acid positions is according to the EU index as set forth in Kabat.In some embodiments, the IgG constant domain contains a substitution of the amino acid at Kabat position 252 with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T); a substitution of the amino acid at Kabat position 254 with threonine (T); a substitution of the amino acid at Kabat position 256 with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T); a substitution of the amino acid at Kabat position 257 with leucine (L); a substitution of the amino acid at Kabat position 309 with proline (P); the amino acid at Kabat position 428 with threonine (T), leucine (L), phenylalanine (F) or serine (S); the amino acid at Kabat position 433 with arginine (R), serine (S), isoleucine (I), proline (P) or glutamine (Q); or the amino acid at Kabat position 434 with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F) or tyrosine. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a YTE variant. The term "YTE" or "YTE variant" refers to a mutation in IgG1 Fc that results in increased binding to human FcRn and improves the serum half-life of antibodies carrying this mutation. The YTE variant contains a combination of three mutations introduced into the heavy chain of IgG1: M252Y / S254T / T256E (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC). See U.S. Patent No. 7,658,921, which is incorporated herein by reference.The YTE variants have been shown to increase the serum half-life of antibodies by approximately four-fold when compared to the wild-type version of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57, 6147-6153). See also U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety.

[0120] In some embodiments, the anti-FRα antibody, or antigen-binding fragment thereof, L at the N-terminus of VH (e.g., position 1); E at the N-terminus (e.g., position 1) of VH; or Q at the N-terminus of VH (e.g., position 1) Includes.

[0121] Definitions and Antibody Formats As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen.

[0122] The antibody of the present invention is generally an isolated antibody or a recombinant antibody. "Isolated" as used herein refers to a polypeptide, e.g., an antibody, that is identified, separated and / or recovered from the cell or cell culture in which it is expressed. Usually, an isolated antibody is prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificity. For example, an isolated antibody that specifically binds to FRα is substantially free of antibodies that specifically bind to antigens other than FRα.

[0123] Generally, an antibody comprises at least two "light chains" (LC) and two "heavy chains" (HC). The light and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region (abbreviated herein as "CH"). The heavy chain constant region comprises heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD and IgG), and optionally a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (abbreviated herein as "VL") and a light chain constant region (abbreviated herein as "CL").

[0124] In some embodiments, the antibody is a full-length antibody. An "intact" or "full-length" antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0125] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable region VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). Preferably, each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of an antibody may further comprise all or a portion of a heavy chain constant region or a light chain constant region.

[0126] The binding between an antibody and its target antigen or epitope is mediated by CDRs. The term "epitope" refers to a region of a target protein (e.g., a polypeptide) that can be bound (e.g., bound) to an antibody or antigen-binding fragment of the present invention. CDRs are the primary determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variation (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, the art may use a combination of these two approaches to determine CDRs.

[0127] The sequences of the CDRs can be identified with reference to any numbering system known in the art, such as the Kabat system (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia &,Lesk, "Canonical Structures for the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., "IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig superfamily V-like domains," Dev. Comp. Immunol. 27, 55-77 (2003)) (see Table 6).

[0128] [Table 10]

[0129] The "constant domains" (or "constant regions") of the heavy and light chains are not directly involved in binding of the antibody to the target, but exhibit various effector functions. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0130] There are five major classes of heavy chain constant regions, classified as IgA, IgG, IgD, IgE, and IgM, each with characteristic effector functions designated by the isotype. Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific for the IgG class of antibodies: FcγRI, FcγRII, and FcγRIII. Binding of antibodies to cell surface Fc receptors induces many important and diverse biological responses, such as engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental passage, and control of immunoglobulin production. Sequences important for IgG binding to FcγR receptors have been reported to be located in the CH2 and CH3 domains.

[0131] In a preferred embodiment, the anti-FRα antibody or antigen-binding fragment thereof is an IgG isotype. The anti-FRα antibody or antigen-binding fragment thereof can be any IgG subclass, for example, IgG1, IgG2, IgG3 or IgG4 isotype. In a preferred embodiment, the anti-FRα antibody or antigen-binding fragment thereof is based on the IgG1 isotype. The use of wild-type human IgG1 molecules, which are closer to natural IgG, can reduce developability and other risks. For example, the inventors have devised an ADC that uses a human IgG1 mAb structure, which is believed to be less immunogenic than other anti-FRα ADCs being developed, such as IMGN151, without being bound by theory.

[0132] The numbering of the heavy chain constant region amino acid positions discussed in the antibodies of the present invention is according to the EU index as originally described in Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63(1969):78-85). Edelman's EU numbering is also described in Kabat et al. (1991) (supra). Thus, the term "EU index as described in Kabat", "EU index", "Kabat's EU index" or "EU numbering" in the context of heavy chains refers to the residue numbering system based on the human IgG1 EU antibody of Edelman et al. as described in Kabat et al. (1991). The numbering system used for light chain constant region amino acid sequences is similarly described in Kabat et al. (supra). Thus, as used herein, "numbered according to Kabat" refers to the Kabat numbering system as described in Kabat et al. (supra).

[0133] The terms "Fc region", "Fc portion" and "Fc" are used interchangeably herein and refer to the portion of a native immunoglobulin formed by two Fc chains. Each "Fc chain" comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. Native Fc regions are homodimers. In some embodiments, Fc regions may comprise modifications to force Fc heterodimerization and therefore may be heterodimers. Fc regions comprise carbohydrate moieties and binding sites for complement and Fc receptors (including the FcRn receptor) and do not have antigen binding activity. Fc may refer to this region independently or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at several positions in the Fc domain, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, so there may be slight differences between the sequences presented in this application and those known in the art. As a result, all naturally occurring IgG Fc regions are referred to as "wild type IgG Fc domains" or "WT IgG Fc domains" (i.e., any allele). The sequences of the heavy chains of human IgG1, IgG2, IgG3 and IgG4 can be obtained in various sequence databases, such as the Uniprot database (www.uniprot.org) under the accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN) and P01861 (IGHG4_HUMAN), respectively.

[0134] In some embodiments, the anti-FRα antibody of the present invention is a monoclonal antibody. A "monoclonal antibody" (mAb) refers to a homogeneous antibody population involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which typically includes various antibodies directed against various antigenic determinants. The term "monoclonal antibody" encompasses both full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced in various ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. More preferably, the anti-FRα antibody of the present invention is an isolated monoclonal antibody. In a more preferred embodiment, the antibody is a fully human monoclonal antibody. In another embodiment, the method of the present invention may use a polyclonal antibody.

[0135] The anti-FRα antibodies and antigen-binding fragments thereof of the present invention can be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments can be mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in human administration, antibodies or antigen-binding fragments of non-human origin can be genetically or structurally modified to be less antigenic when administered to a human patient. Human or humanized antibodies, particularly recombinant human or humanized antibodies, are particularly preferred.

[0136] The term "human antibody" refers to an antibody produced in a human or an antibody having an amino acid sequence corresponding to an antibody produced in a human, made using techniques known in the art. Human antibodies can include intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, such as an antibody comprising a murine light chain and a human heavy chain polypeptide. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or in genetic recombination, or by somatic mutation in vivo). Human antibodies can be made in human cells (via recombinant expression), non-human animals, or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (such as heavy and light chain) genes. Linker peptides not present in naturally occurring human antibodies can be included in single-chain human antibodies. For example, Fv can have a linker peptide, such as 2 to about 8 glycine or other amino acid residues, linking the variable region of the heavy chain and the variable region of the light chain. These linker peptides are believed to be of human origin. Human antibodies can be made using a variety of techniques, including phage display technology using antibody libraries derived from human immunoglobulin sequences. Transgenic mice which are incapable of expressing functional native immunoglobulins, but which can express human immunoglobulin genes, can also be used to make human antibodies (see, e.g., WO 1998 / 24893, WO 1992 / 01047, WO 1996 / 34096, WO 1996 / 33735, U.S. Pat. No. 5,313,631, and U.S. Pat. No. 5,313,631). See, for example, US Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771 and 5,939,598 (each of which is incorporated herein by reference).Human antibodies can also be prepared directly using various techniques known in the art. Immortalized human B lymphocytes can be generated by in vitro immunization or by isolation from immunized individuals that produce antibodies against the target antigen. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147(1): 86-95 (1991); U.S. Patent No. 5,750,373.

[0137] The term "humanized antibody" refers to an antibody in which the framework or CDRs have been modified to include CDRs of an immunoglobulin of different specificity compared to those of the parent immunoglobulin. For example, murine CDRs can be grafted into the framework regions of a human antibody to prepare a "humanized antibody" (see, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270). In some embodiments, a "humanized antibody" is one in which the constant regions have been additionally modified or altered from those of the original antibody to generate desired properties.

[0138] Humanized antibodies can optionally be prepared by a process of analysis of parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display predicted three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen, such as FRα. In this way, FR residues can be selected and combined from the consensus and import sequences to achieve the desired antibody characteristic, such as increased affinity for the target antigen.

[0139] Humanized antibodies can be further modified by substituting additional residues in the Fv framework regions and / or in the replaced non-human residues to refine and optimize antibody specificity, affinity and / or capacity. Generally, a humanized antibody will contain substantially all of at least one, typically two or three, of the variable domains that contain all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. A humanized antibody can also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. Nos. 5,225,539 and 5,639,641, each of which is incorporated herein by reference.

[0140] The term "chimeric antibody" refers to an antibody, usually prepared by recombinant DNA technology, that contains a variable region, i.e., a binding region, from one source or species, and at least a portion of a constant region from a different source or species. Chimeric antibodies containing a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or altered from that of the original antibody to generate desired properties. Such chimeric antibodies are also called "class-switched antibodies." Chimeric antibodies are the product of expressed immunoglobulin genes that contain DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies, including conventional recombinant DNA and gene transfection techniques, are well known in the art. See, e.g., Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244. Each of these documents is incorporated herein by reference.

[0141] In some embodiments, the antibody of the present invention is the above-mentioned full-length antibody. Alternatively, the antibody can be an antigen-binding fragment. The term "antigen-binding fragment" as used herein includes an antigen-binding polypeptide consisting of a naturally occurring or artificially constructed structure that includes one, two or three light chain CDRs and / or one, two or three heavy chain CDRs, where the polypeptide can bind to an antigen.

[0142] In some embodiments, the antigen-binding fragment of the invention is a Fab fragment.An antibody according to the invention can also be a Fab', Fv, scFv, Fd, VNAR domain, IgNAR, intrabody, IgG CH2, minibody, single domain antibody, Fcab, scFv-Fc, F(ab')2, di-scFv, bispecific T cell engager (BiTE®), F(ab')3, tetrabody, triabody, diabody, DVD-Ig, (scFv)2, mAb2 or DARPin.

[0143] The terms "Fab fragment" and "Fab" are used interchangeably herein and comprise a single light chain (e.g., constant domains CL and VL) and a single heavy chain (e.g., constant domains CH1 and VH). The heavy chain of a Fab fragment cannot form disulfide bonds with another heavy chain.

[0144] A "Fab' fragment" contains a single light chain and a single heavy chain, but in addition to CH1 and VH, the "Fab' fragment" contains the region of the heavy chain between the CH1 and CH2 domains that is required for interchain disulfide bond formation. Thus, two "Fab' fragments" can associate through disulfide bond formation to form a F(ab')2 molecule.

[0145] A "F(ab')2 fragment" contains two light chains and two heavy chains, each of which contains a portion of the constant region necessary for interchain disulfide bond formation between the two heavy chains.

[0146] An "Fv fragment" contains only the variable regions of the heavy and light chains. It does not contain the constant regions.

[0147] A "single domain antibody" is an antibody fragment containing a single antibody domain unit (eg VH or VL).

[0148] A "single-chain Fv" ("scFv") is an antibody fragment that contains the VH and VL domains of an antibody linked together to form a single chain. A polypeptide linker is generally used to connect the VH and VL domains of an scFv.

[0149] "Tandem scFv," also known as TandAb®, is a single-chain Fv molecule formed by covalently linking two scFvs in a tandem orientation using a flexible peptide linker.

[0150] "Bispecific T cell engagers" (BiTEs®) are fusion proteins consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to a tumor cell antigen.

[0151] "Diabodies" are small bivalent bispecific antibody fragments in which a heavy chain variable domain (VH) is linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with complementary domains on another chain and promotes the assembly of a dimeric molecule with two functional antigen-binding sites.

[0152] "DARPins" are bispecific ankyrin repeat molecules. DARPins are derived from the natural ankyrin protein, which can be found in the human genome and is one of the most abundant types of binding proteins. DARPin library modules are defined by the natural ankyrin repeat protein sequence, using 229 ankyrin repeats for the initial design and another 2200 ankyrin repeats for the subsequent refinement. Modules serve as building blocks of the DARPin library. The library modules resemble the human genome sequence. DARPins are composed of 4-6 modules. Each module is about 3.5 kDa, so the size of an average DARPin is 16-21 kDa. The selection of binders is performed by ribosome display, which is completely cell-free and described in He M.and Taussig MJ.,Biochem Soc Trans. 2007,Nov;35(Pt5):962-5.

[0153] In some embodiments, the anti-FRα antibodies and antigen-binding fragments thereof of the present invention are naked antibodies. As used herein, the term "naked antibody" refers to an antibody that is not conjugated to a therapeutic agent, such as a cytotoxic agent or a radiolabel. In an embodiment, the antibody or antigen-binding fragment thereof is a naked monospecific antibody. In an alternative preferred embodiment, the antibody or antigen-binding fragment thereof is conjugated to one or more heterologous agents (e.g., cytotoxic agents).

[0154] In some embodiments, the antibody or antigen-binding fragment thereof may be further modified to contain additional chemical moieties that are not normally part of the protein. These derivatized moieties may improve the solubility, biological half-life, or absorption of the protein. These moieties may also reduce or eliminate any undesirable side effects of the protein, etc. A review of such moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0155] FRα binding In a preferred embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the present invention specifically binds to FRα. The term "specifically binds to FRα" refers to an antibody that can bind to a defined target with sufficient affinity such that the antibody is useful as a therapeutic agent targeting FRα. In some embodiments, an antibody that specifically binds to FRα does not bind to other antigens or does not bind to other antigens with sufficient affinity to produce a physiological effect. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention specifically binds to human FRα (UniProt ID: P15328) and / or cynomolgus monkey FRα (UniProt ID: A0A2K5U044). In a particularly preferred embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the present invention specifically binds to human FRα. In a preferred embodiment, the anti-FRα antibody or antigen-binding fragment thereof of the present invention specifically binds to human FRα and cynomolgus monkey FRα.

[0156] In some embodiments of any aspect of the invention, FRα has the sequence of SEQ ID NO: 112 or SEQ ID NO: 113. In preferred embodiments, FRα has the sequence of SEQ ID NO:112. SEQ ID NO: 112: Human FRα protein (predicted mature secreted polypeptide) [ka] SEQ ID NO: 113: Cynomolgus monkey FRα protein (predicted mature secreted polypeptide) [ka]

[0157] In some embodiments, the antibody or antigen-binding fragment thereof does not bind to one or more selected from mouse FRα (UniProt ID: P35846), rat FRα (UniProt ID: G3V8M6), human FRβ (UniProt ID: P14207), human FRγ (UniProt ID: P41439), or a combination thereof.

[0158] The term "does not bind" means that the antibody or antigen-binding fragment thereof of the present invention does not substantially bind to one or more of the above molecules (e.g., mouse FRα, rat FRα, human FRβ, human FRγ, or a combination thereof). The term "substantially free" as used herein in the context of binding can mean that less than 5%, 2%, 1%, 0.5%, or 0.1% of cells expressing one or more of the above molecules in a cell culture become bound by (when contacted with) the antibody or antigen-binding fragment thereof of the present invention. Suitably, the term "substantially free" as used herein in the context of binding can mean that no such cells become bound.

[0159] binding affinity "Binding affinity" generally refers to the overall strength 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 inherent binding affinity that reflects a 1:1 interaction between binding pair members (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high affinity antibodies generally bind antigens more rapidly and tend to remain bound longer.

[0160] Preferably, the antibodies or antigen-binding fragments of the invention bind to the FRα molecule with sufficient affinity such that the antibodies are useful as therapeutic or diagnostic agents targeted to FRα.

[0161] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof binds to human FRα with a KD of about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, or about 1 nM or less.

[0162] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof binds to human FRα with a KD of about 0.5 to about 50 nM, about 0.5 to about 40 nM, about 0.5 to about 30 nM, about 0.5 to about 20 nM, about 1 to about 50 nM, about 1 to about 40 nM, about 1 to about 30 nM, about 1 to about 20 nM, about 2 to about 50 nM, about 2 to about 40 nM, about 2 to about 30 nM, about 2 to about 20 nM, about 5 to about 50 nM, about 5 to about 40 nM, about 5 to about 30 nM, about 5 to about 20 nM, about 10 to about 50 nM, about 10 to about 40 nM, about 10 to about 30 nM, or about 10 to about 20 nM.

[0163] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof binds to cynomolgus FRα with a KD of about 100 nM or less, about 80 nM or less, about 60 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, or about 2 nM or less.

[0164] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof is about 1 to about 100 nM, about 1 to about 80 nM, about 1 to about 60 nM, about 1 to about 40 nM, about 2 to about 100 nM, about 2 to about 80 nM, about 2 to about 60 nM, about 2 to about 40 nM, about 5 to about 100 nM, about 5 to about 80 nM, about 5 to about 60 nM, about 5 to about 40 nM, It binds to cynomolgus monkey FRα with a KD of about 10 to about 100 nM, about 10 to about 80 nM, about 10 to about 60 nM, about 10 to about 40 nM, about 20 to about 100 nM, about 20 to about 80 nM, about 20 to about 60 nM, about 20 to about 40 nM, about 30 to about 100 nM, about 30 to about 80 nM, about 30 to about 60 nM, or about 30 to about 40 nM.

[0165] The affinity or avidity of an antibody or antigen-binding fragment thereof for an antigen can be determined experimentally using any suitable method well known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) or kinetics (e.g., KINEXA® or BIACORE™ analysis). Direct binding assays and competitive binding assay formats can be readily used (see, e.g., Berzofsky et al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992); and methods described herein).

[0166] According to the present invention, the binding affinity of the anti-FRα antibody or antigen-binding fragment thereof of the present invention can be determined using the FRα binding affinity assay described herein. In some embodiments, the binding affinity of the anti-FRα antibody or antigen-binding fragment thereof of the present invention is determined by Biacore, e.g., Biacore T200, at 25° C. For example, the affinity of recombinant human FRα ECD for the anti-FRα antibody or antigen-binding fragment thereof can be measured at 25° C. using Biacore T200, e.g., using the following protocol. Protein A is covalently immobilized on the CM5 chip surface at a concentration of 50 μg / ml in 10 mM sodium acetate pH 4.0 using standard amine coupling techniques. The antibody or antigen-binding fragment thereof is captured on the Protein A surface in HBS-EP+ buffer pH 7.4 at 10 μl / min to allow FRα ECD binding. FRα ECD is serially diluted in HBS-EP+ buffer pH 7.4 (0.4 nM-100 nM human FRα ECD; 0.8 nM-200 nM cynomolgus monkey FRα ECD; 30 nM-4000 nM mouse FRα ECD and rat FRα ECD) and flowed over the chip at 50 μl / min for 2 min binding and 8 min dissociation. The chip surface is thoroughly regenerated with a pulse of 3 M MgCl2 to remove captured antibodies or their antigen-binding fragments along with bound FRα ECD. Multiple injections of buffer alone under the same conditions are performed to allow double reference subtraction of the final sensorgram set and analyzed using Biacore T200 analysis software.

[0167] Alternatively, the binding affinity of the anti-FRα antibody or its antigen-binding fragment of the present invention can be determined by Octet, for example Octet red. For example, the binding affinity of anti-FRα antibody can be assayed by Octet red at 25°C, for example using the following protocol. Binding assay is performed with Octet RED384 (ForteBio) in an assay buffer containing PBS, 0.1v / v% BSA (Sigma, A9576), 0.01v / v% Tween-20 (Sigma, P9416) (pH 7.4) at 25°C using a slanted bottom black 384-well plate (ForteBio, 18-5076). The assay is set up according to the manufacturer's instructions using Protein A or anti-human capture biosensor (AHC) (ForteBio, 18-5089). 10 μg / ml of anti-rat FRα IgG (Sino Biological, 81073-RP01) is coated onto a Protein A biosensor (ForteBio, NC9490476) and 10 μg / ml of test human IgG is loaded onto an anti-human capture biosensor (AHC) (ForteBio, 18-5089) for 180 s. Binding is measured by incubating the biosensor loaded with 500 nM human FRα (in-house) or 500 nM rat FRα (Sino Biological, 81073-R08H). Dissociation is measured after transfer into assay buffer. Data are analyzed using Octet data analysis software version 7.0.

[0168] antibody preparation In a further aspect, the present invention provides a method for producing the anti-FRα antibodies and antigen-binding fragments thereof of the present invention, comprising culturing a recombinant host cell expressing the heavy and light chains and isolating the antibody or antigen-binding fragment produced by the cell.

[0169] In a preferred embodiment, a method for producing an antibody or antigen-binding fragment thereof comprises the steps of (a) culturing a host cell and (b) isolating the expressed antibody or antigen-binding fragment thereof from the cell.

[0170] The antibodies of the present invention can be produced by transfecting a host cell with one or more vectors containing polynucleotides encoding the respective antibody or fragment, culturing the host cell under conditions allowing the synthesis of said antibody molecule, and recovering said antibody molecule from said culture.

[0171] In some embodiments, the method comprises: a) transfecting a host cell with one or more vectors comprising polynucleotides encoding a set of heavy and light chains of an antibody of the invention; b) culturing host cells under conditions allowing the synthesis of said antibody molecules; and c) recovering the antibody molecules from the culture. Includes.

[0172] In a preferred embodiment, the method comprises the steps of: a) transfecting a host cell with a vector comprising a polynucleotide encoding the light and heavy chains of the antibody of the present invention; b) culturing host cells under conditions allowing the synthesis of said antibody molecules; and c) recovering the antibody molecules from the culture. Includes.

[0173] The present invention further encompasses antibodies or antigen-binding fragments thereof obtainable by the above-described methods for producing antibodies or antigen-binding fragments thereof that bind to an FRα polypeptide (eg, an FRα polypeptide epitope).

[0174] Antibodies or antigen-binding fragments thereof (e.g., as monoclonal antibodies) can also be produced using recombinant DNA methods as described in U.S. Pat. No. 4,816,567, which is incorporated herein by reference. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which when transfected into host cells that do not normally produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, the monoclonal antibodies are produced by the host cells. Alternatively, recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing the CDRs of the desired species as described in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0175] Affinity maturation and chain shuffling strategies are known in the art and can be utilized to generate high affinity human antibodies or antigen-binding fragments thereof, see Marks et al., BioTechnology 10:779-783 (1992), incorporated by reference in its entirety.

[0176] Various techniques are known for producing antibody fragments. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies, for example as described in Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In some embodiments, anti-FRα antibody fragments are produced recombinantly. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, allowing for the production of large amounts of these fragments. Such anti-FRα antibody fragments can also be isolated from the antibody phage libraries mentioned above. Anti-FRα antibody fragments can also be linear antibodies, as described in U.S. Pat. No. 5,641,870, which is incorporated herein by reference. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0177] According to the present invention, the technique can be adapted to generate single chain antibodies specific for FRα (see, for example, U.S. Pat. No. 4,946,778). In addition, the method can be adapted to construct Fab expression libraries that allow rapid and efficient identification of monoclonal Fab fragments, or derivatives, fragments, analogs, or homologs thereof, with the desired specificity for FRα. See, for example, Huse et al., Science 246:1275-1281 (1989). Antibody fragments can be generated by techniques known in the art, including, but not limited to, F(ab')2 fragments generated by pepsin digestion of antibody molecules; Fab fragments generated by reduction of disulfide bridges of F(ab')2 fragments; Fab fragments generated by treating antibody molecules with papain and a reducing agent; or Fv fragments.

[0178] Antibody-Drug Conjugates (ADCs) Also provided herein is an ADC comprising an anti-FRα antibody or an antigen-binding fragment thereof.

[0179] Different drugs In some embodiments, the antibody or antigen-binding fragment of the invention is linked to a heterologous agent. In a preferred embodiment, the antibody or antigen-binding fragment is conjugated to the heterologous agent. Suitably, "conjugated" means linked via a covalent or ionic bond.

[0180] In some embodiments, the antibody or antigen-binding fragment is conjugated to one or more heterologous agents selected from the group consisting of a topoisomerase I inhibitor (TOPOi), a tubulysin derivative, a pyrrolobenzodiazepine, an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a xenogenous antibody, a fragment of a xenogenous antibody, a detectable label, polyethylene glycol (PEG), a radioisotope, or a combination thereof.

[0181] In some embodiments, the heterologous polypeptide can be a drug. Preferably, the heterologous drug is a cytotoxin. For example, an antibody or antigen-binding fragment can be conjugated to such a heterologous drug to provide an "antibody drug conjugate" (ADC).

[0182] In another aspect of the present invention, an ADC is provided that comprises an anti-FRα antibody or antigen-binding fragment thereof of the present invention, wherein the anti-FRα antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0183] A heterologous drug is typically linked to or "loaded" onto an antibody or antigen-binding fragment. The drug load (p) is the amount of drug per antibody or antigen-binding fragment. It will be appreciated by those of skill in the art that more than one of the above agents (eg, TOPOi) may be conjugated to an antibody or antigen-binding fragment thereof.

[0184] In some embodiments, the average number of drugs per antibody (or antigen-binding fragment thereof) ranges from 1 to 20. In some embodiments, the range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In some embodiments, there is one drug per antibody (or antigen-binding fragment thereof). In some embodiments, the number of drugs per antibody (or antigen-binding fragment thereof) can be expressed as a ratio of drugs (i.e., drugs) to antibodies. This ratio is called the drug-antibody ratio (DAR). The DAR is the average number of drugs (i.e., drugs) linked to each antibody. In some embodiments of the invention, the DAR ranges from 1 to 20. In some embodiments, the range of the DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In a preferred embodiment, the DAR is about 4 (eg, 3.8 to 4.2) or about 8 (eg, 7.6 to 8.4), more preferably about 8 (eg, 7.6 to 8.4).

[0185] Linker The antibody or antigen-binding fragment can be conjugated to a heterologous agent (eg, a cytotoxic agent) by a linker.

[0186] The term "linker" or "spacer" as used herein means a bivalent chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody or antigen-binding fragment thereof to a heterologous agent (e.g., a cytotoxin) to form a conjugate (e.g., an ADC). In some embodiments, the linker or spacer is a peptide spacer. In some embodiments, the spacer is a non-peptide (e.g., chemical) spacer. A suitable linker has two reactive ends, one for conjugation of the antibody and the other for conjugation of the heterologous agent. One or both of the reactive ends are absent or incomplete (e.g., only a carbonyl of a carboxylic acid) to form bonds between the linker and / or the heterologous agent (e.g., a cytotoxin) and between the linker and / or the antibody or antigen-binding fragment thereof. These conjugation reactions are discussed in more detail below.

[0187] In a preferred embodiment, the linker is cleavably linked (eg, conjugated) to an amino residue, such as an amino acid of an antibody or antigen-binding fragment described herein.

[0188] In some embodiments, the linker is cleavable in the intracellular environment, causing the drug unit to be released from the antibody in the intracellular environment.

[0189] Alternatively, the linker unit may not be cleavable.In such an embodiment, the drug is released, for example, by antibody degradation.However, non-cleavable payloads require complete mAb digestion in lysosomes, and the resulting drug-containing product may be too polar, for example, to achieve bystander effect.

[0190] An antibody (e.g., ADC) linked to a heterologous agent is preferably stable and intact before being transported or delivered into a cell. That is, the antibody must be conjugated to a drug moiety. Outside the target cell, the linker is stable, but inside the cell, the linker can be rapidly cleaved. An effective linker (i) maintains the specific binding properties of the antibody; (ii) allows delivery of the conjugate or drug moiety into the cell; (iii) remains stable and intact (i.e., is not cleaved) until the conjugate is delivered or transported to its target site; and (iv) maintains the cell-killing or cell-proliferation suppressing effect of the cytotoxic moiety. Standard analytical methods such as mass spectrometry, HPLC, and the separation / analysis technique LC / MS can be used to assess the stability of an antibody (e.g., ADC) linked to a heterologous agent.

[0191] The linker can be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0192] Linkers that are hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc. (see, for example, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Linkers that are cleavable under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene) (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987)).

[0193] In preferred embodiments, the linker is susceptible to enzymatic hydrolysis. Such linkers are preferred over pH-sensitive cleavable linkers, which may not be sufficiently stable before reaching the target cell and may be prematurely cleaved, and thus potential off-target toxicity may be observed. An enzymatically cleavable linker may be, for example, a peptide-containing linker that is cleaved by an intracellular peptidase or protease enzyme, such as, but not limited to, a lysosomal protease or an endosomal protease. One advantage of utilizing intracellular proteolytic release of a therapeutic agent is that the drug is usually attenuated when conjugated, and the serum stability of the conjugate is usually high. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Peptides containing the amino acids valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine are examples of suitable peptides. Natural amino acids, minor amino acids, and non-naturally occurring amino acid analogs such as citrulline are all examples of amino acid residues that may make up the amino acid linker moiety. Examples of dipeptides include valine-citrulline (VC or Val-Cit) and alanine-phenylalanine (AF or Ala-Phe). Examples of tripeptides include glycine-valine-citrulline (Gly-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly). In some embodiments, the linker comprises a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit.

[0194] In some embodiments, the linker comprises PEG. A stable protease cleavable linker containing PEG can limit the hydrophobicity of the payload and selectively cleave and release the free drug in the target cancer cells. The less hydrophobic nature of the linkers described herein allows for high loading of the drug onto the antibody or antigen-binding fragment (e.g., DAR8) without aggregation, which would be significantly higher than mirvetuximab soravtansine (DAR3-4) or its derivatives, such as IMGN151 (DAR3.5). This allows the ADC to deliver very high concentrations of cytotoxins to the target cancer cells via binding to FRα on the cancer cells.

[0195] In some embodiments, the linker comprises a maleimide. The use of maleimide in the linker may allow the generation of ADCs with DAR8 and DAR4 using the natural interchain disulfides in the antibody. This is advantageous over conjugation of surface amines from lysine residues, which may lead to mixtures of DAR species and batch-to-batch variability. If the conjugation site interferes with antigen binding, there may also be reproducibility issues that affect the efficacy of the ADC. Furthermore, other conjugation methods, such as azide-alkyne click chemistry, including engineered antibodies, cannot easily achieve DARs greater than 4.

[0196] In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the invention comprises a linker R selected from the following: L is linked to a heterologous drug, preferably a cytotoxin, via: [ka] [In the formula, Q is, [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and; X is [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0)). and; G L is a linker for conjugating to an antibody or antigen-binding fragment thereof herein; [ka] [In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1; or [ka] [In the formula, R L1 and R L2 is as defined above].

[0197] For example, G L , X, Q X (eg, in the linker of Ia above) and preferred embodiments of the linker of Ib are outlined.

[0198] The following preferences may apply to all aspects of the invention described herein or may relate to a single aspect. The preferences may be combined together in any combination.

[0199] Various definitions for specific terms in this section are provided under the heading "Chemical Definitions," provided below.

[0200] G L G L may be selected from the following:

[0201] [Table 11]

[0202] [Table 12]

[0203] In the formula, Ar represents a C5-6 arylene group, for example, phenylene, and X represents a C1-4 alkyl group.

[0204] In some embodiments, G L is G L1-1 and G L1-2 In some of these embodiments, G L is G L1-1 It is.

[0205] X X is preferably [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, and d=0 to 5, where at least b1 or b2=0, and at least c1 or c2=0.) It is. a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0. b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. Preferably, only one of b1 and b2 may not be 0. c1 can be 0 or 1. c2 can be 0 or 1. Preferably, only one of c1 and c2 may be non-0. d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In further embodiments, d is 5.

[0206] In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be 0-8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1-5. In some of these embodiments, the other of a and d is 1. In others of these embodiments, the other of a and d is 5. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0207] Q X In some embodiments, Q is an amino acid residue. The amino acid may be a natural or unnatural amino acid. For example, Q may be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0208] In some embodiments, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. In that case, the dipeptide is the recognition site for cathepsin.

[0209] In some embodiments, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O and NH -Gly-Val- C=O Selected from; Here, Cit is citrulline.

[0210] Preferably, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O and NH -Val-Cit- C=O is selected from.

[0211] More preferably, Q is NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0212] Other suitable dipeptide combinations include: NH -Gly-Gly- C=O , NH -Gly-Val- C=O , NH -Pro-Pro- C=O and NH -Val-Glu- C=O Examples include:

[0213] Other dipeptide combinations, such as those described in Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference, may also be used.

[0214] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. In that case, the tripeptide is the recognition site for cathepsin. Particularly interesting tripeptide linkers are: NH -Glu-Val-Ala- C=O NH -Glu-Val-Cit- C=O NH -αGlu-Val-Ala- C=O NH -αGlu-Val-Cit- C=O It is.

[0215] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. In that case, the tetrapeptide is the recognition site for the cathepsin. Tetrapeptide linkers of particular interest are: NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O It is. In some embodiments, the tetrapeptide is NH -Gly-Gly-Phe-Gly C=O It is.

[0216] In the above representation of peptide residues: NH - represents the N-terminus of the residue, - C=Orepresents the C-terminus of the residue, which is attached to the NH of the Drug Unit (e.g., A*, discussed below). Glu is a glutamic acid residue, i.e. [ka] Represents. αGlu is a glutamic acid residue when bound via the α chain, i.e. [ka] Represents.

[0217] In some embodiments, where appropriate, amino acid side chains are chemically protected. Side chain protecting groups can be as described above. Protected amino acid sequences can be cleaved by enzymes. For example, dipeptide sequences containing Boc side chain protected Lys residues can be cleaved by cathepsin.

[0218] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem catalogue, as discussed above.

[0219] Linker Ib R L1 and R L2 are independently selected from H and methyl, or can form, together with the carbon atom to which they are attached, a cyclopropylene or cyclobutylene group.

[0220] In some embodiments, R L1 and R L2 and R are H. In some embodiments, R L1 is H and R L2 is methyl. In some embodiments, R L1 and R L2 Both are methyl.

[0221] In some embodiments, R L1 and R L2together with the carbon atom to which they are attached form a cyclopropylene group. In some embodiments, R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0222] In group Ib, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group can be at any available position on the ring. In some of these embodiments, it is at the ortho position. In others of these embodiments, it is at the para position.

[0223] R L In some embodiments, R L is selected from the following: [ka] [ka] [ka]

[0224] Preferably, R L teeth, [ka] It is.

[0225] For example, a conjugate (e.g., an antibody-drug conjugate) of the invention can be represented by the general formula IV: L-(D L ) p (IV) Things, or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof of the present invention, L is a linker R linked to the antibody or antigen-binding fragment thereof of the present invention. LL and the linker is preferably a "drug unit" (e.g., a cytotoxin such as a TOPOi) having the structure [ka] (wherein Q and X are as defined above; G LL is a linker linked to the antibody or antigen-binding fragment thereof of the invention, and [ka] (In the formula, R L1 and R L2 is as defined above) is selected from p is an integer from 1 to 20. It could be.

[0226] The drug loading is expressed as p, the number of "drug units" (e.g., cytotoxins such as TOPOi) per antibody or antigen-binding fragment thereof. The drug loading can range from 1 to 20 drug units (D) per antibody or antigen-binding fragment thereof. In the case of a composition, p represents the average drug loading of the conjugates in the composition, and p ranges from 1 to 20. In some embodiments, the range of p is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10, and preferably p is 8.

[0227] G LL G LL may be selected from the following: [ka] [ka] In the formula, Ar is C 5~6 represents an arylene group, e.g., phenylene; X is C 1~4 Represents alkyl.

[0228] In some embodiments, G LL is G LL1-1 and G LL1-2 In some of these embodiments, G LL is GLL1-1 It is.

[0229] In some embodiments, R LL is the above R L It is a group derived from the group.

[0230] It will be appreciated by those of skill in the art that any one or more of the chemical groups, moieties and features disclosed herein can be combined in multiple ways to form linkers useful for conjugating the antibodies and cytotoxins disclosed herein.

[0231] In some embodiments where the compounds described herein are provided in a single enantiomer or in an enantiomerically enriched form, the enantiomerically enriched form has an enantiomeric ratio of greater than 60:40, 70:30, 80:20, or 90:10. In further embodiments, the enantiomeric ratio is greater than 95:5, greater than 97:3, or greater than 99:1.

[0232] cytotoxin In a preferred embodiment, the heterologous agent is a cytotoxin (also called a cytotoxic agent). A cytotoxic agent or cytotoxin can be any molecule known in the art that inhibits or prevents the function of a cell and / or causes the destruction of a cell (cell death) and / or exerts an anti-neoplastic / anti-proliferative effect. Several classes of cytotoxic agents are known to have potential utility in ADC molecules. Suitable cytotoxic agents of the present invention include, but are not limited to, topoisomerase I inhibitors (TOPOi), amanitin, auristatin, daunomycin, doxorubicin, duocarmycin, dolastatin, enediyne, lexitropsin, taxane, puromycin, maytansinoid, vinca alkaloid, tubulysin, and pyrrolobenzodiazepines (PBD). Examples of such cytotoxic agents are AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, calicheamicin, maytansine, DM-1, vinblastine, methotrexate and netropsin, and derivatives and analogs thereof. Further disclosure regarding cytotoxins suitable for use in ADCs can be found, for example, in WO 2015 / 155345 and WO 2015 / 157592, which are incorporated herein by reference in their entireties.

[0233] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxins selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof. For example, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxins selected from the group consisting of the topoisomerase I inhibitors SG3932 (also known as AZ14170133), SG4010, SG4057, or SG4052 (the structures of which are shown below), or a combination thereof. The antibody or antigen-binding fragment thereof may be conjugated to a topoisomerase I inhibitor, preferably the topoisomerase I inhibitor SG3932.

[0234] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention are not conjugated to, or the anti-FRα ADCs of the invention do not comprise, microtubule inhibitors, such as tubulin inhibitors (e.g., maytansinoids, auristatins). The microtubule inhibitor class of molecules is associated with dose-limiting, potentially difficult-to-treat toxicities.

[0235] Topoisomerase I Inhibitors The present invention demonstrates the first anti-FRα ADC that uses a topoisomerase I inhibitor (TOPOi) payload. Furthermore, using in vivo and in vitro models, the inventors have demonstrated the stability and efficacy of an anti-FRα mAb when conjugated to a TOPOi payload and deployed as an ADC.

[0236] Thus, in one aspect, an ADC is provided that comprises an anti-FRα antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment thereof of the invention) conjugated to a TOPOi payload. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is conjugated to a topoisomerase I inhibitor, or the anti-FRα ADC of the invention comprises a topoisomerase I inhibitor.

[0237] Topoisomerase inhibitors are chemical compounds that block the action of topoisomerases (topoisomerase I and II), a type of enzyme that controls changes in DNA structure by catalyzing the cleavage and recombination of the phosphodiester backbone of DNA strands during normal cell cycle. Topoisomerase I inhibitors are advantageous because they mediate tumor cell killing very effectively while at the same time causing very little toxicity to patients. In particular, alternative payloads such as microtubule inhibitors, which have been commonly used to date to develop anti-FRα ADCs, are known to have toxicity issues (Hinrichs, et al. AAPS J. 2015 Sep; 17(5): 1055-1064). Furthermore, the use of less hydrophobic linkers with less potent warheads (e.g., TOPOi) would promote bystander killing in heterogeneous tumors. Bystander activity can be achieved by increasing potency and / or improving the permeability of the warhead by increasing hydrophobicity, but this may result in increased toxicity due to non-specific uptake.

[0238] General examples of suitable topoisomerase I inhibitors are represented by the following compounds: [ka] Said compound is designated as A* and may be referred to herein as a "drug unit."

[0239] The compound (e.g., A*) is preferably provided with a linker for linking (preferably conjugating) to an antibody or antigen-binding fragment of the invention. In a preferred embodiment, the linker is cleavably linked (e.g., conjugated) to an amino residue, e.g., an amino acid of an antibody or antigen-binding fragment of the invention.

[0240] More particularly, examples of suitable topoisomerase I inhibitors include compounds having the following formula "I" [ka] and salts and solvates thereof, Lis defined above).

[0241] Thus, general formula IV: L-(D L ) p (IV) or a pharma- ceutically acceptable salt or solvate thereof, wherein L and p are defined above, D L is represented by formula III: [ka] and salts and solvates thereof, each of which has a linker represented by the formula: LL is defined above).

[0242] In some embodiments, the compound of formula I has formula I P : [ka] and salts and solvates thereof, wherein R LP is a linker for linking to an antibody or antigen-binding fragment thereof of the invention, said linker being selected from: [ka] [In the formula, Q P teeth, [ka] (In the formula, Q XP Q P is an amino acid residue, a dipeptide residue or a tripeptide residue. and; X P teeth, [ka] (In the formula, aP=0 to 5, bP=0 to 16, cP=0 or 1, and dP=0 to 5). and; G L is defined above]; [ka] [In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1.

[0243] aP can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP is 0 to 3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0.

[0244] bP can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is 0 to 12. In some of these embodiments, bP is 0 to 8, and can be 0, 2, 4, or 8.

[0245] cP can be 0 or 1.

[0246] dP can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is 0 to 3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

[0247] X P In some embodiments, aP is 0, cP is 1, dP is 2, and bP can be 0 to 8. In some of these embodiments, bP is 0, 4, or 8.

[0248] Q above for compounds of formula I X Preferred for (e.g., where appropriate) Q XP can be applied to.

[0249] For compounds of formula I,L , R L1 , R L2 and e are preferably of formula I P The present invention can be applied to compounds of the above formula (I).

[0250] In some embodiments, the conjugate of formula IV is P : L-(D LP ) p (IV P ) or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof of the present invention; LP is represented by formula III P : [ka] is a topoisomerase I inhibitor (e.g., a drug linker unit) of LLP is a linker that is linked to an antibody or antigen-binding fragment thereof, said linker comprising: [ka] (In the formula, Q P , X P and G LL is defined above) and [ka] (In the formula, R L1 and R L2 is as defined above) is selected from p is an integer from 1 to 20.

[0251] In some embodiments, the compound of formula I has formula I P2 : [ka] and salts and solvates thereof, wherein R LP2 is a linker for linking to an antibody or antigen-binding fragment thereof of the present invention, the linker comprising: [ka] [In the formula, Q is, [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and X P2 teeth, [ka] (In the formula, aP2=0 to 5, b1P2=0 to 16, b2P2=0 to 16, cP2=0 or 1, dP2=0 to 5, and at least b1P2 or b2P2=0 (i.e., only one of b1 and b2 may not be 0)). and GL is a linker for linking to the antibody or antigen-binding fragment thereof of the invention]; [ka] [In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1. is selected from.

[0252] aP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0.

[0253] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1P2 is 0 to 12. In some of these embodiments, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0254] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0255] Preferably, only one of b1P2 and b2P2 may be non-zero.

[0256] cP2 can be 0 or 1.

[0257] dP2 can be 0, 1, 2, 3, 4 or 5. In some embodiments, dP2 is 0-3. In some of these embodiments, dP2 is 1 or 2. In further embodiments, dP2 is 2. In further embodiments, dP2 is 5.

[0258] X P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 1, dP2 is 2, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, aP2 is 1, b2P2 is 0, cP2 is 0, dP2 is 0, and b1P2 can be 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 0, dP2 is 1, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. P2In some embodiments, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1 to 5. In some of these embodiments, the other of aP2 and d is 1. In others of these embodiments, the other of aP2 and dP2 is 5.

[0259] Q above for compounds of formula I X Preferred for (e.g., where appropriate) are compounds of formula Ia P2 Q X can be applied to.

[0260] For compounds of formula I, L , R L1 , R L2 and e are preferably of formula I P2 The present invention can be applied to compounds of the above formula (I).

[0261] In some embodiments, the conjugate of formula IV is P2 : L-(D LP2 ) p (IV P2 ) or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof of the present invention; LP2 is represented by formula III P2 : [ka] is a topoisomerase I inhibitor (e.g., a drug linker unit) of R LLP2 is a linker that is linked to an antibody or antigen-binding fragment thereof, said linker comprising: [ka] (Wherein, Q and X P2 is as defined above, and G LL is a linker linked to the antibody or antigen-binding fragment thereof of the invention, and [ka] (In the formula, R L1 and R L2 is as defined above) is selected from p is an integer from 1 to 20.

[0262] Particularly suitable Topoisomerase I inhibitors include those having the formula: [ka] [ka]

[0263] SG3932 is particularly preferred. Thus, in a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is conjugated to a topoisomerase I inhibitor (e.g., SG3932) having the following formula: [ka]

[0264] Synthetic methods for making topoisomerase I inhibitors are described, for example, in WO 2020 / 200880, which is incorporated herein by reference.

[0265] As outlined above, although topoisomerase I inhibitors are preferred, it should be noted that any suitable agent (e.g., drug / cytotoxin) may be linked to the antibody or antigen-binding fragment thereof of the invention. Examples of other suitable agents are outlined below.

[0266] Tubulysin and pyrrolobenzodiazepines In some embodiments, the cytotoxin is tubulysin or a tubulysin derivative, hi some embodiments, the cytotoxin is tubulysin A, which has the following chemical structure: [ka]

[0267] Tubulysin is a member of a class of natural products isolated from myxobacterial species. As a cytoskeleton interacting agent, tubulin polymerization is a mitotic poison that leads to cell cycle arrest and apoptosis. As used herein, the term "tubulysin" refers collectively and individually to naturally occurring tubulysins as well as analogs and derivatives of tubulysins. Specific examples of tubulysins are disclosed, for example, in WO 2004 / 005326 A2, WO 2012 / 019123 A1, WO 2009 / 134279 A1, WO 2009 / 055562 A1, WO 2004 / 005327 A1, U.S. Patent No. 7776841, U.S. Patent No. 7754885, U.S. Patent Application Publication No. 2010 / 0240701, U.S. Patent No. 7816377, U.S. Patent Application Publication No. 2011 / 0021568, and U.S. Patent Application Publication No. 2011 / 0263650, which are incorporated herein by reference. It should be understood that such derivatives include, for example, tubulysin prodrugs or tubulysins containing one or more protection groups or protecting groups, one or more linking moieties.

[0268] In another embodiment, the cytotoxin can be a pyrrolobenzodiazepine (PBD) or a PBD derivative. PBDs translocate to the nucleus where they crosslink DNA, block replication during mitosis, and damage DNA by inducing single-strand breaks, which then lead to apoptosis. Some PBDs have the ability to recognize and bind to specific sequences of DNA, with the preferred sequence being PuGPu. PBDs have the following general structure: [ka]

[0269] PBDs vary in the number, type and position of substituents on both their aromatic A-ring and pyrrolo C-ring, and in the degree of saturation of the C-ring. The B-ring contains an imine (N=C), carbinolamine (NH-CH(OH)) or carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center involved in DNA alkylation. All of the known natural products have the (S) configuration at the chiral C11a position, which confers a right-handed twist to the natural products when viewed from the C-ring towards the A-ring. This provides the proper three-dimensional shape for isohelicity to the minor groove of B-form DNA, resulting in a snug fit at the binding site. Their ability to form adducts in the minor groove allows for interference with DNA processing and therefore their use as antitumor drugs.

[0270] The first PBD antitumor antibiotic, anthramycin, was discovered in 1965. Since then, numerous naturally occurring PBDs have been reported and over 10 synthetic routes to various analogues have been developed. Family members include abeimicin, ticamycin, DC-81, mazethramycin, neothramycin A and B, polothramycin, prothracarcin, sivanomycin (DC-102), sibiromycin and tomamycin. PBDs and ADCs containing them are also described in WO 2015 / 155345 and WO 2015 / 157592, which are incorporated by reference in their entireties.

[0271] Specific ADC embodiments In one aspect, the invention provides an anti-FRα antibody, or antigen-binding fragment thereof, conjugated to a cytotoxin, or an ADC comprising an anti-FRα antibody, or antigen-binding fragment thereof, conjugated to a cytotoxin.

[0272] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to a topoisomerase I inhibitor, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to a topoisomerase I inhibitor represented by the following compound having the formula "I": [ka] (In the formula, R L is defined above); The DAR ranges from about 1 to 20, and optionally the DAR range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 8.

[0273] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to a topoisomerase I inhibitor, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to a topoisomerase I inhibitor represented by the following compound having the formula "I": [ka] (In the formula, R L is defined above); The DAR ranges from about 1 to 20, and optionally the DAR range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 4.

[0274] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG3932, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG3932. [ka] Typically, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 8.

[0275] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG3932, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG3932. [ka] In general, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 4.

[0276] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG4010, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG4010. [ka] Typically, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 8.

[0277] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG4010, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG4010. [ka] In general, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 4.

[0278] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG4057, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG4057. [ka] Typically, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 8.

[0279] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG4057, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG4057. [ka] In general, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 4.

[0280] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG4052, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG4052. [ka] Typically, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 8.

[0281] In some embodiments, an anti-FRα antibody, or antigen-binding fragment thereof, of the invention is conjugated to the topoisomerase I inhibitor SG4052, or an anti-FRα ADC of the invention comprises an anti-FRα antibody, or antigen-binding fragment thereof, of the invention conjugated to the topoisomerase I inhibitor SG4052. [ka] In general, the DAR is in the range of about 1 to 20, and optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, and is preferably about 4.

[0282] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) the cytotoxin is a topoisomerase I inhibitor represented by the following compound having formula "I" [ka] , (where R L is defined above); and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 8.

[0283] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) the cytotoxin is a topoisomerase I inhibitor represented by the following compound having formula "I" [ka] , (where R L is defined above); and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 4.

[0284] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG3932. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 8.

[0285] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG3932. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 4.

[0286] In some embodiments; (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG4010. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 8.

[0287] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG4010. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 4.

[0288] In some embodiments; (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG4057. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 8.

[0289] In some embodiments; (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG4057. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 4.

[0290] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG4052. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 8.

[0291] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 of SEQ ID NO: 1 (SDSATWN); a heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS); a heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY); a light chain CDR1 of SEQ ID NO: 4 (RASQSISSWLA); a light chain CDR2 of SEQ ID NO: 5 (KASGLES); and a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT); optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO: 37, and and a VL comprising an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:38, and optionally the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:49, and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical, or identical to the amino acid sequence of SEQ ID NO:50; (ii) The cytotoxic agent is the topoisomerase I inhibitor SG4052. [ka] and (iii) The DAR is in the range of about 1 to 20, optionally the DAR is in the range selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10, preferably about 4.

[0292] Internal migration Internalization can be a useful property of ADCs. For example, internalization allows for the delivery of a payload into cells. The present inventors have shown that the antibodies and ADCs of the present invention exhibit rapid internalization and lysosomal trafficking.

[0293] In some embodiments, an antibody or antigen fragment thereof, or an anti-FRα ADC of the invention binds to FRα on the cell surface and is internalized into the cell, hi some embodiments, internalization of an antigen or antibody fragment, or an anti-FRα ADC of the invention into an FRα-expressing cell is saturated within about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.

[0294] cytotoxicity In some embodiments, the anti-FRα ADCs of the invention inhibit or suppress growth (e.g., of a tumor) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% (preferably at least 40%), compared to the level of inhibition or suppression in the absence of the antibody or antigen-binding fragment thereof (e.g., an anti-FRα ADC). Cell proliferation can be assayed using art-recognized techniques that measure the rate of cell division, and / or the proportion of cells within a cell population undergoing cell division, and / or the rate of cell loss from a cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

[0295] In some embodiments, the anti-FRα ADCs of the invention are cytotoxic to cells expressing FRα with an EC50 value of about 1000 ng / ml or less, about 500 ng / ml or less, about 400 ng / ml or less, about 300 ng / ml or less, about 290 ng / ml or less, about 280 ng / ml or less, about 270 ng / ml or less, about 260 ng / ml or less, or about 250 ng / ml or less.

[0296] In some embodiments, the anti-FRα ADCs of the invention are cytotoxic to cells expressing FRα with an IC50 value of about 100 μg / ml or less, about 50 μg / ml or less, about 25 μg / ml or less, about 10 μg / ml or less, about 5 μg / ml or less, about 2.5 μg / ml or less, about 1 μg / ml or less, about 0.75 μg / ml or less, about 0.5 μg / ml or less, about 0.25 μg / ml or less, about 0.1 μg / ml or less, about 0.075 μg / ml or less, about 0.05 μg / ml or less, about 0.025 μg / ml or less, or about 0.01 μg / ml or less.

[0297] In some embodiments, the anti-FRα ADCs of the invention inhibit or suppress the proliferation of cell populations that exhibit heterogeneous expression of FRα and / or low expression of FRα. In some embodiments, the anti-FRα ADCs of the invention inhibit or suppress the proliferation of cell populations that moderately express FRα (e.g., Jeg-3, OVCAR-3 cell lines, or cells with similar or equivalent expression levels of FRα), that moderately to highly express FRα (e.g., Igrov-1 cell lines, or cells with similar or equivalent expression levels of FRα), or that highly express FRα (e.g., KB cell lines, or cells with similar or equivalent expression levels of FRα).

[0298] Preparation of ADCs or antibodies linked to heterologous agents Antibodies linked to heterologous agents (e.g., ADCs) of the present disclosure can be made in a variety of ways using known organic chemistry reactions, conditions, and reagents, such as by (1) reacting a reactive substituent on an antibody or antigen-binding fragment with a bivalent linker reagent, which is then reacted with a heterologous agent (e.g., a cytotoxin, preferably a topoisomerase I inhibitor); or (2) reacting a reactive substituent on a heterologous agent (e.g., a cytotoxin, preferably a topoisomerase I inhibitor) with a bivalent linker reagent, which is then reacted with a reactive substituent on an antibody or antigen-binding fragment thereof of the present disclosure.

[0299] Reactive substituents that may be present in the antibodies or antigen-binding fragments thereof disclosed herein include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) nucleophilic groups such as hydroxyl or amino groups of sugars when the antibody is glycosylated. Reactive substituents that may be present in the antibodies or antigen-binding fragments thereof disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of unnatural amino acids. In some embodiments, reactive substituents present in the antibodies or antigen-binding fragments thereof disclosed herein include amine or thiol moieties. Certain antibodies have cysteine ​​bridges that are reducible interchain disulfides. Antibodies can be reacted with linker reagents for conjugation by treating them with reducing agents, such as DL-dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). Each cysteine ​​bridge theoretically results in the formation of two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies using the reaction of lysine with 2-iminothiolane (Traut's reagent), which results in the conversion of an amine to a thiol. One, two, three, four or more cysteine ​​residues can be used to insert reactive thiol groups into an antibody (or a fragment thereof) (e.g., to prepare a mutant antibody containing one or more non-natural cysteine ​​amino acid residues). Engineering antibodies with reactive cysteine ​​amino acids is described in U.S. Pat. No. 7,521,541, which is incorporated herein by reference.

[0300] In another embodiment, an antibody or antigen-binding fragment thereof can have one or more carbohydrate groups that can be chemically altered to contain one or more sulfhydryl groups. An antibody linked to a heterologous agent (e.g., an ADC) is then formed by conjugation through the sulfhydryl sulfur atom.

[0301] In yet another embodiment, the antibody may contain one or more carbohydrate groups that can be oxidized to generate aldehyde (-CHO) groups (see, e.g., Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). Conjugation via the corresponding aldehyde forms an antibody linked to a heterologous agent (e.g., an ADC). Further protocols for modifying proteins to attach or conjugate cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002). Methods for conjugation of linker-drug moieties to cell-targeting proteins such as antibodies, immunoglobulins or fragments thereof can be found, for example, in U.S. Pat. No. 5,208,020, U.S. Pat. No. 6,441,163, WO 2005 / 037992, WO 2005 / 081711 and WO 2006 / 034488, which are incorporated herein by reference.

[0302] Conventional conjugation strategies for antibodies or antigen-binding fragments thereof rely on random or stochastic conjugation of payloads to the antibodies or fragments via lysines or cysteines. In some embodiments, antibodies or antigen-binding fragments thereof are stochastically conjugated to heterologous agents (e.g., cytotoxins, preferably topoisomerase I inhibitors) by, for example, partially reducing the antibody or fragment and then reacting with the desired agent, with or without a linker moiety attached. The antibody or fragment can be reduced with DTT or other reducing agents, such as TCEP, to perform a similar reduction. The agent, with or without a linker moiety attached, can then be added in molar excess to the reduced antibody or fragment in the presence of DMSO. After conjugation, a quenching agent, such as N-acetyl-L-cysteine, can be added to quench unreacted agent. The reaction mixture can then be purified (e.g., by TFF, SEC-FPLC, CHT, spin filter centrifugation) and buffer exchanged into PBS or other relevant formulation buffers.

[0303] In some embodiments, an agent (e.g., a cytotoxin) is conjugated to an antibody or antigen-binding fragment thereof by site-specific conjugation. In some embodiments, site-specific conjugation of a therapeutic moiety to an antibody using reactive amino acid residues at specific positions results in homogenous preparations of antibodies linked to heterologous agents (e.g., ADCs) in uniform stoichiometric ratios.

[0304] Site-specific conjugation can be via cysteine ​​residues or unnatural amino acids. In a preferred embodiment, a heterologous agent (preferably a cytotoxin) is conjugated to an antibody or antigen-binding fragment thereof via at least one cysteine ​​residue. Cysteine ​​amino acids that are in reactive sites of an antibody (or antigen-binding fragment thereof) and preferably do not form intrachain or intermolecular disulfide bonds can be engineered (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO 2009 / 052249). In some embodiments, the agent (e.g., a cytotoxin) is conjugated to the antibody or antigen-binding fragment thereof by cysteine ​​substitution at least one of positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443, and 446 (numbering corresponds to EU index of Kabat). In some embodiments, the specific Kabat positions are 239, 442, or both. In some aspects, the specific positions are Kabat position 442, an amino acid insertion between Kabat positions 239 and 240, or both. In some embodiments, the heterologous agent (preferably a cytotoxin) is conjugated to the antibody or antigen-binding fragment thereof through a thiol-maleimide bond. In some aspects, the amino acid side chain is a sulfhydryl side chain.

[0305] When multiple nucleophilic or electrophilic groups on an antibody or antigen-binding fragment thereof react with a drug, the resulting product can be a mixture of heterogeneous drug-linked antibodies (e.g., ADCs) with a distribution of drug units attached to the antibody, e.g., 1, 2, 3, etc. Liquid chromatography methods such as hydrophobic interaction (HIC) can separate compounds in a mixture by drug loading value. Preparations of heterogeneous drug-linked antibodies (e.g., ADCs) with a single drug loading value (p) can be isolated.

[0306] The average number of drugs per antibody (or antigen-binding fragment) when preparing ADCs from conjugation reactions can be characterized by conventional means such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of ADCs with respect to p can also be determined. ELISA can determine the average value of p in a particular preparation of antibodies (e.g., ADCs) linked to heterologous drugs (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11:843-852). In some instances, separation, purification, and characterization of homogeneous antibodies (e.g., ADCs) linked to heterologous drugs (where p is a particular value from antibodies containing other drugs) can be achieved by means such as reversed-phase HPLC, electrophoresis, TFF, SEC-FPLC, CHT, spin filter centrifugation, and the like. Such techniques are also applicable to other types of conjugates.

[0307] chemistry definition The following definitions are particularly relevant to the description of Topoisomerase I inhibitors above.

[0308] C 5~6 Arylene: The term "C 5~6 "Arylene," as used herein, pertains to a divalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of an aromatic compound.

[0309] In this context, prefixes (e.g., C 5~6) indicates the number of ring atoms or range of number of ring atoms, whether carbon atoms or heteroatoms.

[0310] The ring atoms may all be carbon atoms, such as in a "carbarylene group," in which case the group is phenylene (C6).

[0311] Alternatively, the ring atoms may contain one or more heteroatoms, as in "heteroarylene groups." Examples of heteroarylene groups include, but are not limited to, those derived from: N1: Pyrrole (azole) (C5), pyridine (azine) (C6); O1: Furan (oxol) (C5); S1: thiophene (thiol) (C5); N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6); N2O1: oxadiazole (furazan) (C5); N3O1: oxatriazole (C5); N1S1: thiazole (C5), isothiazole (C5); N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); and N3: Triazole (C5), triazine (C6). C 1~4 Alkyl: The term "C 1~4 "Alkyl," as used herein, refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated or fully unsaturated). 1~n"Alkyl," as used herein, pertains to a monovalent moiety obtained by removal of a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

[0312] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), and butyl (C4).

[0313] Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), and n-butyl (C4).

[0314] Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4) and tert-butyl (C4).

[0315] C 2~4 Alkenyl: The term "C 2~4 "Alkenyl," as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

[0316] Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1-methylvinyl, -C(CH3)=CH2), and butenyl (C4).

[0317] C 2~4 Alkynyl: The term "C 2~4 "Alkynyl," as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

[0318] Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (propargyl, -CH2-C≡CH).

[0319] C 3~4 Cycloalkyl: The term "C 3~4 "Cycloalkyl," as used herein, refers to an alkyl group that is also a cyclyl group, i.e., a monovalent moiety obtained by removal of a hydrogen atom from a single alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, said moiety having 3 to 7 carbon atoms containing 3 to 7 ring atoms.

[0320] Examples of cycloalkyl groups include, but are not limited to, those derived from: Saturated monocyclic hydrocarbon compounds: Cyclopropane (C3) and cyclobutane (C4); and Unsaturated monocyclic hydrocarbon compounds: Cyclopropene (C3) and cyclobutene (C4).

[0321] Connecting indicator: Expression [ka] In the superscript sign C(=O) and NH indicates the group to which the atom is attached. For example, an NH group is shown attached to a carbonyl (not part of the depicted moiety) and a carbonyl is shown attached to an NH group (not part of the depicted moiety).

[0322] salt It may be convenient or desirable to prepare, purify, and / or handle the corresponding salt of the active compound / agent, e.g., a pharma- ceutically acceptable salt. Examples of pharma-ceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0323] For example, the compound may be anionic or have a functional group that may be anionic (e.g., -COOH may be -COO - In the case where the salt is a cation, the salt may be formed with a suitable cation. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as Al +3 Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are included, but are not limited to. Some suitable examples of substituted ammonium ions include ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0324] The compound is cationic or has a functional group that can be cationic (e.g., -NH2 can be -NH3 + In some cases, salts may be formed with suitable anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0325] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0326] solvate It may be convenient or desirable to prepare, purify and / or handle the corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g., active compound, salt of active compound) and solvent. When the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, etc.

[0327] Isomers Certain compounds / agents of the present invention may exist in one or more particular geometric isomers, optical isomers, enantiomers, diastereomers, epimers, atropes, stereoisomers, tautomers, conformers or anomeric forms, including, but not limited to, cis and trans forms; E and Z forms; c, t and r forms; endo and exo forms; R, S and meso forms; D and L forms; d and l forms; (+) and (-) forms; keto, enol and enolate forms; syn and anti forms; synclinal and anticlinal forms; α and β forms; axial and equatorial forms; boat, chair, twist, envelope and half-chair forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[0328] Unless otherwise specified, reference to a particular compound encompasses all such isomeric forms, including (fully or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by applying the methods taught herein or known methods in a known manner.

[0329] Polynucleotides, Vectors and Host Cells In some embodiments, a polynucleotide is provided that encodes an anti-FRα antibody or antigen-binding fragment thereof of the invention. The polynucleotide can have any of the nucleotide sequences in Tables 7-8. In case of differences, the sequence in the tables takes precedence.

[0330] In another aspect, the polynucleotide comprises a sequence encoding (a) a VL that is at least 80%, 85%, 90% or 95% identical to or identical to the reference VL nucleotide sequence of any one of the constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 7, and (b) a VH that is at least 80%, 85%, 90% or 95% identical to or identical to the reference VH nucleotide sequence of any one of the constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 7.

[0331] In another embodiment, the polynucleotide comprises a sequence encoding (a) a light chain that is at least 80%, 85%, 90% or 95% identical to or identical to the reference light chain nucleotide sequence of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 8, and (b) a heavy chain that is at least 80%, 85%, 90% or 95% identical to or identical to the reference heavy chain nucleotide sequence of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 listed in Table 8.

[0332] [Table 13]

[0333] [Table 14]

[0334] [Table 15]

[0335] [Table 16]

[0336] [Table 17]

[0337] [Table 18]

[0338] [Table 19]

[0339] [Table 20]

[0340] [Table 21]

[0341] [Table 22]

[0342] [Table 23]

[0343] [Table 24]

[0344] In some embodiments, the polynucleotide is an isolated polynucleotide.

[0345] The sequences (e.g., polynucleotide sequences) of the present invention include sequences removed from their natural environment, recombinant or cloned (e.g., DNA) isolates, and chemically synthesized analogs or biologically synthesized analogs produced by heterologous systems.

[0346] The sequences (e.g., polynucleotide sequences) of the invention can be prepared by any means known in the art. For example, large amounts of the sequences can be produced by replication and / or expression in a suitable host cell. Natural or synthetic DNA fragments encoding the desired fragments are typically incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in prokaryotic or eukaryotic cells. Typically, the DNA constructs are suitable for autonomous replication in unicellular hosts such as yeast or bacteria, but may also be intended for introduction into and integration into the genomes of cultured bacteria, insects, mammals, plants or other eukaryotic cell lines.

[0347] The sequences (e.g., polynucleotide sequences) of the present invention can also be produced by chemical synthesis, for example, by phosphoramidite or triester polynucleotide synthesis, which can be carried out in commercially available automated oligonucleotide synthesizers. Double-stranded (e.g., DNA) fragments can be obtained from the single-stranded products of chemical synthesis by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0348] As applied to sequences (e.g., polynucleotide sequences) of the present invention, the term "isolated" preferably indicates that the sequence has been removed from its natural genetic environment, and thus is free of other extraneous or unwanted coding sequences (but may include natural 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within a genetically engineered protein production system. Such isolated molecules have been separated from their natural environment.

[0349] Variants of the polynucleotides described herein are encompassed by the present invention. Polynucleotide variants may contain modifications in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants include modifications that produce silent substitutions, additions, or deletions, but do not change the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are made by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants may be made for a variety of reasons, such as to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by bacterial hosts, such as E. coli).

[0350] In some embodiments, a vector comprising the polynucleotide is also provided. The vector can be an expression vector. The expression vector can include one or more additional sequences, such as, but not limited to, regulatory sequences (e.g., promoters, enhancers), selection markers and polyadenylation signals. Vectors for transfecting a wide variety of host cells are well known, including, but not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), and other bacterial, yeast and viral vectors.

[0351] A vector may comprise a nucleic acid sequence that controls the expression of a polynucleotide. In some embodiments, a vector comprises a polynucleotide of the invention operably linked to a promoter. For example, a vector comprises a polynucleotide encoding a VH region of an antibody of the invention and a polynucleotide encoding a VL region of an antibody of the invention, said polynucleotides being operably linked to one or more promoters. As used herein, the term "promoter" refers to any nucleic acid sequence that controls the expression of a polynucleotide by driving the transcription of the polynucleotide. As used herein, the terms "operably linked" and "operably linked" refer to the promoter being in the correct functional location and / or orientation relative to the polynucleotide it regulates to control transcription initiation and / or expression of the sequence. In some embodiments, the polynucleotides encoding the VH region and the VL region are operably linked to the same promoter. In some embodiments, the polynucleotides encoding the VH region and the VL region are each operably linked to a separate promoter. In some embodiments, the separate promoters are the same type of promoter. In some embodiments, the separate promoters are different types of promoters.

[0352] In some embodiments, for example, the vector comprises one or more of an enhancer and a repressor sequence. As used herein, the term "enhancer" refers to a nucleic acid sequence that binds to one or more proteins that increase the transcriptional activation of a polynucleotide. As used herein, the term "repressor" refers to a nucleic acid sequence that binds to one or more proteins that decrease the transcriptional activation of a polynucleotide.

[0353] In some aspects, the invention provides expression vectors that include one or more of the following operably linked elements: a transcription promoter, a polynucleotide encoding the heavy chain of an antibody or antigen-binding fragment of the invention, a polynucleotide encoding the light chain of an antibody or antigen-binding fragment of the invention, and a transcription terminator.

[0354] Another aspect provided herein is a host cell comprising a polynucleotide, said polynucleotide comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof of the invention.

[0355] In a further aspect, host cells are provided that express and are capable of expressing the vectors of the invention. These cells can be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Spodoptera frugiperda cells), yeast cells (such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris), plant cells, or bacterial cells (such as E. coli). In a preferred embodiment, the cells are mammalian cells, preferably CHO cells.

[0356] Pharmaceutical Compositions The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient is exerted and which does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile and may contain a pharma-ceutically acceptable carrier, such as saline. Suitable pharmaceutical compositions may include one or more of a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer (e.g., human albumin), a preservative (e.g., benzyl alcohol), and an absorption enhancer to enhance bioavailability, and / or other conventional solubilizers or dispersants.

[0357] The term "pharmaceutical acceptable," as used herein, means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

[0358] The anti-FRα antibody of the present invention or an antigen-binding fragment thereof, or the ADC of the present invention can be administered to a subject as a pharmaceutical composition. Thus, the present invention also provides a pharmaceutical composition comprising the anti-FRα antibody of the present invention or an antigen-binding fragment thereof, or the ADC of the present invention, and a pharma- ceutical acceptable excipient.

[0359] In another aspect, pharmaceutical compositions according to the invention and for use according to the invention may contain, in addition to the active ingredient (e.g., an anti-FRα antibody or antigen-binding fragment thereof, or an ADC of the invention), a pharma- ceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials will depend on the route of administration, which may be oral, or by injection, e.g., cutaneous, subcutaneous, or intravenous.

[0360] In some embodiments, pharmaceutical compositions of the invention may include pharma- ceutically acceptable, non-toxic, sterile carriers, such as saline, non-toxic buffers, preservatives, etc. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0361] Examples of suitable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, or sodium chloride in the composition.

[0362] One of ordinary skill in the art will understand that the appropriate choice of excipient for use with the anti-FRα antibodies or antigen-binding fragments thereof, or ADCs of the invention, will depend on the desired properties of the pharmaceutical composition.

[0363] In some embodiments, the pharmaceutical compositions of the present invention may be contained within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, a lyophilized powder that can be reconstituted to make a suspension or solution prior to use, or combinations thereof.

[0364] In some embodiments, a pharmaceutical composition comprises more than one type of antibody or antigen-binding fragment of the invention. For example, a pharmaceutical composition may comprise two or more selected from an antibody, an antigen-binding fragment, an antibody or antigen-binding fragment thereof conjugated to a cytotoxin, or a combination thereof.

[0365] In some embodiments, the pharmaceutical composition may include a buffer (e.g., an acetate, phosphate or citrate buffer), a surfactant (e.g., a polysorbate), optionally a stabilizer (e.g., human albumin), and the like.

[0366] The pharmaceutical compositions disclosed herein are used, including but not limited to, for the diagnosis, detection or monitoring of a disorder, the prevention, treatment, management or amelioration of a disorder or one or more symptoms thereof, and / or research. The pharmaceutical compositions disclosed herein may be suitable for veterinary or pharmaceutical use in humans.

[0367] The pharmaceutical compositions of the invention can be administered to a patient by any suitable systemic or local route of administration, for example, administration can be oral, buccal, sublingual, ocular, intranasal, intratracheal, pulmonary, topical, transdermal, genitourinary, rectal, subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumor.

[0368] The pharmaceutical compositions of the present invention can be formulated for administration by any suitable means, for example, by epidermal or transdermal patch, ointment, lotion, cream, or gel; by atomizer, vaporizer, or inhaler; by injection or infusion; or in the form of capsules, tablets, solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route of administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation.

[0369] The pharmaceutical composition for oral administration may be in tablet, capsule, powder or liquid form. Tablets may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Capsules may contain a solid carrier such as gelatin.

[0370] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art are fully capable of preparing suitable solutions using isotonic vehicles such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0371] treatment The present invention encompasses therapeutic methods comprising administering to a subject an anti-FRα antibody or antigen-binding fragment thereof, ADC, or pharmaceutical composition of the present invention to prevent, treat, or ameliorate symptoms associated with a disease, disorder, or infection.

[0372] "Treating" refers to a therapeutic measure that cures, slows, alleviates symptoms, and / or halts progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already with the disorder. In some embodiments, a subject has been successfully "treated" by the methods provided herein for a disease or disorder (preferably, cancer) if the subject exhibits, for example, complete, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder (preferably, cancer).

[0373] "Preventing" refers to prophylactic or preventative measures that prevent and / or delay the onset of the targeted pathological condition or disorder. Thus, those in need of prevention include those prone or susceptible to developing the disorder. In some embodiments, a disease or disorder (preferably cancer) has been successfully prevented by the methods provided herein if the patient has, for example, fewer or less severe symptoms associated with the disease or disorder, or has a later onset of symptoms associated with the disease or disorder, either transiently or permanently, than a patient who has not been subjected to the methods of the invention.

[0374] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammalian subject. In some embodiments, the "subject" is a human, a farm animal, a livestock animal, a sport animal and a zoo animal, such as a human, a non-human primate, a dog, a cat, a guinea pig, a rabbit, a rat, a mouse, a horse, a cow, etc. In some embodiments, the subject is a cynomolgus monkey (Macaca fascicularis). In a preferred embodiment, the subject is a human. In the methods of the invention, the subject may not have been previously diagnosed with cancer. Alternatively, the subject may have been previously diagnosed with cancer. The subject may also exhibit disease risk factors or have no symptoms of cancer. The subject may also have cancer or be at risk for developing cancer. In some embodiments, the subject has previously undergone cancer treatment.

[0375] Thus, in some aspects, there is provided an anti-FRα antibody or antigen-binding fragment thereof, an ADC of the invention, or a pharmaceutical composition, for example, for use in therapy to treat a disease or disorder (e.g., cancer). Also provided is a method of treating a disease or disorder (e.g., cancer), comprising administering to a subject a therapeutically effective amount of an anti-FRα antibody or antigen-binding fragment thereof, an ADC, or a pharmaceutical composition of the invention. In one aspect, there is provided a method of preventing the onset of a disease or disorder (e.g., cancer), comprising administering to a subject a therapeutically effective amount of an anti-FRα antibody or antigen-binding fragment thereof, an ADC, or a pharmaceutical composition of the invention.

[0376] The term "therapeutically effective amount" is an amount sufficient to provide a benefit to a patient. Such benefit may be at least an amelioration of at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., decisions regarding dosage, are within the responsibility of general practitioners and other physicians.

[0377] In one aspect, the anti-FRα antibody or antigen-binding fragment thereof, ADC, or pharmaceutical composition of the present invention is used to treat cancer associated with FRα expression. In another aspect, a method for treating cancer associated with FRα expression is provided, comprising administering to a subject an anti-FRα antibody or antigen-binding fragment thereof, ADC, or pharmaceutical composition of the present invention. In other words, the cancer referred to herein may comprise cancerous cells expressing FRα. The cancerous cells may be contained within a tumor. In some embodiments, the cancer comprises cancer cells that exhibit heterogeneous expression of FRα and / or low expression of FRα.

[0378] Preferably, the cancer is selected from ovarian cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, breast cancer (e.g., TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinoma (HNSCC) and malignant pleural mesothelioma. More preferably, the cancer is ovarian cancer or lung cancer. In some embodiments, the cancer is one or more non-small cell lung cancers (NSCLC), preferably selected from squamous cell NSCLC, adenocarcinoma NSCLC or a combination thereof.

[0379] Further examples of cancer include, but are not limited to, benign, pre-malignant and malignant cell growths, including, but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancer (e.g., ovarian cancer, lung cancer, non-small cell lung cancer (squamous cell carcinoma or adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, colorectal cancer, squamous cell carcinoma of the head and neck, malignant pleural mesothelioma, breast cancer (e.g., TNBC), and renal cancer. Any type of cell can be treated, including, but not limited to, lung, gastrointestinal, mammary (breast), ovarian, kidney (renal) and pancreatic cells.

[0380] In one aspect, the anti-FRα antibody or antigen-binding fragment thereof, ADC, or pharmaceutical composition of the invention is used in a method for depleting a population of FRα-positive cells in a subject, the method comprising administering the anti-FRα antibody or antigen-binding fragment thereof, ADC, or pharmaceutical composition to the subject. In another aspect, a method for depleting a population of FRα-positive cells in a subject is provided, the method comprising administering a therapeutically effective amount of the anti-FRα antibody or antigen-binding fragment thereof, ADC, or pharmaceutical composition of the invention to the subject. In some embodiments, the FRα-positive cells exhibit heterogeneous expression of FRα and / or underexpression of FRα.

[0381] Other uses for antibodies The antibodies or antigen-binding fragments exhibit high affinity for FRα both in vitro and in vivo and therefore may be advantageously used in methods for detecting FRα epitopes and related diagnostic methods.

[0382] The antibodies or antigen-binding fragments thereof of the present invention may be used in assays for immunospecific binding by any method known in the art, including but not limited to competitive and non-competitive assay systems using techniques such as Western blot, RIA, ELISA, ELISPOT, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.

[0383] The antibody or antigen-binding fragment thereof of the present invention may be used histologically, for example, for in situ detection of FRα or its conservative variants or peptide fragments, such as in immunofluorescence, immunoelectron microscopy, or non-immunological assays. In situ detection can be achieved by removing a histological specimen from a patient and applying a labeled antibody or antigen-binding fragment thereof of the present invention thereto, for example by overlaying the labeled antibody or antigen-binding fragment thereof on the biological sample. Using such a procedure, it is possible to determine not only the presence of FRα or its conservative variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention, one skilled in the art will readily understand that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

[0384] For example, to aid in detection of cell binding (in vitro or in vivo), the antibody or antigen-binding fragment thereof, or antibody-drug conjugate may be labeled. The label may be a biotin label. In another embodiment, the label may be a radioisotope. In another embodiment, the label may be a fluorophore.

[0385] In one aspect, a method is provided for detecting the presence or absence of an FRα polypeptide (e.g., an FRα polypeptide epitope) in a sample, the method comprising: (a) contacting the sample with an antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the invention to provide an antibody-antigen complex; and (b) detecting the presence or absence of said antibody-antigen complex. Including, The presence of an antibody-antigen complex confirms the presence of an FRα polypeptide (eg, an FRα polypeptide epitope) or, the absence of an antibody-antigen complex confirms the absence of an FRα polypeptide (eg, an FRα polypeptide epitope).

[0386] Products and Kits In a further aspect, provided herein is an article of manufacture comprising one or more anti-FRα antibodies or antigen-binding fragments thereof of the invention, or an ADC of the invention, or a pharmaceutical composition of the invention.

[0387] In yet a further aspect, a pharmaceutical pack or kit is provided that includes one or more containers filled with one or more of the components of the pharmaceutical composition of the invention, such as one or more antibodies or antigen-binding fragments thereof, or ADCs of the invention. Optionally, such containers may be accompanied by a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which notice indicates approval by the agency of manufacture, use, or sale for administration to humans. Instructions on how to use the provided pharmaceutical composition in the treatment of cancer, such as, for example, ovarian cancer, lung cancer (e.g., NSCLC), endometrial cancer, breast cancer (e.g., TNBC), cervical cancer, pancreatic cancer, gastric cancer, renal cell carcinoma, colorectal cancer, head and neck squamous cell carcinoma (HNSCC), and malignant pleural mesothelioma, may also be included or made available to the patient or health care provider.

[0388] In one aspect, a kit is provided comprising an antibody or antigen-binding fragment, or ADC, or pharmaceutical composition of the invention. Further encompassed is the use of said kit in the methods of the invention.

[0389] In some embodiments, the kit may provide the antigen or antibody-binding fragment and the heterologous agent individually (e.g., a cytotoxin that is not conjugated to the antibody or antigen-binding fragment but is in a form suitable for conjugation thereto); optionally, the kit further comprises instructions and / or reagents for conjugating the heterologous agent to the antibody or antigen-binding fragment. In some embodiments, the kit includes all of the components necessary and / or sufficient to perform a detection assay, including all controls, instructions for performing the assay, and any software necessary for analysis and presentation of the results.

[0390] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.

[0391] The above embodiments should be understood as illustrative examples. Further embodiments are envisioned. It should be understood that any feature described with respect to any of the embodiments can be used alone or in combination with other features described, and can also be used in combination with one or more features of any other embodiment or aspect, or any combination of any other embodiment or aspect. Moreover, equivalents and modifications not described above can also be used without departing from the scope of the invention as defined in the appended claims.

[0392] Other examples and variations of the antibodies and methods described herein with respect to the present invention will be apparent to one of skill in the art and are within the scope of this disclosure as set forth in the appended claims.

[0393] All documents cited herein are hereby incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents. EXAMPLES

[0394] Example 1. Generation of anti-FRα antibodies the purpose A hybridoma campaign was conducted using humanized transgenic mice to obtain a high-affinity, fully human antibody that binds to the folate receptor alpha (FRα).

[0395] Materials and Methods Generation of human FRα for immunization The human FRα gene was inserted into the pDEST12.2 OriP vector. The sequence corresponding to the soluble region of the folate receptor was further cloned with an N-terminal CD33 leader and a C-terminal Avi and His6 tag. The sequence involved in GPI anchoring was removed to obtain a soluble construct.

[0396] Human FRα protein was expressed and purified using standard methods. Briefly, plasmid DNA was prepared and transfected at 4×10 6 An in-house suspension-adapted CHO cell line was transfected using PEI-mediated delivery with a cell density of 10 ...

[0397] immunity Using a repeated multiple site immunization (RIMMS) strategy, Del-1 humanized mice and CD1 wild type mice were immunized as follows: - Minus 4 days: Pre-bleed - Day 0: Prime Immunization - 7th day: 2nd boost - 13th day: First blood draw - 15th: 3rd boost - 20th day: 2nd blood draw - 22nd: 4th boost - 24th: 5th boost - 28th day: Final blood sampling and fusion of spleen (SP) and lymph node (LN)

[0398] For immunization, six humanized Del-1 mice and four CD1 wild-type mice were divided into two groups (group 1: Del-1 mice 1-6, group 2: CD1 mice 7-10). Animals were immunized with recombinant human FRα extracellular domain as described above. Recombinant FRα was diluted in PBS, emulsified with an equal volume of complete Freund's adjuvant, and injected into mice at two sites. In the three subsequent injections, the immunogen was emulsified with incomplete Freund's adjuvant and injected as described above. A final boost was given on day 24 by intraperitoneally injecting the recombinant protein in PBS.

[0399] Tail vein bleeds were obtained from mice pre-immunization, day 13 after the first immunization, and day 20 after the second immunization. IgG titers against human FRα were determined by serum ELISA.

[0400] Assessment of mouse immune responses to FRα (serum ELISA) Serum IgG titers against human FRα and negative protein controls were determined by ELISA in 96-well microtiter plates using standard techniques. Antibodies were detected using a HRP-labeled polyclonal goat anti-mouse IgG specific secondary antibody (Jackson Immunolabs), and the assay was run using TMB substrate (Sigma), followed by addition of 0.5M sulfuric acid to stop the reaction. Plates were then read using a PerkinElmer EnVision 2103 multi-label plate reader.

[0401] Serum titration curves of human FRα and the negative protein control were plotted, and the area under the curve (AUC) of each was calculated.

[0402] Monoclonal mouse IgG isolation (hybridoma generation) Four days after the final boost, lymph nodes were aseptically harvested and cells were isolated by mechanical disruption and then counted. These cells were mixed with SP2 / 0 myeloma cells and fused using an electrofusion apparatus. The resulting fusion was mixed with a methylcellulose-based semi-solid medium and plated on OmniTray plates. The cells in the semi-solid medium were cultured for 13 days at 37°C in a 5% CO2 incubator. During this incubation, clonal colonies form from single precursor hybridoma cells. These colonies secrete IgG that is captured in the vicinity of the colony by FITC-conjugated anti-IgG present in the semi-solid medium. The resulting immune complex formation can be seen as a fluorescent "halo" around the cells when visualized by a ClonePix FL colony picker (Molecular Devices). Colonies with these halos are then picked into 96-well microtiter plates. After 3-5 days of culture, the supernatants of the harvested colonies were collected and screened for human FRα binding.

[0403] DNA sequencing of mouse IgG Messenger RNA (mRNA) was extracted from hybridoma cells using magnetic oligo(dT) particles and reverse transcribed into cDNA. PCR amplification was performed using polyC and constant region VH or VL primers specific for all mouse IgG subclasses. The sequences of the PCR amplicons were determined by Sanger sequencing.

[0404] Phynexus purification of mouse IgG Cells were grown in 24-well plates and after 10 days, the supernatants were transferred to a 96-well master block and all subclasses of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) were purified from overgrown cell culture supernatants on ProPlus resin (Phynexus) using a Perkin Elmer Minitrack. Captured mouse IgG was eluted with 100 mM HEPES, 140 mM NaCl pH 3.0 and then neutralized with an equal volume of 200 mM HEPES pH 8.0. Purified IgG was quantified using absorbance readings at 280 nm in a UV-Star 384-well plate.

[0405] Reformatting Mouse IgG Mouse hybridoma IgG clones were molecularly reformatted to generate constructs expressing mouse VH and VL domains and relevant mouse IgG constant domains for each hybridoma essentially as described in Persic et al., Gene 187:9-18, 1997. The VH domain was cloned into the relevant vector containing mouse heavy chain constant domains and regulatory elements to express full IgG1 heavy chains in mammalian cells. Similarly, the VL domain was cloned into a vector for expression of the appropriate mouse light chain (lambda or kappa) constant domains and regulatory elements to express full IgG light chains in mammalian cells. To obtain IgG, mammalian suspension CHO cells were transiently transfected with heavy and light chain IgG vectors. IgG was expressed and secreted into the culture medium. IgG was purified from the clarified supernatant using a MabSelect SuRe chromatography column (GE Healthcare Lifesciences Catalog No. 11003493 (for 1 ml column); 11003495 (for 5 ml column)) and an AktaXpress™ purification system from GE Healthcare Lifesciences. The eluted material was buffer exchanged into PBS using a PD-10 desalting column (GE Healthcare Lifesciences; Catalog No.; 17085101). The concentration of IgG was measured spectrophotometrically using the extinction coefficient based on the amino acid sequence of IgG (Pace et al., Protein Sci. 4:2411-2423, 1995), and purified IgG was analyzed for purity using SDS-PAGE and HP-SEC analysis.

[0406] result Serum anti-FRα IgG titers after immunization Various approaches are available for mAb discovery, including the use of phage display, immunization, and binding profiles (including identifying mAbs that compete with folate binding to FRα). Here, a dual approach was used to generate anti-FRα antibodies through immunization. The first route involved immunization of human transgenic mice (i.e., Del-1) that contain fully human VH and Vk domains in the Ig locus. This ensured that diverse mAbs were generated, yet fully human and ready for development without the need for humanization. The second route involved immunization of non-transgenic mice (i.e., CD-1), which required humanization of the mAbs.

[0407] Serum samples were collected from all mice on days -4 (pre-bleed), 13 (bleed 1), and 20 (bleed 2) of the immunization protocol and tested for the presence of anti-FRα-specific antibodies. All mice responded to immunization and produced anti-FRα-specific antibodies.

[0408] Generation of hybridomas A total of 10510 hybridoma clones were generated, and 2586 were identified as IgG-secreting colonies. IgG-secreting colonies were picked into 96-well microtiter plates. After 3-5 days of culture, supernatants from picked colonies were collected and screened for human FRα binding. All lead antibodies generated (see Example 2) were obtained from Del-1 transgenic mice.

[0409] Example 2 - Species cross-reactivity of anti-FRα antibodies the purpose The generated antibodies were characterized for binding strength to the target, ortholog specificity, and paralog specificity.

[0410] Materials and Methods Preparation of antigens for assay The species from which the protein originates, the vector in which the construct was cloned, and the signal peptide and epitope tag fused to the protein are shown. Table 10 further shows the sequence of each insert. In each case, the sequence corresponding to the soluble region of the folate receptor was further cloned with an N-terminal CD33 leader and a C-terminal Avi and His6 tag. The sequence involved in GPI anchoring was removed to obtain a soluble construct.

[0411] [Table 25]

[0412] [Table 26]

[0413] [Table 27]

[0414] All proteins in Table 10 were expressed and purified using the methods discussed in Example 1 for human FRα.

[0415] HTRF assay HTRF assays were performed in 384-well white shallow-well non-binding plates (Corning, 4513) in assay buffer containing phosphate-buffered saline (PBS) (Life Technologies, 14190), 0.1 v / v% bovine serum albumin (BSA) (Sigma, A9576), and 0.4 M potassium fluoride (VWR International, 26820). Time-resolved fluorescence at 590 nm and 665 nm was measured after the indicated incubation periods on an Envision (PerkinElmer) plate reader after excitation at 320 nm. The ratio value of (665 nm emission / 590 nm emission) × 10,000 was used to calculate delta F% according to the following formula:

number

[0416] Negative control ratios were obtained from non-specific binding (NSB) control wells. Curves were analyzed by a four-parameter logistic curve fitting equation using GraphPad Prism software.

[0417] HTRF antigen-binding assay Supernatants containing mouse IgG from hybridoma cells were incubated with 0.5 nM streptavidin-cryptate (CisBio, 610SAKLB), anti-mouse IgG AF647 (Jackson ImmunoResearch, 115-605-164), and 1 nM biotinylated human FRα, cynomolgus monkey FRα, mouse FRα, human FRβ or human FRγ at a final assay dilution of 25% for 4 hours at room temperature to give a final assay volume of 10 μl, after which fluorescence was measured.

[0418] Antigen-binding assay of rat FRα Species binding assays were performed with Octet RED384 (ForteBio) in assay buffer containing PBS, 0.1 v / v% BSA (Sigma, A9576), 0.01 v / v% Tween-20 (Sigma, P9416) (pH 7.4) in slanted bottom black 384-well plates (ForteBio, 18-5076) at 25°C. Assays were set up using Protein A or anti-human capture biosensor (AHC) (ForteBio, 18-5089) according to the manufacturer's instructions. 10 μg / ml of anti-rat FRα IgG (Sino Biological, 81073-RP01) was coated onto a Protein A biosensor (ForteBio, NC9490476) and 10 μg / ml of test human IgG was loaded onto the anti-human capture biosensor (AHC) (ForteBio, 18-5089) for 180 seconds. Binding was measured by incubating biosensors loaded with 500 nM human FRα (in-house) or 500 nM rat FRα (Sino Biological, 81073-R08H). Dissociation was measured after transfer into assay buffer. Data was analyzed using Octet data analysis software version 7.0.

[0419] result The supernatants of 2586 hybridomas were tested for binding to human FRα, cynomolgus monkey FRα, mouse FRα, human FRβ or human FRγ in HTRF assay format. This experiment was performed to isolate antibodies that cross-react with cynomolgus monkey FRα, while confirming that no binding to paralogs FRβ and FRγ was observed. To determine the sequence similarity of the human folate receptor to paralogs and orthologs, multiple sequence alignments were determined using the Clustal Omega v1.2.2 algorithm. The sequence identities of human FRα to paralogs (Table 11) and orthologs (Table 12) are shown below.

[0420] [Table 28]

[0421] [Table 29]

[0422] Binders were defined as IgGs with an assay signal of >30 delta F%. A total of 129 IgGs showed binding only to human and cynomolgus FRα, 9 IgGs showed binding only to human, cynomolgus and mouse FRα, and an additional 30 IgGs showed binding only to human, cynomolgus or mouse FRα. Data for the six lead IgGs are shown in Table 13.

[0423] [Table 30]

[0424] 168 hybridoma IgGs identified in the hybridoma supernatant screening were designated as Phynexus purified IgGs. These were tested in antigen binding assays at 4 dilutions of IgG. 115 IgGs were identified as IgGs that were cross-reactive with human and cynomolgus monkey FRα and did not bind to human FRβ and human FRγ. Data for the six lead IgGs are shown in Figures 1A-1F.

[0425] Furthermore, in a rat FRα antigen binding assay, the six lead IgGs also demonstrated binding to human FRα, but not to rat FRα.

[0426] Overall, the extensive, high-throughput antibody generation and screening campaign demonstrated the generation of a variety of different anti-FRα IgG antibodies.

[0427] Example 3 - High-throughput internalization assay the purpose The antibodies generated were characterized for their rate of internalization.

[0428] Materials and Methods PhyNexus purified IgG samples from hybridoma output were evaluated in the internalization assay. Frozen stocks of KB cells were plated overnight before adding IgG samples. The assay was performed using the fixed acquisition method, with a 2.5 hour incubation before fixation. The assay was performed using pHrodo Green labeling detection reagent and Cell Mask Red (Thermo Fisher Scientific).

[0429] Day 1 Frozen vials of KB cells were thawed, diluted with medium, centrifuged, and resuspended in fresh medium before counting. Cells were plated at 10,000 cells / well in MEM+NEAA+10% FCS (Thermo Fisher Scientific) and the prepared plates were incubated overnight at 37° C., 5% CO2 in a humidified incubator.

[0430] Day 2 Antibodies and secondary detection reagents were prepared and pre-incubated for 30 minutes at room temperature. The medium was removed from the cells and pre-mixed antibodies and detection reagents were added. The plates were incubated at 37°C for 2.5 hours. After incubation, 30 μl / well of 7.2% formaldehyde (Thermo Fisher Scientific) + Hoechst (Thermo Fisher Scientific) 1:5000 diluted solution was added to obtain a 3.7% formaldehyde fixative and a 1:10000 diluted nuclear stain. The plates were incubated at room temperature for 20 minutes and then washed with 1×HBSS (Thermo Fisher Scientific) before adding 30 μl / well of cell mask red (1:5000 diluted in 0.1% Triton X-100 (Merck)). The plates were incubated at room temperature for 20 minutes in the dark, washed twice with HBSS and imaged on the Opera High Content Imaging system (see Table 14 for Opera Acquisition parameters).

[0431] [Table 31]

[0432] result A high-throughput internalization assay was performed to assay antibody internalization rates into KB cells. Phynexus purified antibodies were labeled and added to KB cells, and cells were fixed after 2.5 hours. Image analysis was performed using Columbus and output analyzed within Spotfire's High Content Profiler, allowing multiparameter analysis of the resulting image data and the creation of a "hit list" containing mAbs that showed improved uptake rates (Figure 2). All six lead antibodies demonstrated internalization above the cutoff determined by positive control samples.

[0433] Example 4: Epitope binning of generated anti-FRα antibodies the purpose The purpose of this experiment was to epitope bin the anti-FRα antibodies generated, specifically the six exemplary antibodies.

[0434] Materials and Methods Three benchmark anti-FRα IgGs were labeled for use in homogeneous time-resolved fluorescence (HTRF) assays using a microscale DyLight 650 antibody labeling kit (Thermo Scientific, 84536) according to the manufacturer's instructions (see Example 2 for general set-up of the HTRF assay).

[0435] HTRF epitope competition assays were set up in an assay volume of 10 μl with titrations of test IgG, 1 nM streptavidin cryptate, biotinylated human FRα and DyLight 650-labeled IgG. For comparative antibody 1 and comparative antibody 2 assays, 0.5 nM biotinylated human FRα and 0.5 nM DyLight 650-labeled comparative antibody 1 or comparative antibody 2 were used. For comparative antibody 3 assays, 1.25 nM biotinylated human FRα and 1.25 nM DyLight 650-labeled comparative antibody 3 were used. The assay plates were incubated at room temperature for 3-4 hours before fluorescence measurements.

[0436] result 115 human / cynomolgus folate receptor-specific hybridoma IgGs were profiled in three HTRF epitope competition assays to determine epitope diversity: 8 IgGs were inhibited in all assays, 41 in Comparator 1 and Comparator 2 assays, 39 in Comparator 2 and Comparator 3 assays, 12 in Comparator 1 and Comparator 3 assays, only 4 in Comparator 1 assay, only 6 in Comparator 3 assay, and 5 IgGs were not inhibited in any assay.

[0437] Six exemplary antibodies were reformatted as human IgG1, expressed, purified, and profiled in HTRF epitope competition assay to confirm potency. Notably, all lead exemplary antibodies could inhibit in comparative antibody 2 competition assay. AB1370117 and AB1370035 inhibited in all three epitope competition assays. AB1370026, AB1370083, and AB1370095 inhibited only in comparative antibody 1 and comparative antibody 2 epitope competition assays. AB1370049 inhibited only in comparative antibody 2 and comparative antibody 3 competition assays (see Table 15 and Figure 3).

[0438] [Table 32]

[0439] conclusion Six antibodies were profiled by epitope competition assay. Various behaviors and epitope diversity were observed, with some molecules competing with all three comparative antibodies, and others competing with only a subset. AB1370049 showed no competition with comparative antibody 1, the weakest competition with comparative antibody 2, but did compete with comparative antibody 3.

[0440] Example 5 - Antigen binding affinity of generated anti-FRα antibodies the purpose To determine the binding kinetics and equilibrium dissociation constants of the panel of generated antibodies, particularly six exemplary antibodies, against human FRα and cynomolgus FRα using surface plasmon resonance analysis.

[0441] Materials and Methods Antibody affinity to human and cynomolgus monkey FRα proteins was measured at 25°C using a Biacore T200 surface plasmon resonance system (Cytiva). Protein A was covalently immobilized on a CM5 chip surface using standard amine coupling techniques at a concentration of 50 μg / ml in 10 mM sodium acetate (pH 4.0). To allow FRα ECD binding, antibodies were captured on the Protein A surface at 10 μl / min in HBS-EP+ buffer (pH 7.4). FRα ECD was serially diluted in HBS-EP+ buffer pH 7.4 (0.4 nM-100 nM human FRα ECD; 0.8 nM-200 nM cynomolgus monkey FRα ECD; 30 nM-4000 nM mouse FRα ECD and rat FRα ECD) and flowed over the chip at 50 μl / min, bound for 2 min, and dissociated for 8 min. The chip surface was thoroughly regenerated with a pulse of 3M MgCl2 to remove the capture antibody along with any bound FRαECD. Multiple buffer-only injections were performed under the same conditions to allow for double reference subtraction of the final set of sensorgrams and analyzed using Biacore T200 analysis software to derive the equilibrium dissociation constants.

[0442] result The antibody affinity of the antibody panel to human and cynomolgus monkey FRα proteins was measured by SPR. All antibodies bound to both human and cynomolgus monkey proteins. The affinity to human FRα ranged from 1-16 nM, and the affinity to cynomolgus monkey FRα ranged from 1-37 nM. The kinetic binding parameters of six exemplary antibodies are summarized in Table 16 below.

[0443] [Table 33]

[0444] conclusion All six antibodies have dissociation constants in the low nM range, and in all cases the affinity for binding to cynomolgus monkey FRα is within 2.5-fold of the affinity for binding to human FRα. Therefore, all six exemplary antibodies appear to be suitable from an affinity standpoint for further development as therapeutic agents.

[0445] Example 6 - Physicochemical properties of generated anti-FRα antibodies the purpose To generate therapeutic antibodies that are viable and aligned to manufacturing requirements, the physicochemical properties of the antibodies must be thoroughly evaluated. In this study, a panel of generated antibodies was evaluated for their expression titers, stability, and tendency towards reversible self-association.

[0446] Materials and Methods Hydrophobic Interaction Chromatography (HIC) HPLC UHPLC-HIC analysis was performed on a Shimadzu Prominence system using a Sepax Proteomix HIC Butyl-NP5 5 μM non-porous 4.6 × 35 mm column, on approximately 1 mg / ml samples of each lead mAb, as a 1:1 dilution with 1.5 M ammonium sulfate in 25 mM sodium phosphate, pH 7.40 buffer, eluting with a gradient of 1.5 M to 0 M ammonium sulfate and 0 to 20% acetonitrile in 25 mM sodium phosphate, pH 7.40. The more hydrophobic the species, the later it elutes and therefore the longer the retention time.

[0447] Affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) Capture antibody (goat anti-human IgG Fcγ fragment specific, Jackson ImmunoResearch) was buffer exchanged into binding buffer (20 mM potassium acetate, pH 4.3) and capture nanoparticles (citrate stabilized 20 nm gold nanoparticles (OD=1), Innova Biosciences) were prepared by incubating with 0.4 mg / ml of antibody for 1 h at room temperature. Nanoparticles were blocked by incubation with 100 nM PEG2000 (Merck) for 1 h at room temperature followed by 10-fold concentration. Antibodies for analysis were prepared at a final concentration of 45 μg / ml in a solution containing 12 μl of capture nanoparticles and 20 mM histidine, 120 mM sucrose, 80 mM arginine, pH 6 (HSA) buffer for a total volume of 120 μl in a 96-well plate. Samples were incubated at room temperature for 20 minutes, then 50 μl aliquots were transferred to 384-well polystyrene plates (Nunc 384-well clear polystyrene plates, Thermo Scientific). Sample absorbance was measured on a plate reader and the wavelength red shift of each sample was determined relative to that of buffer-only control wells. A shift of >5 nm was flagged as a risk of self-association.

[0448] Baculovirus ELISA Non-specific binding of antibodies to baculovirus particles was assayed by ELISA as described by Hotzel et al (Hotzel et al 2012 mAbs 4:6, 753-760). Preparations of each antibody were made at 100 nM or 10 nM in PBS (Gibco 14190-086) + 0.5% BSA (Sigma A9576) and used in duplicate in an ELISA assay on 96-well Nunc Maxisorp F plates coated overnight at 4°C with 50 μL / well of 1% baculovirus extract in 50 mM sodium carbonate (BV plates) or 50 mM sodium carbonate (blank plates). After washing with PBS, plates were blocked with 300 μL / well of PBS + 0.5% BSA for 1 hour at room temperature and washed three times with PBS. 50 μL / well of PBS + 0.5% BSA (background) or test antibody dilutions were added and incubated at room temperature for 1 hour. After washing three times with PBS, detection antibody (anti-human Fc specific-HRP Sigma A0170) diluted 1:5000 in PBS+0.5% BSA was added at 50 μL / well. Samples were incubated for 1 hour at room temperature and the plate was washed three times with PBS. HRP substrate TMB (SureBlue Reserve KPL 53-00-03) was then added at 50 μL / well and after color change, the reaction was stopped by adding 50 μL / well of 0.5 M sulfuric acid. Absorbance was measured at 450 nm. The BV score was calculated by averaging the 450 nm absorbance at 10 nM and 100 nM concentrations of each antibody sample and then dividing by the second control sample only. A BV score of >5 may indicate a risk of increased clearance due to non-specific binding.

[0449] Accelerated thermostability assay IgG was diluted to 1 mg / ml in PBS and then incubated at 4° C. or 45° C. for 2 weeks, then filtered using a filter spin column (Millipore, UFC30HVNB). High performance size exclusion chromatography (HP-SEC) was performed by loading 70 μl of IgG onto a TSKgel G3000SWXL; 5 μM, 7.8 mm×300 mm column with a flow rate of 1 ml / min and 0.1 M anhydrous disodium phosphate and 0.1 M sodium sulfate (pH 6.8) as the isocratic running buffer. Larger molecules are excluded from the pores of the size exclusion column to a greater extent than smaller molecules, and therefore elute earlier. Peaks eluting earlier than the monomer peak are recorded as aggregates. Peaks eluting after the monomer peak (excluding buffer-related peaks) are recorded as fragments. In parallel, antibodies were profiled in HTRF epitope competition assays for potency changes.

[0450] result Hydrophobic Interaction Chromatography (HIC) HPLC Higher HIC retention times correspond to increased hydrophobicity, which may indicate increased risk of aggregation and clearance due to non-specific uptake. Furthermore, more hydrophobic mAbs may result in more hydrophobic ADCs, which may lead to aggregation during conjugation, instability as ADCs, and potentially more non-specific uptake into normal tissues and therefore toxicity.

[0451] The panel of antibodies generated exhibited retention times ranging from approximately 2.0 to 2.8 minutes by HPLC-HIC. Notably, six exemplary antibodies exhibited acceptably low retention times compared to the panel of antibodies tested (see Table 17).

[0452] [Table 34]

[0453] Affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) The antibodies were also tested for their tendency to self-interact by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). This behavior is the basis for or associated with undesirable properties such as reversible self-association, aggregation, viscosity, milkiness and phase separation. AC-SINS indirectly measures the tendency of proteins to self-interact in environments with high protein concentrations by monitoring the wavelength of peak absorbance (plasmon wavelength) of antibody-coated gold particles. The red shift of the exemplary antibodies showed negligible levels of self-interaction in HSA buffer for all tested antibodies, while the negative and positive control antibodies behaved as expected (Figure 4). These results indicate a low risk of self-association for all six exemplary antibodies tested.

[0454] Baculovirus ELISA To test for non-specific binding, which may indicate an increased clearance rate in vivo, the level of binding of antibody to baculovirus particles was assayed in ELISA format.In all cases, antibody showed negligible levels of non-specific binding, all of which were below the cutoff assay threshold of 5.Advantageously, this suggests a low risk of insufficient clearance in vivo.

[0455] Accelerated thermostability assay A panel of six exemplary antibodies was assessed for their thermal stability by both HP-SEC and a parallel HTRF epitope competition assay for change in potency (Table 18). In all cases, the change in potency was less than 2-fold and the loss of monomer was less than 3%, indicating that all antibodies were thermally stable under the conditions tested.

[0456] [Table 35]

[0457] conclusion All six lead mAbs exhibited good thermal stability, low self-association risk, negligible non-specific binding, and low hydrophobicity, and therefore all lead mAbs exhibit acceptable developability parameters.

[0458] Example 7 - Pharmacokinetics of generated anti-FRα antibodies in SCID mice the purpose To examine the pharmacokinetics of the generated anti-FRα antibodies in wild-type SCID mice following a single intravenous administration.

[0459] Materials and Methods Wild-type SCID mice (n=3 per group) were intravenously (bolus) administered exemplary anti-FRα antibodies (5 mg / kg). Blood samples were taken 15 minutes, 4 hours, 1, 2, 3, 6, 10, 14 and 21 days after administration, centrifuged to obtain plasma, and analyzed for total antibody. Pharmacokinetic parameters were determined using Phoenix64 software (Certara).

[0460] Plasma antibody concentrations were determined by a universal ELISA IgG assay.

[0461] result Although exemplified by the screening process based on the above examples, a wide range of PK profiles was observed within this broad panel of mice. PK screening eliminated leads with undesirable high clearance and short half-lives.

[0462] The panel of antibodies tested gave clearances ranging from about 5 to 25 ml / kg / day and half-lives ranging from about 4 to 20 days. Among the antibodies tested, six exemplary anti-FRα antibodies showed relatively low clearance and long half-lives. The pharmacokinetic parameters of the six exemplary anti-FRα antibodies in mice are shown in Table 19.

[0463] [Table 36]

[0464] conclusion The exemplary anti-FRα antibodies exhibited different pharmacokinetic properties in wild-type SCID mice, with AB1370035 exhibiting the longest half-life in this model, closely followed by AB1370049, which also provided the lowest clearance along with AB1370117.

[0465] Example 8 - Pharmacokinetics of AB1370049 in hFcRn Tg32 mice the purpose To investigate the pharmacokinetics of AB1370049 in hFcRn Tg32 mice after a single intravenous administration.

[0466] Materials and Methods Human FcRn Tg32 mice (n=3 per group) were intravenously (bolus) dosed with AB1370049 (5 mg / kg). Blood samples were taken 15 min, 4 h, 1, 2, 3, 6, 10, 14 and 21 days after dosing, centrifuged to obtain plasma, and total antibody was analyzed. Pharmacokinetic parameters were determined using Phoenix64 software (Certara).

[0467] Antibody concentrations were measured using an immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μl of plasma samples were then diluted in TBS and incubated with the magnetic beads. After capture, the magnetic beads were washed multiple times and then digested with trypsin in the presence of an internal standard. Acid was added to quench the digestion. An aliquot of the trypsin digested liquid contents was then transferred to an injection plate for antibody analysis by LC-MS / MS.

[0468] A signature tryptic peptide (VVSVLTVLHQDWLNGK) on the human antibody Fc region was used to calculate the concentration of total antibody in the selected matrix. Tryptic digests from immunoaffinity enriched samples were separated using reversed-phase chromatography (RPLC) and the signature peptides were subsequently detected using multiple reaction monitoring (MRM). The internal standard used in this experiment is an isotope-labeled peptide. The peak area ratio of the analyte to the internal standard was used to calculate against a standard curve created by spiking the ADC reference material into the desired matrix.

[0469] Standard curves and QCs are prepared by spiking the target compound (ADC reference material) at different levels into the same matrix as the sample matrix. The quantification range covers 100ng / ml to 12,000ng / ml, and the diluted QCs cover dilutions up to 50-fold. The standard curves were fitted with linear regression with a weighting of 1 / x2.

[0470] result The pharmacokinetic parameters of AB1370049 in hFcRn Tg32 mice are shown in Table 20.

[0471] [Table 37]

[0472] conclusion AB1370049 showed similar pharmacokinetic properties in hFcRn Tg32 and wild-type SCID mice. hFcRn Tg32 mice are transgenic mouse models, in which mouse FcRn is knocked out and human FcRn is knocked in. Therefore, it represents a model that is particularly suitable for predicting the human pharmacokinetics of AB1370049. Based on the pharmacokinetics of hFcRn Tg32 mice, it is expected that the human clearance of AB1370049 is likely to be low, and the half-life is similar to other clinically used antibodies, thus allowing convenient dosing.

[0473] Example 9 - Internalization / lysosomal trafficking of exemplary anti-FRα antibodies in SCID mice the purpose To evaluate the internalization of the lead FRα mAb into FRα-positive KB and JEG-3 cells.

[0474] Materials and Methods KB and Jeg-3 cells were stained with cell trace violet (Thermo scientific), plated in 96-well plates, and incubated overnight. Monoclonal antibodies were incubated with 50 nM Fab-pHast human (ATS bio, Carlsbad) at RT and then added to the plated cells. Cell images (20×) were taken and the fluorescent area was automatically analyzed every 30 min for up to 48 h on a CellInsight CX7 high content screening platform (Thermo Scientific).

[0475] result All lead antibodies tested were rapidly internalized and saturated in the FRα-expressing cell lines at approximately 6-7 hours in JEG-3 cells (Figure 5A) and 5 hours in KB cells (Figure 5B).

[0476] conclusion Despite lower FRα expression on the JEG-3 (choriocarcinoma) cell surface (990,000 FRα receptors / cell) compared to KB cells (37 million FRα receptors / cell), the antibodies tested were rapidly internalized into both cell types, demonstrating their potential utility in an ADC format.

[0477] Example 10 - ADC Generation in DAR8 Format the purpose To generate DAR8 ADC for lead mAb with SG3932 payload for in vitro and in vivo activity evaluation for lead selection.

[0478] Materials and Methods A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP, Pierce) in phosphate buffered saline, pH 7.4 (PBS, Gibco) (50 molar equivalents / antibody, 16.7 micromoles, 333 μL) was added to a 20 mL solution of antibody (AB1370049 lead, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117; 50 mg each, 333 nmoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA, Molekula) for a final antibody concentration of 2.5 mg / mL. The reduction mixture was heated at +37° C. for 2 hours (or until complete reduction was observed by UHPLC) on an incubated orbital shaker with gentle shaking (60 rpm). After cooling to room temperature, excess reducing agent was removed by spin filter centrifugation into PBS+1 mM EDTA using a 15 ml Amicon Ultracell 50 kDa MWCO spin filter. SG3932 was added at 2.0-2.5 mg / ml as a DMSO solution (12-24 molar equivalents / antibody, 4.8-9.0 micromoles in 2.0-2.3 mL DMSO) to 18-19 mL of this reduced antibody solution (37.0-46.9 mg, 247-313 nanomoles) for a final DMSO concentration of 10% (v / v). The solutions were mixed at room temperature for 1-18 hours, then complexation was quenched by the addition of N-acetylcysteine ​​(22.2-45.2 micromolar, 222-452 μl at 100 mM) for >30 minutes at room temperature, sterile filtered, and then purified by spin filtration (PBS followed by 20 mM histidine + 240 mM sucrose, pH 6.0), concentrated using a 15 ml Amicon Ultracell 30 KDa MWCO spin filter, sterile filtered, and analyzed.

[0479] result Using a Thermo Scientific MAbPac 50 mm × 2.1 mm column on a Shimadzu Prominence system, eluting with a gradient of water and acetonitrile, and performing UHPLC-RP analysis of the reduced sample of the ADC at 214 nm and 330 nm (specific for SG3932), a mixture of uncomplexed light chain (L0), light chain bound to a single molecule of SG3932 (L1), uncomplexed heavy chain (H0), and heavy chains bound to a maximum of three molecules of SG3932 (H1, H2, H3) was shown (where the main species are L1 and H3, corresponding to a drug-antibody ratio (DAR) of 7.58 - 7.94 molecules of SG3932 per antibody calculated from the following formula using chromatogram peak integration at 214 nm). The payload quenched with free NAC was <LOD / LOQ. Representative UHPLC-RP chromatograms of AB1370049-SG3932 DAR8 are shown in Figures 6A - 6B, which demonstrated a DAR of 7.82.

Number

[0480] Using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 × 150 mm column (with a 4 μm 3.0 × 20 mm guard column) on a Shimadzu Prominence system, eluting with a sterile filtered SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride, and 10% isopropanol (v / v) at 0.3 mL / min, and performing UHPLC-SEC analysis of the sample of the ADC at 280 nm, a monomer purity of 98.5 - 99.5% was shown. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR8 is shown in Figure 6C.

[0481] UHPLC-HIC analysis of the ADC samples on a Shimadzu Prominence system using a Sepax Proteomix HIC Butyl-NP5 5 μM nonporous 4.6 × 35 mm column eluted with a gradient of 1.5 M to 0 M ammonium sulfate and 0 to 20% acetonitrile in 25 mM sodium phosphate, pH 7.40 at 214 nm showed a complex single peak corresponding to the DAR8 species with retention times of 2.98 to 3.11 min. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR8 is shown in Figure 6D.

[0482] UV (Nanodrop, Thermo) analysis gave final ADC concentrations of 0.92–3.10 mg / ml in 5.0–6.0 ml, with the resulting mass of ADC being 11.0–37.0 mg (yield 23–74%).

[0483] conclusion All lead antibodies are suitable for DAR8 conjugation, exhibiting high conjugation efficiency with DAR >7.5, no loss of DAR during purification, no aggregation / fragmentation during manufacturing, and monomer purity >98%. Thus, the ADCs of the present invention can specifically deliver very high concentrations of cytotoxic payloads to target cancer cells by binding to FRα on cancer cells. DAR8 ADCs are also homogeneous, offering the advantage of reproducibility and limited batch-to-batch variation during manufacturing. This allows for the delivery of more of less potent drugs (such as TOPOi) to target cancer cells while maintaining tolerability.

[0484] Furthermore, the relatively less hydrophobic nature of the resulting ADC may result in less nonspecific uptake by normal tissues and therefore improved tolerability compared to comparative ADCs delivering more hydrophobic drugs, such as mirvetuximab soravtansine or IMGN151.

[0485] The exemplary lead ADCs were then further evaluated for their thermal and serum stability, in vivo mouse PK, and in vitro and in vivo efficacy (see Examples below).

[0486] Example 11 - DAR4 format ADC generation the purpose To generate a DAR4 ADC for lead mAb with SG3932 payload for in vitro and in vivo activity evaluation and for comparison with the DAR8 ADC for lead selection.

[0487] Materials and Methods A 5 mM solution (2.7–2.9 molar equivalents / antibody, 416–588 nmoles, 83.2–117.7 μL) of tris(2-carboxyethyl)phosphine (TCEP, Pierce) in phosphate-buffered saline, pH 7.4 (PBS, Gibco) was added to 9.2–12 mL of a final antibody concentration of 2.5 mg / mL of antibody (AB1370049 lead, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117; 23–30 mg each, 153–200 nmoles) dissolved in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA, Molekula). The reduction mixture was heated at +37 °C for 3 h on an orbital shaker incubated with gentle (60 rpm) shaking. After cooling to room temperature, SG3932 was added as a DMSO solution (7 molar equivalents / antibody, 2.91-4.12 micromoles in 1.02-1.33 mL DMSO) to the reduced antibody solution (23-30 mg, 153-200 nanomoles) to a final DMSO concentration of 10% (v / v). The solution was mixed at room temperature for 17 hours, then conjugation was quenched by the addition of N-acetylcysteine ​​(5.4-7.1 micromoles, 54-71 μL at 100 mM) for >30 minutes at room temperature, sterile filtered, and then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoad™ 26 / 600 column packed with Superdex 200PG, eluted with PBS at 2.6 mL / min. Fractions corresponding to the ADC monomer peak were pooled, concentrated using a 15 ml Amicon Ultracell 30KDa MWCO spin filter, sterile filtered, and analyzed.

[0488] Result When the reduced sample of the ADC was analyzed by UHPLC-RP using a Thermo Scientific MAbPac 50 mm × 2.1 mm column on a Shimadzu Prominence system and eluting with a gradient of water and acetonitrile at 214 nm and 330 nm (specific for SG3932), a mixture of uncomplexed light chain (L0), light chain bound to a single molecule of SG3932 (L1), uncomplexed heavy chain (H0), and heavy chains bound to a maximum of three molecules of SG3932 (H1, H2, H3) was shown, which corresponded to a drug-to-antibody ratio (DAR) of 4.13 - 4.27 molecules of SG3932 per antibody as calculated in Example 10. The payload quenched with free NAC was <LOD. Representative UHPLC-RP chromatograms of AB1370049-SG3932 DAR4 are shown in Figures 7A - 7B, which demonstrated a DAR of 4.24.

[0489] When the sample of the ADC was analyzed by UHPLC-SEC using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 × 150 mm column (with a 4 μm 3.0 × 20 mm guard column) on a Shimadzu Prominence system and eluting with a sterile filtered SEC buffer containing 200 mM potassium phosphate (pH 6.95), 250 mM potassium chloride and 10% isopropanol (v / v) at 0.3 mL / min at 280 nm, it showed a monomer purity of greater than 99.5%. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR4 is shown in Figure 7C.

[0490] UHPLC-HIC analysis of the ADC samples at 214 nm using a Sepax Proteomix HIC Butyl-NP5 5 μM non-porous 4.6 × 35 mm column on a Shimadzu Prominence system eluted with a gradient of 1.5 M to 0 M ammonium sulfate and 0 to 20% acetonitrile in 25 mM sodium phosphate, pH 7.40, showed a mixture of DAR species corresponding to an average DAR of approximately 4. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR4 is shown in Figure 7D.

[0491] UV (Stunner, Unchained Labs) analysis gave final ADC concentrations of 3.59–4.06 mg / ml in 5.0–6.0 ml, with resulting ADC masses of 18.0–24.0 mg (68–79% yield).

[0492] conclusion All lead antibodies were suitable for adjusting TCEP equivalents for stochastic DAR4 conjugation, showed no DAR loss during purification, no aggregation / fragmentation during processing, and showed >99% monomer purity and high yields.

[0493] Exemplary lead ADCs were then further evaluated for their in vivo mouse PK, as well as in vivo efficacy (see Examples below).

[0494] Example 12 - In vitro serum stability / deconjugation of exemplary ADCs the purpose To determine the stability of AB1370049-SG3932DAR8 and other lead DAR8 ADCs to various sera.

[0495] Materials and Methods IgG depletion of human serum Human serum (100 ml) was passed twice through a column packed with MabSelect SuRe™ LX Protein A resin (42 ml, 1 CV) equilibrated in PBS, and the human serum was collected each time. The column was regenerated each time by washing with citrate buffer (100 mM sodium citrate, pH 3.0, 210 ml, 5 CV).

[0496] Incubation of ADC in serum ADC (1.00 mg / ml, diluted in PBS as required) was added to serum (mouse (ab7486, abcam), rat (C13SDZ, BioRad), IgG-depleted human (DHP-2001, Access Biologicals), and cynomolgus monkey (custom-ordered, BioIVT), ADC diluted 20-fold in serum). The resulting mixture (1.8 ml) was dispensed (200 μl) into 96-well plates (Sterile Nunc™). Plates were sealed with AeraSeal™ film, covered with microplate lids (Nunc™), and incubated at 37° C. in a 5% CO2 incubator. Sample aliquots were removed at various time points (days 0, 1, 3, and 7) and samples were stored at −80° C. prior to analysis.

[0497] Preparation of capture resin PureProteome™ Streptavidin Magnetic Beads (4.90 ml) were washed in PBS (5 times) and CaptureSelect™ Human IgG-Fc PK Biotin Complex (750 μl of a 1.0 mg / ml solution) was added. The mixture was further diluted with PBS (60 μl, pH 7.4) and then mixed on an end-over-end mixer (3 h, 40 rpm). Residual CaptureSelect was removed and the beads were washed in PBS (5 times), made up to the original volume with PBS, and stored at 2-8°C before further use.

[0498] Capture of ADC from serum Thawed serum samples (200 μl each) were diluted with PBS (7.2 μl per sample) and treated with PNGaseF (P0704L, NEB) (0.8 μl per sample) for 6 h. Meanwhile, capture resin (50 μl per sample) was washed successively with SN1 buffer (1×TBS (pH 7.4), 0.05% TWEEN20, 0.1% BSA, 2×300 μl per sample), Pierce™ IgG Elution Buffer (100 μl per sample), and again with SN1 buffer (3×300 μl per sample) on a KingFisher Flex magnetic bead handling system. ADC capture was performed by incubating the samples with the prepared resin aliquots (15 min). The resin was then washed with PBS (3 × 300 μl per sample), the ADC was eluted by incubating the resin in IgG elution buffer (25 min, 50 μl per sample), and the eluted ADC solution was filtered using a MultiScreenHTS HV filter plate (0.45 μm, clear, non-sterile).

[0499] Samples were reduced (17 μl per sample) for 15 min at 37° C. using a master mix of reduction mixture (1.0 M sodium phosphate (pH 6.0), 150 mM sodium borate (pH 8.4), 1.0 M DTT, and LC-MS grade water in a ratio of 15:20:6:10). Quenching was performed by adding 2% formic acid in 50% v / v acetonitrile (in water) (40 μl per sample) to obtain final prepared samples prior to LC-MS analysis.

[0500] LC-MS analysis Samples were subjected to analysis on an LC-MS system consisting of an UltiMate 3000 HPLC stack equipped with a variable wavelength detector (VWD) coupled to an Exactive Plus EMR Orbitrap mass spectrometer (Thermo Scientific). Samples (78 μl) were injected onto a MAbPac RP column (2.1 × 50 mm, 4 μm, Thermo Scientific, 088648) and separated with a segmented gradient (acetonitrile (in water) with 0.03% trifluoroacetic acid, 5–25% for 30 s, then 25–60% for 2 min). Analytes were detected at 280 nm on the VWD and in EMR Full MS mode on the mass spectrometer (MS). MS tune file parameters: sheath gas flow rate: 35, auxiliary gas flow rate: 10, sweep gas flow rate: 0, spray voltage: 3.5 kV, capillary temperature: 300 °C, S-lens RF level: 200, auxiliary gas heater temperature: 300. MS method parameters: polarity: positive, CID in source: 20.0 eV, microscans: 10, resolution 17500, AGC target: 3e6, max IT: auto, scan range: 1000-4000 m / z.

[0501] MS Data Analysis Mass spectra were deconvoluted using Thermo BioPharma Finder. Peaks in the deconvoluted spectra that were deemed chemically significant had their height percentage values ​​extracted, allowing the determination of the DAR and percentage of significant chemical modification for each sample. Comparing the DAR values ​​of a given ADC in a given serum over time allowed the determination of the deconjugation rate for each sample.

[0502] result All ADCs showed low payload deconjugation in all sera (Figure 8A). This is partially supported by all ADCs showing significant levels of maleimide hydrolysis, at least in mouse, rat and cynomolgus monkey sera (Figure 8B). Maleimide hydrolysis chemically stabilizes conjugated payloads and prevents them from being deconjugated from the ADC's antibodies. However, in IgG-depleted human serum, the incidence of maleimide hydrolysis was low. Thus, the reduced protective effect conferred by maleimide hydrolysis indicates the inherent stability of these ADCs against deconjugation.

[0503] conclusion All ADCs showed good overall stability against all sera.

[0504] Example 13 - AB1370049-SG3932 DAR8 in vivo stability / deconjugation the purpose To evaluate AB1370049-SG3932 DAR8 antibody integrity and warhead deconjugation under in vivo conditions over the entire cynomolgus monkey DRF study time frame.

[0505] Materials and Methods Male cynomolgus monkeys (N=3) were administered a single dose of AB1370049-SG3932 DAR8 intravenously twice (3 weeks apart, i.e., on days 1 and 22).

[0506] Blood samples were collected by venipuncture and centrifuged to obtain plasma. Total antibody and ADC assays were performed by LBA-LCMS method. AB1370049-SG3932 DAR8 was immunocaptured by biotinylated anti-human antibody conjugated to magnetic beads. This was further subjected to multiple washing steps. Trypsin digestion was then performed and one aliquot of the supernatant was injected into the LCMS for total antibody assay. Another aliquot was subjected to further enzymatic cleavage with papain and the solution was used for ADC assay. The injected samples were separated using reversed phase chromatography (RPLC) and then detected by multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard was used to calculate against a standard curve made by spiking the ADC reference material into the desired matrix.

[0507] For the total antibody assay, two signature tryptic peptides on the AB1370049-SG3932 DAR8 antibody were used to calculate the concentration of total antibody in the selected matrix (heavy chain (VVSVLTVLHQDWLNGK) and light chain (DSTYSLSSTLTLSK)). For the ADC assay, a papain releasing warhead was used to calculate the concentration. Isotopically labeled peptides or warheads were used as internal standards.

[0508] result The ADC to total antibody ratio was approximately 1, indicating that there was no significant deconjugation of the warhead in vivo.

[0509] conclusion Under in vivo conditions, the AB1370049-SG3932DAR8 ADC was stable and no significant degradation was observed.

[0510] Example 14 - Pharmacokinetics of exemplary DAR8 ADCs in SCID mice the purpose To examine the pharmacokinetics of six exemplary anti-FRα DAR8 ADCs in wild-type SCID mice following a single intravenous dose.

[0511] Materials and Methods Wild-type SCID mice (n=3 per group) were intravenously (bolus) dosed with exemplary anti-FRα ADC (5 mg / kg). Blood samples were taken 15 minutes, 4 hours, 1, 2, 3, 6, 10, 14 and 21 days after dosing, centrifuged to obtain plasma, and total ADC was analyzed. Pharmacokinetic parameters were determined using Phoenix64 software (Certara).

[0512] Total ADC concentrations were measured using an immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μl of plasma samples were then diluted in TBS and incubated with magnetic beads. After capture, the magnetic beads were washed multiple times and then digested with trypsin. An aliquot of the trypsin-digested supernatant was subjected to enzymatic digestion with papain overnight in the presence of an internal standard. The digested samples were quenched with the addition of acid and then analyzed by LC-MS / MS.

[0513] The concentration of ADC was calculated using the papain-released warhead. The internal standard used in this experiment is an isotope-labeled warhead. Papain digests from immunoaffinity-enriched samples were separated using reversed-phase chromatography (RPLC), and the released warheads were subsequently detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard was used to calculate against a standard curve made by spiking the ADC reference material into the desired matrix.

[0514] Standard curves and QCs were prepared by spiking the target compound (ADC reference material) at different levels into the same matrix as the sample matrix. For rodent studies (FcRn and Tg32), the quantification range covers 100ng / ml to 12,000ng / ml, and the diluted QCs cover dilutions up to 50-fold. The standard curves were fitted with linear regression with a weighting of 1 / x2.

[0515] result Pharmacokinetic parameters of exemplary anti-FRα DAR8 ADCs in SCID mice are shown in Table 21.

[0516] [Table 38]

[0517] conclusion The exemplary anti-FRα ADCs exhibited different pharmacokinetic properties in wild-type SCID mice. SCID mouse pharmacokinetics of all exemplary antibodies showed low plasma clearance and long plasma half-life in SCID mice.

[0518] Example 15 - Pharmacokinetics of DAR4 and DAR8 ADCs formed from AB1370049 and SG3932 in hFcRn Tg32 mice the purpose To investigate the pharmacokinetics of DAR4 and DAR8 ADCs formed from AB1370049 and SG3932 in hFcRn Tg32 mice after a single intravenous administration.

[0519] Materials and Methods Human FcRn Tg32 mice (n=3 per group) were intravenously (bolus) dosed with DAR4 or DAR8 ADC (5 mg / kg) formed from AB1370049 and SG3932. Blood samples were taken 15 minutes, 4 hours, 1, 2, 3, 6, 10, 14, and 21 days after dosing, centrifuged to obtain plasma, and total ADC was analyzed. Pharmacokinetic parameters were determined using Phoenix64 software (Certara).

[0520] The concentration of AB1370049-SG3932 DAR4 or AB1370049-SG3932 DAR8 was measured by immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μl of plasma samples were then diluted in TBS and incubated with magnetic beads. After capture, the magnetic beads were washed multiple times and then digested with trypsin. An aliquot of the trypsin-digested supernatant was subjected to enzymatic digestion with papain overnight in the presence of an internal standard. The digested samples were quenched with the addition of acid and then analyzed by LC-MS / MS.

[0521] The concentration of DAR4 or DAR8 ADC was calculated using the papain-released warhead. The internal standard used in this experiment is an isotope-labeled warhead. Papain digests from immunoaffinity-enriched samples were separated using reversed-phase chromatography (RPLC), and the released warheads were subsequently detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard was used to calculate against a standard curve generated by spiking the DAR4 or DAR8 ADC reference material into the desired matrix.

[0522] Standard curves and QCs are prepared by spiking DAR4 or DAR8 ADC (ADC reference material) at different levels into the same matrix as the sample matrix. The quantification range covers 100ng / ml to 12,000ng / ml, and the diluted QCs cover dilutions up to 50-fold. The standard curves were fitted with linear regression with a weighting of 1 / x2.

[0523] result The pharmacokinetic parameters of DAR4 and DAR8 ADC in hFcRn Tg32 mice are shown in Table 22.

[0524] [Table 39]

[0525] conclusion AB1370049-SG3932 DAR8 showed slightly higher clearance and shorter half-life in hFcRn Tg32 than in wild-type SCID mice. hFcRn Tg32 mice are transgenic mouse models, with mouse FcRn knocked out and human FcRn knocked in. Therefore, it represents a suitable model for predicting the human pharmacokinetics of AB1370049-SG3932 DAR8. Based on the hFcRn Tg32 mouse pharmacokinetics, AB1370049-SG3932 DAR8 will likely have a low human clearance and a half-life in common with other clinically used ADCs, allowing for convenient dosing. This data is consistent with the pharmacokinetic data obtained with the exemplary mAb of the present invention. DAR4 ADC shows a slightly longer half-life in hFcRn Tg32 mice, driven by a slightly lower plasma clearance.

[0526] Example 16 - In vitro cell killing activity of exemplary DAR8 ADCs the purpose To evaluate the cytotoxic activity of exemplary anti-FRα DAR8 ADCs using cancer cell lines expressing various levels of FRα.

[0527] Materials and Methods Cancer cell lines with various levels of FRα expression were obtained from ATCC (American Tissue Culture collection). KB cells are a cervical cancer cell line with HeLa contamination, while IGROV-1 is an ovarian cancer cell line and JEG-3 is a choriocarcinoma cell line. Cells were plated in duplicate in 96-well plates. After 24 h of incubation, cells were treated with lead ADC or non-targeting ADC control NIP228 ranging from 0 to 66.66 nM (0 to 10 μg / ml) for 6 days. Then, on day 6, cells were incubated with CellTiter-Glo reagent for 10 min and luminescence was measured using a 96-well plate reader. Background luminescence was measured in medium without cells and subtracted from experimental values. IC50 values ​​were calculated with Graph Pad Prism.

[0528] result The exemplary ADCs were tested in various cancer cell lines with different levels of FRα expression (KB (high), IGROV-1 (high to medium), and JEG-3 (medium)). As shown in Figures 9A-9C, all exemplary ADCs demonstrated cytotoxic activity in the three FRα positive cell lines tested. The ADC lead candidates had stronger potency in the high FRα expressing cell lines KB (IC50 = 0.13-0.23 μg / ml) and medium JEG-3 (IC50 = 0.01-0.14 μg / ml).

[0529] conclusion All ADCs showed high potency in these in vitro cell killing assays, indicating their potential efficacy in treating cancers that overexpress FRα. In particular, AB1370049-SG3932 DAR8 had the best cytotoxic activity across the three FRα-expressing cell lines.

[0530] Example 17 - Bystander killing of AB1370049-SG3932 DAR8 the purpose To evaluate the bystander activity of AB1370049-SG3932 DAR8 in co-culture experiments with FRα positive and negative KB cells.

[0531] Materials and Methods To evaluate the bystander activity of AB1370049-SG3932 DAR8, a mixture of antigen-positive KB cells (KB WT) and antigen-negative KB FRα knockout cells with GFP tag (KB FRα k / o GFP) cells was incubated in the presence of AB1370049-SG3932DAR8. After 6 days of culture, the remaining cell population was harvested using trypsin and analyzed by flow cytometry. Antigen-positive and antigen-negative live cells were identified, counted, and compared to untreated samples.

[0532] result AB1370049-SG3932 DAR8 exhibited efficient bystander killing activity when mixed in a 1:1 coculture experiment of FRα-expressing and FRα-negative KB cells (FIG. 10).

[0533] conclusion AB1370049-SG3932 DAR8 can induce apoptosis and ultimately cell death in tumors that express FRα homogeneously as well as in tumors that express it heterogeneously, as shown in co-culture experiments where 50% of cells express FRα and 50% do not. The ADC is taken up into FRα-positive cells, and the liberated warhead is released to kill the target cancer cells. The liberated warhead can also spread to neighboring cancer cells that do not express FRα and kill them as well.

[0534] Example 18 - In vivo anti-cancer activity of exemplary ADCs in cell-derived xenograft models the purpose To evaluate the anti-tumor activity of exemplary ADCs, particularly AB1370049-SG3932 DAR8, in various CDX models of human cancers exhibiting different target expression levels.

[0535] Materials and Methods In the KB xenograft model, female CB17-SCID mice (Charles River Laboratories) were inoculated with 6 × 10 6 Cells / mouse were inoculated subcutaneously. Tumors were approximately 150–200 mm 3 Once the mice reached 100 mg / mL, mice were randomly assigned to groups where each of the six lead DAR8 ADCs was administered intravenously at a single dose (day 7) of 0.3125 mg / kg, 0.625 mg / kg, 1.25 mg / kg, 2.5 mg / kg, 5 mg / kg, and 10 mg / kg, and the DAR4 ADCs at 0.625 mg / kg, 1.25 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg, along with the isotype control NIP228 at the corresponding dose levels.

[0536] For the Caco-2 xenograft model, 5 × 106 Cells / mouse were inoculated subcutaneously into female athymic nude mice (Harlon Laboratories). Tumors were approximately 150 mm 3 Once the 17 days had been reached, mice were randomly assigned to groups: AB1370049-SG3932 DAR8 was administered intravenously as a single dose (day 17) at 1.25 mg / kg, 2.5 mg / kg, 5 mg / kg, corresponding isotype control NIP228 at 1.25 mg / kg, 5 mg / kg, and FRα-DM4 ADC ("biosimilar to mirvetuximab soravtansine", DAR approx. 3) at 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg, and corresponding isotype control NIP228 at 1.25 mg / kg and 5 mg / kg.

[0537] For the IGROV-1 xenograft model, 1 × 10 7 Cells / mouse were inoculated subcutaneously into female severe combined immunodeficient (SCID) mice (Envigo). Tumors grew to approximately 285 mm 3 Upon reaching tumor maturity (day 32), mice were randomly assigned into groups based on tumor volume. Control animals received 100 μl vehicle intravenously, whereas treated animals received AB1370049-SG3932 DAR8 at 1.25 mg / kg, 2.5 mg / kg, 5 mg / kg, 4 ml / kg, corresponding isotype control NIP228 at 1.25 mg / kg, 2.5 mg / kg, 5 mg / kg, FRα-DM4 ADC ("biosimilar of mirvetuximab soravtansine", DAR approximately 3) at 5 mg / kg, and corresponding isotype control NIP228 at 5 mg / kg intravenously.

[0538] In the OVCAR3 xenograft model, AB1370049-SG3932 DAR8 was administered as a single dose at 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg, as well as FRα-DM4 ADC ("biosimilar to mirvetuximab soravtansine," DAR approximately 3) at 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg.

[0539] Tumor volumes were measured twice weekly with calipers.

[0540] result Complete tumor regression was observed at 5 mg / kg (KB and OVCAR-3) and 1.25 mg / kg (OVCAR-3 only) for all DAR8 ADCs tested (Figures 11-12). In IGROV-1 xenografts, which show high-to-moderate FRα expression, AB1370049-SG3932 DAR8 was active at 5 mg / kg and showed partial tumor regression over 70 days before tumors began to regrow (Figure 13). Lead ADC AB1370049-SG3932 DAR8 was selected because it showed more durable responses compared to other lead candidates, especially in the KB and OVCAR-3 xenograft models.

[0541] The lead DAR4 ADC was also tested in the KB model (Figure 14). All ADCs tested achieved complete tumor regression at 5 mg / kg, indicating that the DAR4 ADC also has good efficacy.

[0542] AB1370049-SG3932 DAR8 was also tested against FRα-DM4 ADC (a biosimilar of mirvetuximab soravtansine, DAR approximately 3) in xenograft models OVCAR-3 and CaCo-2 with medium to medium-low FRα expression (Figures 15A-15B). AB1370049-SG3932 DAR8 was more effective at lower dose concentrations (1.25 and 2.5 mg / kg) and had more sustained antitumor activity than FRα-DM4 in these models.

[0543] conclusion All of the exemplary lead ADCs demonstrated robust antitumor activity at different doses in a variety of different xenograft models, which was comparable or better than the comparator molecule, FRα-DM4.

[0544] Example 19 - In vivo anti-cancer activity of AB1370049-SG3932 DAR8 in Champions and in-house patient-derived xenograft models the purpose To evaluate the antitumor activity of AB1370049-SG3932DAR8 in Low Passage Champions PDX models and in-house PDX models of human non-small cell lung, ovarian, colorectal and endometrial cancer representing various target expression levels.

[0545] Materials and Methods Champions Model Thirty PDX models (19 NSCLC models, 9 ovarian cancer models, and 2 endometrial cancer models) were selected for investigation. Tumor tissue fragments were subcutaneously implanted into 6-8 week-old female athymic nude-Foxn1nu stock mice. A sufficient number of stock animals were selected to be 1000-1500 mm 3 Once tumors reached the mean volume of 150–300 mm, tumors were harvested and fragments were transplanted into pre-test animals. 3 Once tumor volume reached 100%, animals were randomized into 5 groups according to tumor volume. Group 1 received no treatment, while groups 2 and 4 received AB1370049-SG3932 DAR8 at 5 mg / kg or 2.5 mg / kg, respectively. Groups 3 and 5 received the isotype control antibody NIP228-SG3932 at 5 mg / kg or 2.5 mg / kg, respectively. All treated animals received one dose intravenously on day 0.

[0546] Animals were observed daily, and tumor dimensions and body weights were measured and recorded twice weekly. Tumor volumes were measured with digital calipers and calculated using the following formula: Tumor volume = length (mm) x width (mm). 2 × 0.52, where length and width are the longest and shortest diameters of the tumor, respectively.

[0547] The study endpoint for all models was a mean tumor volume of 1200mm in the control group. 3 The study was performed when the maximum tumor volume was reached, or up to 60 days if the maximum tumor volume was not reached. 3 If tumor volumes increased before day 28, treatment groups were monitored until day 28. After randomization of animals to the study, tumor samples were collected from three animals per model. Tumors were grown to approximately 400-600 mm 3and half of the tumors were processed for immunohistochemical analysis and the other half for genomic analysis.

[0548] In-house model We performed in-house in vivo patient-derived xenograft (PDX) efficacy studies using various tumor models including ovarian, colon, endometrial, and non-small cell lung cancer. Tumor fragments from these PDX models were implanted subcutaneously using a trocar into female NOD-SCID mice (Envigo). Tumors were approximately 150–200 mm 3 Once the tumor size reached 100 mg / kg, mice were randomly assigned to groups. AB1370049-SG3932 DAR8 was administered intravenously as a single dose at 2.5 mg / kg, 5 mg / kg, and with the corresponding dose level of isotype control NIP228. Tumor volumes were measured twice weekly with a caliper. Tumor growth was calculated using the formula 1 / 2×L×W2 (L=length; W=width). Body weight was measured twice weekly to assess the tolerability of the treatment.

[0549] Measurement of antitumor responses The response of final tumor volume (FTV) from the initial tumor volume (ITV) was calculated at the time point that resulted in the maximum reduction from the initial tumor volume. The anti-tumor response was calculated for each tumor-bearing mouse using the following formula: Tumor proliferation rate (%) = [(FTV-ITV) / ITV] × 100 The percent tumor growth was calculated for each animal in the test group and the group response was calculated as the median response of all treated mice.

[0550] For each treatment group, a response or lack of response to the study drug was determined. Response criteria were based on RECIST 1.1, with response based on a 30% reduction in tumor volume from baseline tumor measurement.

[0551] result Table 23 and Figure 16A summarize the median tumor growth rates resulting from a single dose of 5.0 mg / kg AB1370049-SG3932 DAR8 or NIP228-SG3932 in 51 PDX models. Tumor growth inhibition was observed after a single dose of 5.0 mg / kg AB1370049-SG3932 DAR8, with 54% (29 of 54) of models showing a 30% or greater reduction in tumor volume from baseline. A single dose of 5.0 mg / kg NIP228-SG3932 reduced tumor volume from baseline by 30% or greater in 22% (11 of 49) of models tested. In the ovarian cancer PDX models tested, AB1370049-SG3932 DAR8 at 5.0 mg / kg reduced tumor volume by 30% or more from baseline in 78.3% (18 of 23: 95% CI 58% to 91%) of models tested. NIP228-SG3932 at 5.0 mg / kg reduced tumor volume by 30% or more from baseline in 43.5% (10 of 23: 95% CI 26% to 63%) of models tested.

[0552] [Table 40]

[0553] [Table 41]

[0554] Table 24 and Figure 16B summarize the median tumor growth rates resulting from a single dose of 2.5 mg / kg AB1370049-SG3932 DAR8 or NIP228-SG3932 in 39 PDX models. Specific tumor growth inhibition was observed in 49% (19 out of 39) of the PDX models tested after a single intravenous dose of 2.5 mg / kg AB1370049-SG3932 DAR8, while 51% (20 out of 39) of the models did not respond. Only 2 out of 37 models tested responded to the same dose of non-targeted ADC NIP228-SG3932 DAR8. The activity of non-targeted ADC is dose-dependent, as fewer models responded to NIP228-SG3932 DAR8 (5%) at lower dose concentrations.

[0555] [Table 42]

[0556] [Table 43]

[0557] AB1370049-SG3932DAR8 demonstrated antitumor responses in multiple models across four indications: ovarian cancer, NSCLC, CRC and endometrial cancer. Figures 17A-17C show representative studies from models CTG-0711 (ovarian cancer), CTG-2367 (NSCLC) and CTG-2268 (endometrial).

[0558] The objective response rate in ovarian cancer was 80%, NSCLC 30%, CRC 20%, and endometrium 50%. There was a positive correlation between FRα expression and the activity of AB1370049-SG3932 DAR8.

[0559] conclusion AB1370049-SG3932 DAR8 is highly effective in FRα expressing tumors with high, medium and medium-low expression levels. In general, there is a positive correlation between the antitumor activity of AB1370049-SG3932 DAR8 and FRα expression level.

[0560] Example 20 - In Vitro Safety Studies with Exemplary ADCs the purpose The in vitro safety of AB1370049-SG3932 DAR8 was evaluated using primary hematopoietic stem and progenitor cells (HSPCs) that differentiate into erythroid, myeloid and megakaryocytic cells.

[0561] Materials and Methods Cryopreserved human bone marrow CD34 + Progenitor cells (Lonza) were thawed and allowed to recover overnight in maintenance medium (StemSpan SFEM II (Stem Cell Technologies) containing 25ng / ml SCF, 50ng / ml TPO and 50ng / ml Flt3-L human recombinant protein (all Peprotech)) in a humidified incubator at 37°C with 5% CO2. The next day, cells were resuspended in cell growth medium appropriate for erythroid (Preferred Cell Systems, SEC-BFU1-40H), myeloid (Preferred Cell Systems, SEC-GM1-40H) or megakaryocytic (Stem Cell Technologies, 09707) differentiation in the presence of drugs at a concentration of 5000 cells / ml for erythroid and myeloid cells or 15000 cells / ml for megakaryocytic cells.

[0562] Cells (100 μl) were plated in triplicate into white-walled, clear-bottom 96-well tissue culture plates (Corning) supplemented with an exemplary ADC (e.g., AB1370049-SG3932 DAR8) or NIP228 isotype control ADC (200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03 and 0 μg / ml) and cultured for 5 days in a humidified incubator at 37° C. with 5% CO2.

[0563] Additionally, the effect of exemplary ADCs on proliferating and differentiated cells was evaluated. To do so, cells were plated at a concentration of 5000 cells / ml for 5 days of erythroid differentiation, followed by plating in triplicate white-walled, clear-bottom 96-well tissue culture plates supplemented with the following concentrations (200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03, and 0 μg / ml) for 5 days for erythroid differentiation. For myeloid or megakaryocytic differentiation, cells were seeded at 10000 and 15000 cells / ml, respectively, for 5 days, then sedimented and seeded in fresh medium at 10000 and 30000 cells / ml, respectively, for another 5 days, and then plated in myeloid differentiation medium or megakaryocytic at 20000 and 30000 cells / ml for 4 days in the presence of AB1370049-SG3932 DAR8 (200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03 and 0 μg / ml).

[0564] Viability was determined using CellTiter-Glo 2.0 from Promega (using an optimized volume of 10 μl / well) and luminescence was detected using an Envision plate reader (Perkin Elmer). Relative luminescence signals were normalized to the percentage of control (control equals 100, maximum cell death equals 0) in GraphPad software (Prism).

[0565] result 2. Primary Human CD34 Cells Using a 2D In Vitro Culture System +Toxicity was measured in hematopoietic stem and progenitor cells (HSPCs). HSPCs proliferate and differentiate along erythroid, megakaryocytic and myeloid (granulocytic / monocytic) lineages in the presence of the compounds. Inhibitory effects were determined using ATP quantification as a surrogate for cell viability. This method was used to evaluate the toxicity induced by AB1370049-SG3932 DAR8 and its related non-FRα-targeted ADC control to assess any exacerbated toxicity of AB1370049-SG3932 DAR8.

[0566] AB1370049-SG3932 DAR8 shows similar toxicity levels to non-FRα-targeted ADC molecules with the same payload (Figures 18A-18F). This observation was consistent with the results of primary CD34 cells differentiated into any lineage, regardless of the level of differentiation. + In bone marrow cells. Similar results were observed with other exemplary ADCs of the present invention, including mAbs AB1370095, AB1370026 and AB1370117, when conjugated to the same TOPOi payload. When administered under similar culture conditions as described for AB1370049-SG3932 DAR8, unconjugated mAb (AB1370049) did not induce a significant decrease in ATP levels in HSPCs. Furthermore, HSPCs treated with AB1370049 conjugated to the same TOPOi payload at DAR4 showed a similar level of toxicity as HSPCs treated with non-FRα-targeted ADC DAR4.

[0567] conclusion The results suggest that there is no target-mediated toxicity or exacerbation of toxicity driven by the lead antibody compared to non-FRα-targeted ADC molecules.

[0568] Example 21 - In vivo pharmacokinetic study with AB1370049 and AB1370049-SG3932 DAR8 the purpose Plasma pharmacokinetic (PK) analysis of AB1370049-SG3932DAR8 in cynomolgus monkeys was performed, including peak and total exposure, clearance, and half-life. PK samples were collected from cynomolgus monkeys across various dose levels of the lead ADC candidate and unconjugated antibody. Non-compartmental analysis was performed to estimate PK parameters.

[0569] Materials and Methods Administration of AB1370049 and AB1370049-SG3932 DAR8 to cynomolgus monkeys Male cynomolgus monkeys (N=3) were intravenously administered two single doses (3 weeks apart, i.e., on days 1 and 22) of AB1370049 at 15 mg / kg or AB1370049-SG3932 DAR8 at 15 mg / kg and 25 mg / kg.

[0570] Blood samples were collected by venipuncture pre-dose, 0.04, 0.25, 1, 3, 7, 14 and 21 days post-dose on day 1, and on day 22, centrifuged to obtain plasma, and analyzed for total antibody, total ADC, and unconjugated payload. Pharmacokinetic parameters were determined using Phoenix64 software (Certara).

[0571] Total antibody and ADC assays Total ADC and total antibody concentrations were measured using an immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 40 μl of plasma samples were then diluted in TBS and incubated with magnetic beads. After capture, the magnetic beads were washed multiple times and then digested with trypsin in the presence of an internal standard. Acid was added to quench the digestion. An aliquot of the trypsin digested liquid contents was then transferred to an injection plate for total Ab analysis.

[0572] For the total ADC assay, an aliquot of the trypsin digested supernatant was subjected to enzymatic digestion with papain overnight in the presence of an internal standard. The digested samples were analyzed by LC-MS / MS after quenching with the addition of acid. The papain released warheads were used to calculate the concentrations. The internal standard used in this experiment is an isotope-labeled warhead. Papain digests from immunoaffinity enriched samples were separated using reversed-phase chromatography (RPLC) and the released warheads were subsequently detected using multiple reaction monitoring (MRM). The peak area ratios of the analytes relative to the internal standard were used to calculate against a standard curve created by spiking the ADC reference material into the desired matrix.

[0573] For the total Ab assay, a signature tryptic peptide (VVSVLTVLHQDWLNGK) on the human antibody Fc region was used to calculate the concentration of total antibody in the selected matrix. Tryptic digests from immunoaffinity enriched samples were separated using reversed-phase chromatography (RPLC) and the signature peptide was subsequently detected using multiple reaction monitoring (MRM). The internal standard used in this experiment is an isotope-labeled peptide. The peak area ratio of the analyte to the internal standard was used to calculate against a standard curve created by spiking the ADC reference material into the desired matrix.

[0574] Standard curves and QCs were prepared by spiking the target compound (ADC reference material) at different levels into the same matrix as the sample matrix. For NHP studies, the quantification range is 100-15000ng / ml with dilution factors up to 100x. The standard curves were fitted to linear regression with a weighting of 1 / x2, and the accuracy and precision of the assay are within 20% at all levels except for the LLOQ (25%).

[0575] Non-complexed warhead assay The uncomplexed warhead assay was performed by a precipitation procedure. The internal standard used in this experiment is an isotope-labeled warhead. Samples were precipitated with a buffer containing a high percentage of organic solvent spiked into the internal standard. The supernatant was then dried under nitrogen before being reconstituted with the appropriate buffer for injection into the LCMS. The samples were then separated using reversed-phase chromatography (RPLC), followed by detection of the uncomplexed warhead using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard was used to calculate against a standard curve created by spiking the ADC reference material into the desired matrix.

[0576] Standard curves and QCs were prepared by spiking uncomplexed warheads at different levels into the same matrix as the sample matrix. In NHP studies, the quantification range of the assay is 0.059-29.5 ng / ml. The standard curves were fitted with linear regression. The accuracy and precision of the assay are within 15% at all levels, except for the LLOQ (20%).

[0577] result Mean PK parameters based on non-compartmental analysis (NCA) are summarized in Table 25. The unconjugated mAb (AB1370049) demonstrated linear PK at 15 mg / kg in cynomolgus monkeys with half-life and clearance consistent with the literature for human IgG in monkeys. The ADC (AB1370049-SG3932 DAR8) demonstrated linear PK at 15 mg / kg and 25 mg / kg in cynomolgus monkeys with dose-proportional exposure (C max and AUC), equivalent CL and t 1 / 2 was observed. The exposure of the unconjugated antibody is higher compared to the dose-matched ADC, as would be expected for the addition of eight cytotoxic warheads, with slower clearance and a longer half-life than the ADC. However, the PK of the ADC is well within the ADC acceptance criteria in cynomolgus monkeys.

[0578] [Table 44]

[0579] No significant accumulation of ADC was seen between the first and second doses, nor was there a drop in exposure after the second dose, indicating that no significant neutralizing anti-drug antibodies were formed (Figure 19). The levels of total antibody and total ADC measured in the AB1370049-SG3932 DAR8 (25 mg / kg) samples were comparable to those of the unconjugated mAb (15 mg / kg) samples from day 0 to day 42. The levels of total antibody and total ADC measured in the AB1370049-SG3932 DAR8 (15 mg / kg) samples were relatively low. In summary, total antibody and total ADC were similar within the groups, and furthermore, minimal free warheads were seen over time - all showing limited deconjugation and high in vivo stability.

[0580] conclusion The DAR8 ADC (AB1370049-SG3932 DAR8) was advanced into a dose-ranging study in monkeys and showed dose-dependent exposure with a consistently long half-life and slow clearance as previously shown in mouse PK studies.

[0581] In particular, AB1370049-SG3932 DAR8 exhibited a longer half-life and slower clearance than the most advanced FRα ADC in the art (Olga, et al. Cancer Res August 15 2020(80)(16Supplement)2890; DOI:10.1158 / 1538-7445.AM2020-2890; WO 2020 / 223221A1). The toxicity profile of the ADCs described in this invention may result in more tolerable treatments and potential clinical advantages.

[0582] Example 22 - In vivo anti-cancer activity of AB1370049-SG3932 DAR8 in patient-derived xenograft models, OVO857_CIS and CTG3226 the purpose To evaluate the antitumor activity of AB1370049-SG3932 DAR8 compared to anti-FRα-DM4 ADC in ovarian PDX models with intermediate and intermediate-low FRα expression.

[0583] Materials and Methods An in-house in vivo patient-derived xenograft (PDX) efficacy study was conducted using two different ovarian PDX models, OVO857_CIS ("OVO0875") and CTG3226. Six to eight week old female NSG (NOD-SCID IL2Rgamma) mice served as seed mice. null Tumor tissue fragments were subcutaneously implanted in the oocytes of the mice. 3 When the tumor volume reached approximately 150-250 mm, the tumor was harvested and fragments were transplanted into the test animals. 3 Once the 100% immunization rate was reached, mice were randomly assigned to different groups (n=3 mice per group). One group received no treatment. Four groups received AB1370049-SG3932 DAR8 at 10, 5, 2.5 and 1.25 mg / kg intravenously, respectively. Two groups received FRα-DM4 ADC ("biosimilar of mirvetuximab soravtansine", DAR approx. 3) at 5 and 2.5 mg / kg intravenously, respectively. All treated animals received the drug intravenously as a single dose of 10 ml / kg on day 39 for OVO857_CIS and day 46 for CTG3226 PDX models.

[0584] Tumor volumes were measured with digital calipers and calculated using the following formula: tumor volume = length (mm) × width (mm). 2 ×0.52 (where length and width are the longest and shortest diameters of the tumor, respectively). Body weights were measured twice weekly to assess the tolerability of the treatment.

[0585] Tumor growth in each experimental group was evaluated based on the mean tumor volume (mm ) of the number of animals used. 3 ) ± SEM.

[0586] Tumor growth inhibition (TGI) was calculated by comparing each treatment group to an untreated control group. Percentage inhibition was calculated from group means using the following formula: TGI(%)=(CT) / C×100. where C is the mean tumor volume in the untreated control group and T is the mean tumor volume in the treated group.

[0587] Immunohistochemical staining of tumor samples for FRα expression Anti-rabbit FRα monoclonal antibody (clone [EPR20277]) used for immunohistochemistry (IHC) was obtained from Abcam (catalog number ab221543) and diluted in antibody diluent with background reducing agent (Agilent, catalog number S3022). IHC was performed on a Ventana Discovery Ultra instrument (Roche Diagnostics, Indianapolis, IN, USA). Sections of formalin-fixed paraffin-embedded (FFPE) tissues were cut at 4 μm, placed on StarFrost® microscope slides, and loaded into an automated stainer. Slides were antigen retrieved with Ultra Cell Conditioning 1 (Roche Diagnostics, catalog number 05424569001) and blocked with inhibitor CM (Roche Diagnostics, catalog number 05266645001). Sections were then incubated with primary antibodies, HRP was blocked with DISCOVERY inhibitor (Roche, Cat. No. 07017944001) and samples were incubated with anti-rabbit IgG HRP secondary antibody (Roche Diagnostics, Cat. No. 05269717001). Staining was visualized with brown 3,3'diaminobenzidine (DAB) using the DISCOVERY ChromoMap DAB kit (Roche Diagnostics, Cat. No. 05266645001). Sections were counterstained with hematoxylin II (Roche Diagnostics, Cat. No. 05277965001) and a bluing reagent (Roche Diagnostics, Cat. No. 05266769001). Finally, sections were dehydrated in graded ethanol, cleared in xylene and coverslipped.

[0588] FRα staining was assessed by a pathologist, quantified, and classified into the following ranges: absent (0+), low (1+), moderate (2+), or high (3+).

[0589] Digital quantification of FRα expression in tumor samples To confirm proper identification of analyzable tumor areas (ATA) containing at least 80% of the invasive tumor present in the tissue samples, digital images from the FRα IHC assay were analyzed by a pathologist, computer quantified, and verified. Cell membrane optical density (OD) values ​​were calculated, including overall mean and median (i.e., 50th quantile) OD values. Membrane OD values ​​range from 0 to 255. For each model, target expression is expressed as the median membrane OD of three tumors collected from independent untreated test mice.

[0590] result OVO875 tumors were classified as intermediate FRα expressers based on an IHC score of 2+ and a median OD of 41.

[0591] CTG3226 tumors were classified as low-to-medium FRα expressers based on IHC scores of 1+ to 2+ and a median OD of 28.

[0592] Two ovarian cancer PDX models, OVO875 (medium FRα expression) and CTG3226 (low-medium FRα expression), were administered a single iv do...

Claims

1. (a) a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, a light chain CDR2 (KASGLES) of SEQ ID NO: 5, and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6; (b) a heavy chain CDR1 (SYAMS) of SEQ ID NO: 7, a heavy chain CDR2 (SISSGRSYIYYADSVKG) of SEQ ID NO: 8, a heavy chain CDR3 (EMQQLALDY) of SEQ ID NO: 9, a light chain CDR1 (RASQGISNFLA) of SEQ ID NO: 10, a light chain CDR2 (AASSLQS) of SEQ ID NO: 11, and a light chain CDR3 (QQYNSYPFT) of SEQ ID NO: 12; (c) a heavy chain CDR1 (SNSAAWN) of SEQ ID NO: 13, a heavy chain CDR2 (RTYYRSNWYNDYTLSVKS) of SEQ ID NO: 14, a heavy chain CDR3 (GVGRFDS) of SEQ ID NO: 15, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 16, a light chain CDR2 (KASSLES) of SEQ ID NO: 17, and a light chain CDR3 (QEYKTYSIFT) of SEQ ID NO: 18; (d) a heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, a heavy chain CDR2 (SISSGSSYIYYADSMKG) of SEQ ID NO: 20, a heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 21, a light chain CDR1 (RASQGISTFLA) of SEQ ID NO: 22, a light chain CDR2 (AASSLQS) of SEQ ID NO: 23, and a light chain CDR3 (QQYISYPLT) of SEQ ID NO: 24; (e) a heavy chain CDR1 of SEQ ID NO: 25 (SYSMN), a heavy chain CDR2 of SEQ ID NO: 26 (SISSRSSYVYYADSVKG), a heavy chain CDR3 of SEQ ID NO: 27 (GMTTLTFDY), a light chain CDR1 of SEQ ID NO: 28 (RASQGISSFLA), a light chain CDR2 of SEQ ID NO: 29 (AASSLQS), and a light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT); or (f) a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 31, a heavy chain CDR2 (RTYYRSKWYSDYAVSVKS) of SEQ ID NO: 32, a heavy chain CDR3 (GGAPFDY) of SEQ ID NO: 33, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 34, a light chain CDR2 (KASSLES) of SEQ ID NO: 35, and a light chain CDR3 (QQYNSYSMYT) of SEQ ID NO:

36. An anti-FRα antibody or an antigen-binding fragment thereof comprising:

2. (a) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 37, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38; (b) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 39, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 40; (c) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 41, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 42; (d) a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 43, and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 44; (e) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 45, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 46; or (f) a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47, and a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48; The anti-FRα antibody or antigen-binding fragment thereof according to claim 1 , comprising:

3. (a) an L at the N-terminus (e.g., position 1) of the VH; (b) an E at the N-terminus (e.g., position 1) of the VH; or (c) adding L to the N-terminus (e.g., position 1) of the VH The anti-FRα antibody or antigen-binding fragment thereof according to claim 2, comprising:

4. (a) a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (b) VH of SEQ ID NO: 39 and VL of SEQ ID NO: 40 (c) a VH of SEQ ID NO: 41 and a VL of SEQ ID NO: 42; (d) a VH of SEQ ID NO: 43 and a VL of SEQ ID NO: 44; (e) a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46; or (f) VH of SEQ ID NO: 47 and VL of SEQ ID NO: 48 The anti-FRα antibody or antigen-binding fragment thereof according to claim 1 , comprising:

5. The anti-FRα antibody or its antigen-binding fragment according to claim 1, wherein the anti-FRα antibody comprises a constant heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 109 or 111, and a constant light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

110.

6. The anti-FRα antibody or antigen-binding fragment thereof of claim 1, wherein the anti-FRα antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 109 or 111, and a constant light chain amino acid sequence comprising the sequence of SEQ ID NO:

110.

7. (a) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 49, and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 50; (b) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 51, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 52; (c) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 53, and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 54; (d) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 55, and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 56; (e) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 57, and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 58; or (f) a heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 59, and a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

60. The anti-FRα antibody of claim 1 , comprising:

8. (a) a heavy chain amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence of SEQ ID NO: 50; (b) a heavy chain amino acid sequence of SEQ ID NO: 51 and a light chain amino acid sequence of SEQ ID NO: 52; (c) a heavy chain amino acid sequence of SEQ ID NO: 53 and a light chain amino acid sequence of SEQ ID NO: 54; (d) a heavy chain amino acid sequence of SEQ ID NO: 55 and a light chain amino acid sequence of SEQ ID NO: 56; (e) a heavy chain amino acid sequence of SEQ ID NO: 57 and a light chain amino acid sequence of SEQ ID NO: 58; or (f) a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO: 60 The anti-FRα antibody of claim 1 , comprising:

9. The antigen-binding fragment of claim 1, which is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

10. The anti-FRα antibody or antigen-binding fragment thereof according to claim 1, which is humanized, chimeric or fully human, preferably fully human.

11. The anti-FRα antibody or antigen-binding fragment thereof of claim 1 , which is monoclonal, polyclonal, recombinant, or multispecific.

12. The anti-FRα antibody or antigen-binding fragment thereof according to claim 1, which is of the IgG1, IgG2, IgG3 or IgG4 type, preferably of the IgG1 type.

13. The anti-FRα antibody or antigen-binding fragment thereof of claim 1 , conjugated to one or more heterologous agents.

14. The anti-FRα antibody or antigen-binding fragment thereof of claim 13, wherein the one or more heterologous agents are selected from the group consisting of cytotoxins, antimicrobial agents, therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, biological response modifiers, pharmaceuticals, lymphokines, heterologous antibodies, fragments of heterologous antibodies, detectable labels, polyethylene glycol (PEG), radioactive isotopes, or combinations thereof.

15. The anti-FRα antibody or antigen-binding fragment thereof of claim 14 , wherein the heterologous agent is a cytotoxin.

16. An antibody-drug conjugate (ADC) comprising the anti-FRα antibody or antigen-binding fragment thereof of claim 1, wherein the anti-FRα antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

17. The cytotoxin is 【Chemistry 1】 wherein Q is 【Chemistry 2】 (In the formula, -NHQ Ｘ -C(=O)- is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. and X is: 【Transformation 3】 (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0)) and G Ｌ is a linker for binding to an anti-FRα antibody or an antigen-binding fragment thereof; 【Chemistry 4】 [In the formula, R Ｌ１ and R Ｌ２ are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1; or 【Transformation 5】 [In the formula, R Ｌ１ and R Ｌ２ are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group. a linker R selected from Ｌ The anti-FRα antibody or antigen-binding fragment thereof of claim 14, or the ADC of claim 16, wherein the anti-FRα antibody or antigen-binding fragment thereof is linked to the anti-FRα antibody or antigen-binding fragment thereof via

18. G Ｌ teeth, 【Transformation 6】 The anti-FRα antibody or antigen-binding fragment thereof, or ADC according to claim 17,

19. R Ｌ teeth, 【Transformation 7】 The anti-FRα antibody or antigen-binding fragment thereof, or ADC according to claim 17,

20. The anti-FRα antibody or antigen-binding fragment thereof of claim 14, or the ADC of claim 16, wherein the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof, and preferably the cytotoxin is a topoisomerase I inhibitor.

21. The anti-FRα antibody or antigen-binding fragment thereof, or ADC of claim 20, wherein the cytotoxin is a topoisomerase I inhibitor.

22. The topoisomerase I inhibitor has the formula (I): 【Transformation 8】 or a salt or solvate thereof, In the formula, R Ｌ teeth, 【Chemistry 9】 wherein Q is 【Chemistry 10】 (wherein —NHQ X —C(═O)— is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue). and X is: 【Chemistry 11】 (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0)) and G L is a linker for conjugating to an anti-FRα antibody or antigen-binding fragment thereof; 【Chemistry 12】 wherein R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; and e is 0 or 1; or 【Chemistry 13】 wherein R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group. The anti-FRα antibody or antigen-binding fragment thereof, or ADC according to claim 20, selected from the group consisting of:

23. The topoisomerase I inhibitor is 【Chemistry 14】 and preferably, the topoisomerase I inhibitor is 【Chemistry 15】 The anti-FRα antibody or antigen-binding fragment thereof, or ADC according to claim 21,

24. The anti-FRα antibody or antigen-binding fragment thereof of claim 14, or the ADC of claim 16, wherein the drug-antibody ratio (DAR) is in the range of about 1 to 20, optionally the DAR range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10.

25. The anti-FRα antibody or antigen-binding fragment thereof, or ADC described in claim 24, wherein the DAR is about 8 or about 4, preferably the DAR is about 8.

26. 1. An ADC comprising an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin, (i) the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2; a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3; a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4; a light chain CDR2 (KASGLES) of SEQ ID NO: 5; and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, optionally wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) the cytotoxin is the topoisomerase I inhibitor SG3932 【Chemistry 16】 and (iii) the DAR is about 8; ADC.

27. An isolated polynucleotide encoding the anti-FRα antibody or antigen-binding fragment thereof of claim 1.

28. (a) the polynucleotide of claim 27 operably linked to a promoter; or (b) a polynucleotide encoding a VH region as defined in claim 2, and a polynucleotide encoding a VL region as defined in claim 2, wherein the polynucleotide is operably linked to one or more promoters. A vector comprising:

29. 29. The vector of claim 28, further comprising a polynucleotide encoding a constant heavy chain region as defined in claim 5 or 6, and a polynucleotide encoding a constant light chain region as defined in claim 5 or 6.

30. 29. A host cell comprising the polynucleotide of claim 27 or the vector of claim 28.

31. A method for preparing the anti-FRα antibody or antigen-binding fragment thereof of claim 1, comprising: (a) transfecting the vector of claim 28; (b) culturing the host cells under conditions that allow synthesis of the antibody or antigen-binding fragment; and (c) recovering the antibody or antigen-binding fragment from the culture. A method comprising:

32. A pharmaceutical composition comprising the anti-FRα antibody or antigen-binding fragment thereof described in claim 1, or the ADC described in claim 16, and a pharmaceutically acceptable excipient.

33. 33. The pharmaceutical composition of claim 32 for depleting a population of FRa-positive cells in a subject, optionally wherein the FRa-positive cells are FRa-positive cancer cells.

34. 33. The pharmaceutical composition of claim 32 for the treatment of cancer associated with FRa expression.

35. 35. The pharmaceutical composition of claim 34, wherein the cancer comprises cancer cells with heterogeneous expression of FRα and / or low expression of FRα, and optionally the cancer cells have FRα expression similar to that of an Igrov-1 cell line.

36. 35. The pharmaceutical composition of claim 34, wherein the cancer is selected from ovarian cancer, lung cancer, endometrial cancer, pancreatic cancer, gastric cancer, renal cell carcinoma (RCC), colorectal cancer, head and neck squamous cell carcinoma (HNSCC), breast cancer (e.g., TNBC), cervical cancer and malignant pleural mesothelioma, preferably the cancer is selected from ovarian cancer and lung cancer.

37. 37. The pharmaceutical composition of claim 36, wherein the lung cancer is non-small cell lung cancer (NSCLC), optionally wherein the NSCLC is selected from squamous cell NSCLC, adenocarcinoma NSCLC, or a combination thereof.

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