Combination Therapies for the Treatment of Cancer Using Therapeutic Binding Molecules - Patent application

Abstract

Provided herein are methods for treating cancer, including combination therapy of (i) an ADC comprising an anti-FRα antibody and (ii) a PARP1 inhibitor, and kits comprising the same.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED PATENT APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Application No. 63 / 485,366, filed February 16, 2023, U.S. Provisional Application No. 63 / 578,233, filed August 23, 2023, and U.S. Provisional Application No. 63 / 619,055, filed January 9, 2024. The above applications are incorporated by reference herein in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present disclosure relates to a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject (a) an antibody-drug conjugate (ADC) comprising an anti-FRα antibody or antigen-binding fragment thereof conjugated to a cytotoxin, and (b) a PARP1 inhibitor. The disclosure also relates to a kit comprising (a) an ADC comprising an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin, and (b) a PARP1 inhibitor. [Background technology]

[0003] Cancer remains one of the most prevalent diseases worldwide, despite years of research into the mechanisms of cancer pathogenesis and the development of numerous potential anticancer drugs. In particular, lung cancer and ovarian cancer are the third and fifth most common cancers in women, respectively. Chemotherapy and radiation therapy are the most common cancer treatments. Nevertheless, these therapies are associated with various negative side effects, including fatigue, nausea, and hair loss. These problems are compounded by the fact that chemotherapy treatments are administered frequently over long periods of time. Over recent decades, many antibody therapies for cancer have been developed and commercialized, resulting in a reduction in the need for traditional forms of chemotherapy for many cancer types. While the availability of methodologies for generating antibodies (e.g., monoclonal antibodies) has greatly improved over this period, relatively few clinically available anti-cancer antibodies remain, and even fewer can be used to target various cancer types. Furthermore, there is a need to increase the efficacy of therapeutic antibodies, which is generally limited by the prevalence of target antigen expression and the subsequent effect on cancer cells after antibody binding. Conjugating monoclonal antibodies to cytotoxic molecules to generate antibody-drug conjugates (ADCs) is a novel approach for delivering targeted therapies for malignant tumors. This approach has been successfully implemented against specific targets (HER2, CD30, and CD79b), resulting in commercially available ADCs such as fam-trastuzumab deruxtecan-NXKI (breast cancer and gastric cancer), brentuximab vedotin (Hodgkin lymphoma), and polatuzumab vedotin-PIIQ (non-Hodgkin lymphoma), respectively.

[0004] Folate receptors (FRs) are membrane-bound proteins present on the cell surface and can therefore be utilized to develop new ADCs. The FR family includes FRα, FRβ, FRγ, and FRδ. FRs bind to folate molecules and transport them into cells, where they are then delivered to the folate cycle to support nucleotide metabolism. In particular, folate is important for DNA synthesis, methylation, and repair (see Cheung, et al., Oncotarget. 2016;7(32):52553-52574).

[0005] 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 (see Kelemen, Int J Cancer. 2006;119(2):243-250). However, folate transport in adults is primarily driven by the ubiquitous expression of reduced folate carriers and proton-coupled folate transporters (Zhao, et al., Annu Rev Nutr. 2011;31:177-201). 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.

[0006] Overexpression of FRα, also known as folate receptor 1 (FOLR1) or folate-binding protein (FBP), is frequently observed in tumor cells, such as ovarian, lung (e.g., non-small cell lung cancer (NSCLC)), and breast cancers (Shi, et al., Drug Des Devel Ther. 2015;9:4989-4996). In particular, previous studies have found elevated levels of soluble FRα in the blood of ovarian cancer patients, supporting the potential application of FRα as a biomarker for early-stage ovarian cancer (Basal, et al., PLoS One. 2009;4(7):e6292). Preclinical ovarian models have also revealed that overexpression of FRα is associated with tumor progression and that folate binding to FRα can mediate activation of the proto-oncogene STAT3 (Hansen, et al., Cell Signal. 2015;27(7):1356-1368).

[0007] Antibodies against FRα in the art have deficiencies such as poor internalization, short half-life, and poor cytotoxicity. Furthermore, FRα-targeting ADCs in the art employ microtubule inhibitors that have been associated with certain toxicities in clinical trials, such as keratitis (mirvetuximab solavancin, consisting of the anti-FRα antibody M9346A conjugated to the maytansinoid warhead DM4 via a sulfo-SPBD linker) (Moore et al. (2017) Cancer 123:3080-7), interstitial lung disease (MORAb-202, consisting of the LK26-derived humanized antibody farletuzumab conjugated to the eribulin warhead) (Sato, et al. (2020) ESMO Abstract https: / / doi.org / 10.1016 / j.annonc.2020.01.026), neuropathy, and neutropenia (STRO-002, consisting of the anti-FRα antibody SP8166 conjugated to the hemiasterlin warhead) (Naumann, et al. 2021) J Clin Oncol 39(Suppl 15 / abstr 5550)https: / / doi10.1200 / JCO.2021.39.15_suppl.5550). Recent studies have also shown that cancer cells can acquire resistance to microtubule inhibitors (Ganguly, et al., Biochim Biophys Acta. 2011 Dec;1816(2):164-171).

[0008] There is a need to develop new cancer therapies based on ADCs that target FRα. Summary of the Invention

[0009] The present disclosure provides, inter alia, a method for treating cancer, which comprises a combination therapy of an ADC comprising an anti-FRα antibody with a PARP1 inhibitor, and a kit comprising the same.

[0010] In one aspect, a method of treating cancer in a human subject in need thereof is provided, comprising administering to the human subject: (a) an antibody-drug conjugate (ADC) comprising an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin, and (b) administering a PARP1 inhibitor.

[0011] In some embodiments of any aspect of the present disclosure, the cancer comprises cancer cells with heterogeneous expression of FRα and / or low expression of FRα. Optionally, the cancer cells have FRα expression similar to the Igrov-1 cell line.

[0012] In some embodiments of any aspect of the present disclosure, the cancer is selected from ovarian cancer, lung cancer (e.g., lung adenocarcinoma), 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. In particular embodiments, the cancer may be selected from ovarian cancer and lung cancer.

[0013] In some embodiments, 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.

[0014] In another aspect, (a) an ADC comprising an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin, and (b) A kit containing a PARP1 inhibitor is provided.

[0015] In some embodiments of any aspect of the disclosure, the PARP1 inhibitor has the following formula:

[0016] [ka] or AZD5305 having a pharmaceutically acceptable salt thereof.

[0017] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is (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, 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, 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, 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, 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, a light chain CDR3 (QQYNSYPLT) of SEQ ID NO: 30, or (f) Contains 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.

[0018] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is (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 at least 90% identical to the amino acid sequence of SEQ ID NO: 39, and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 40; (c) a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 41, and a VL comprising an amino acid sequence 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) VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 47, and VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 48.

[0019] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is (a) L at the N-terminus (e.g., position 1) of VH; (b) an E at the N-terminus (e.g., position 1) of VH, or (c) Contains Q at the N-terminus (e.g., position 1) of VH.

[0020] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is (a) VH of SEQ ID NO: 37 and VL of SEQ ID NO: 38; (b) VH of SEQ ID NO: 39 and VL of SEQ ID NO: 40; (c) VH of SEQ ID NO: 41 and VL of SEQ ID NO: 42; (d) VH of SEQ ID NO: 43 and VL of SEQ ID NO: 44; (e) a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46, or (f) comprising a VH of sequence number 47 and a VL of sequence number 48.

[0021] In some embodiments of any aspect of the disclosure, 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.

[0022] In some embodiments of any aspect of the disclosure, 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.

[0023] In some embodiments of any aspect of the disclosure, the anti-FRα antibody (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.

[0024] In some embodiments of any aspect of the disclosure, the anti-FRα antibody (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) comprising a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO: 60.

[0025] In some embodiments of any aspect of the disclosure, the antigen-binding fragment is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0026] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is humanized, chimeric, or fully human. In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof is fully human.

[0027] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is monoclonal, polyclonal, recombinant, or multispecific.

[0028] In some embodiments of any aspect of the disclosure, the anti-FRα antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 type. In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof is of the IgG1 type.

[0029] In some embodiments of any aspect of the disclosure, the cytotoxin is linked to a linker R selected from: L linked to the anti-FRα antibody or antigen-binding fragment thereof via

[0030] [ka] (Wherein Q is

[0031] [ka] and Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, X is

[0032] [ka] and a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and 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), G L is a linker for connecting to the anti-FRα antibody or antigen-binding fragment thereof), and

[0033] [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, and e is 0 or 1; or

[0034] [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).

[0035] In some embodiments of any aspect of the disclosure, G L teeth

[0036] [ka] is.

[0037] In some embodiments of any aspect of the disclosure, R L teeth

[0038] [ka] is.

[0039] In some embodiments of any aspect of the disclosure, the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof, hi certain embodiments, the cytotoxin is a topoisomerase I inhibitor.

[0040] In some embodiments of any aspect of the disclosure, the topoisomerase I inhibitor has formula (I):

[0041] [ka] and salts and solvates thereof, In the formula, R L is defined above.

[0042] In some embodiments of any aspect of the disclosure, the topoisomerase I inhibitor is

[0043] [ka] is.

[0044] In certain embodiments, the topoisomerase I inhibitor is

[0045] [ka] is.

[0046] In some embodiments of any aspect of the disclosure, the drug-to-antibody ratio (DAR) is in the range of about 1-20, and optionally, the DAR range is selected from about 1-10, about 2-10, about 2-8, about 2-6, and about 4-10.

[0047] In some embodiments of any aspect of the disclosure, the DAR is about 8 or about 4. In particular embodiments, the DAR is about 8.

[0048] In some embodiments of any aspect of the present disclosure, (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; and optionally, 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0049] [ka] and (iii) DAR is about 8.

[0050] In some embodiments of any aspect of the disclosure, the ADC and the PARP1 inhibitor are administered separately or sequentially.

[0051] In some embodiments of any aspect of the disclosure, the ADC and the PARP1 inhibitor are administered together.

[0052] In another aspect, a method of treating cancer in a human subject in need thereof is provided, comprising administering to the human subject: (a) an antibody-drug conjugate (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; and optionally, 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0053] [ka] and (iii) an antibody-drug conjugate (ADC) having a DAR of about 8; and (b) A PARP1 inhibitor, the PARP1 inhibitor having the following formula:

[0054] [ka] or a pharmaceutically acceptable salt thereof.

[0055] In another aspect, (a) an antibody-drug conjugate (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; and optionally, 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0056] [ka] and (iii) an antibody-drug conjugate (ADC) having a DAR of about 8; and (b) A PARP1 inhibitor, the PARP1 inhibitor having the following formula:

[0057] [ka] and a PARP1 inhibitor, wherein the PARP1 inhibitor is AZD5305, or a pharmaceutically acceptable salt thereof.

[0058] Aspects and embodiments of the present disclosure are set forth in the accompanying claims. These and other aspects and embodiments of the present disclosure are also described herein. [Brief explanation of the drawings]

[0059] The invention will now be described in more detail with reference to the accompanying drawings. [Figure 1A] (A) Binding of AB1370026 is shown. [Figure 1B] (B) Binding of AB1370035 is shown. [Figure 1C] (C) Binding of AB1370049 is shown. [Figure 1D] (D) Binding of AB1370083 is shown. [Figure 1E] (E) Binding of AB1370096 is shown. [Figure 1F] Binding of (F)AB1370117 to human FRα, cynomolgus monkey FRα, mouse FRα, human FRβ, or human FRγ by HTRF assay. [Figure 2] High Content Profiler multiparametric analysis of antibody internalization into KB cells is shown, 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 selected and are shown as gray diamonds, while those below the cutoff are shown 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. [Figure 3A] (A) Six exemplary IgGs in an HTRF epitope competition assay with Comparator 1 IgG. [Figure 3B] (B) Six exemplary IgGs in an HTRF epitope competition assay with Comparator 2 IgG. [Figure 3C] (C) Six exemplary IgGs in an HTRF epitope competition assay with comparator 3 IgG. [Figure 4]Results of the AC-SINS self-interaction assay are shown. Antibodies were assayed for their tendency to self-associate in HSA (black columns) buffer. As expected, the negative control antibody showed low levels of self-interaction, while the positive control antibody showed high levels of self-interaction in HSA. The threshold (>5 nm) for flagging antibodies at risk is indicated by the horizontal dotted line. [Figure 5A] (A) Internalization of anti-FRα antibody into Jeg-3 cells (which have moderate FRα expression) over time as measured by increase in fluorescence in a CX7 instrument. [Figure 5B] (B) Internalization of anti-FRα antibody into KB cells (which have high FRα expression) over time as measured by increase in fluorescence in a CX7 instrument. [Figure 6A] (A) Chromatogram of AB1370049-SG3932 DAR8 obtained from UHPLC-RP (reduced form) at 214 nm. [Figure 6B] (B) Chromatogram of AB1370049-SG3932 DAR8 obtained from UHPLC-RP (reduced form) at 330 nm. [Figure 6C] (C) Chromatogram of AB1370049-SG3932 DAR8 obtained from UHPLC-SEC at 280 nm. [Figure 6D] (D) Chromatogram of AB1370049-SG3932 DAR8 obtained from UHPLC-HIC at 214 nm. [Figure 7A] (A) Chromatogram of AB1370049-SG3932 DAR4 obtained from UHPLC-RP (reduced form) at 214 nm. [Figure 7B] (B) Chromatogram of AB1370049-SG3932 DAR4 obtained from UHPLC-RP (reduced form) at 330 nm. [Figure 7C] (C) Chromatogram of AB1370049-SG3932 DAR4 obtained from UHPLC-SEC at 280 nm. [Figure 7D](D) Chromatogram of AB1370049-SG3932 DAR4 obtained by UHPLC-HIC at 214 nm. [Figure 8A] The progression of chemical conversion to a series of DAR8 ADCs upon exposure to various sera is shown. The observed chemical processes are (A) deconjugation; [Figure 8B] The progression of chemical conversion to a series of DAR8 ADCs upon exposure to various serums is shown. The observed chemical process is (B) maleimide hydrolysis. [Figure 9A] (A) Six-day cytotoxicity assay using the lead DAR8 ADC against KB cells (with high FRα expression). [Figure 9B] (B) A 6-day cytotoxicity assay using the lead DAR8 ADC against Jeg-3 cells (with medium to high FRα expression). [Figure 9C] (C) Six-day cytotoxicity assay using the lead DAR8 ADC against Igrov-1 cells (with moderate FRα expression). [Figure 10] Figure 1 shows the bystander killing activity of AB1370049-SG3932 DAR8. FRα-positive and FRα-negative KB cells were treated with either alone or with 1 nM AB1370049-SG3932 DAR8 ADC at a 1:1 ratio for 6 days. Cytotoxic activity was measured after 6 days using flow cytometry. [Figure 11A] KB xenograft study with lead DAR8 ADC. On day 6, (A) a single intravenous dose of 1.25 mg / kg was administered. [Figure 11B] KB xenograft study with lead DAR8 ADC. (B) A single intravenous dose of 5 mg / kg was administered on day 6. [Figure 12A] OVCAR-3 xenograft study with lead DAR8 ADC. (A) A single intravenous dose of 1.25 mg / kg was administered on day 33. [Figure 12B]OVCAR-3 xenograft study with lead DAR8 ADC. (B) A single intravenous dose of 5 mg / kg was administered on day 33. [Figure 13]

[0033] Figure 2 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]

[0033] Figure 1 shows a KB xenograft study with the lead DAR4 ADC. A single intravenous dose of 5 mg / kg was administered on day 7. [Figure 15A] (A) OVCAR-3 with single doses of 1.25, 2.5, and 5 mg / kg AB1370049-SG3932 DAR8 or FRα-DM4 ADC. [Figure 15B] (B) CaCo-2 xenograft study using single doses of 1.25, 2.5, and 5 mg / kg of AB1370049-SG3932 DAR8 or FRα-DM4 ADC. [Figure 16A] (A) Median percent tumor growth resulting from a single dose of 5 mg / kg of AB1370049-SG3932 DAR8 in 51 PDX models. [Figure 16B] (B) Median percent tumor growth resulting from a single dose of 2.5 mg / kg AB1370049-SG3932 DAR8 in 39 PDX models. [Figure 17A] (A) PDX model study using a single dose of 2.5 mg / kg or 5 mg / kg of AB1370049-SG3932 DAR8 on the ovarian PDX model CTG-0711. [Figure 17B] PDX model studies using a single dose of 2.5 mg / kg or 5 mg / kg of AB1370049-SG3932 DAR8 are shown for (A) ovarian PDX model CTG-0711, (B) NSCLC PDX model CTG-2367, and (C) endometrial PDX model CTG-2268. [Figure 17C]PDX model studies using a single dose of 2.5 mg / kg or 5 mg / kg of AB1370049-SG3932 DAR8 are shown for (A) ovarian PDX model CTG-0711, (B) NSCLC PDX model CTG-2367, and (C) endometrial PDX model CTG-2268. [Figure 18A] (A) Cell viability signals measured for progenitor cells in the megakaryocytic lineage. The X-axis represents drug concentration, and the Y-axis represents viability (values ​​are mean - / +SD of triplicates). AB1370049-SG3932 DAR8 does not exhibit worse toxicity than the non-targeting ADC control in primary CD34+ bone marrow-derived hematopoietic stem cell progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages. [Figure 18B] (B) Cell viability signals measured for myeloid progenitor cells. The X-axis represents drug concentration, and the Y-axis represents viability (values ​​are mean - / +SD of triplicates). AB1370049-SG3932 DAR8 does not exhibit worse toxicity than the non-targeting ADC control in primary CD34+ bone marrow-derived hematopoietic stem cell progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages. [Figure 18C] (C) Cell viability signals measured for erythroid lineage progenitor cells. The X-axis represents drug concentration, and the Y-axis represents viability (values ​​are mean - / +SD of triplicates). AB1370049-SG3932 DAR8 does not exhibit worse toxicity than the non-targeting ADC control in primary CD34+ bone marrow-derived hematopoietic stem cell progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages. [Figure 18D] (D) Cell viability signals measured for megakaryocytic lineage proliferating and differentiated cells. The X-axis represents drug concentration, and the Y-axis represents viability (values ​​are mean - / +SD of triplicates). AB1370049-SG3932 DAR8 does not exhibit worse toxicity than the non-targeting ADC control in primary CD34+ bone marrow-derived hematopoietic stem cell progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages. [Figure 18E](E) Cell viability signals measured for myeloid proliferating and differentiated cells. The X-axis represents drug concentration, and the Y-axis represents viability (values ​​are mean - / +SD of triplicates). AB1370049-SG3932 DAR8 does not exhibit worse toxicity than the non-targeting ADC control in primary CD34+ bone marrow-derived hematopoietic stem cell progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages. [Figure 18F] (F) Cell viability signals measured for erythroid proliferating and differentiated cells. The X-axis represents drug concentration, and the Y-axis represents viability (values ​​are mean - / +SD of triplicates). AB1370049-SG3932 DAR8 does not exhibit worse toxicity than the non-targeting ADC control in primary CD34+ bone marrow-derived hematopoietic stem cell progenitor cells induced to differentiate into erythroid, myeloid, or megakaryocytic lineages. [Figure 19] Shown are mean (±SD) unconjugated mAb vs. AB1370049-SG3932 DAR8 concentration-time profiles in cynomolgus monkeys. PK profiles for 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 (including ADC or unconjugated mAb, in the case of ADC). Total ADC is a measure of intact ADC only. [Figure 20A] (A) Results of a 6-day cytotoxicity assay on KB cells treated with 0.000457-3 nM AZD5335, 123.45-10,000 nM AZD5305, or a combination of both. Data points with AZD5305 alone are included on the graph for comparison; they do not correspond to the concentrations provided on the x-axis. [Figure 20B] (B) Results of a 6-day cytotoxicity assay on IGROV-1 cells treated with 0.00152-10 nM AZD5335, 123.45-10,000 nM AZD5305, or a combination of both. Data points with AZD5305 alone are included on the graph for comparison; they do not correspond to the concentrations provided on the x-axis. [Figure 20C](C) Results of a 6-day cytotoxicity assay on OVCAR-3 cells treated with 0.00152-50 nM AZD5335, 0.0247-2 nM AZD5305, or a combination of both. Data points with AZD5305 alone are included on the graph for comparison; they do not correspond to the concentrations provided on the x-axis. [Figure 20D] (D) Results of a 6-day cytotoxicity assay on SKOV-3 cells treated with 0.1 nM to 700 nM AZD5335 and 30 nM to 30 μM AZD5305. Data points with AZD5305 alone are included on the graph for comparison; they do not correspond to the concentrations provided on the x-axis. [Figure 21A] (A) Synergy matrix heat map of AZD5335 and AZD5305 in KB cells. Synergy was calculated using Bliss model analysis. A higher positive score indicated greater synergy. A score of 0 means additive, and a negative value means antagonism. The darker the color, the greater the synergy score. [Figure 21B] (B) Synergy matrix heat map of AZD5335 and AZD5305 in IGROV-1 cells. Synergy was calculated using Bliss model analysis. A higher positive score indicated greater synergy. A score of 0 means additive, and a negative value means antagonism. The darker the color, the greater the synergy score. [Figure 21C] (C) Synergy matrix heat map of AZD5335 and AZD5305 in OVCAR-3 cells. Synergy was calculated using Bliss model analysis. A higher positive score indicated greater synergy. A score of 0 means additive, and a negative value means antagonism. The darker the color, the greater the synergy score. [Figure 21D] (D) Synergy matrix heat map of AZD5335 and AZD5305 in SKOV-3 cells. Synergy was calculated using Bliss model analysis. A higher positive score indicated greater synergy. A score of 0 means additive, and a negative value means antagonism. The darker the color, the greater the synergy score. [Figure 22] This paper presents an OVCAR-3 CDX model study using monotherapy with AZD5335 or AZD5305, or combination therapy with AZD5335 and AZD5305. OVCAR-3 xenograft tumors were grown in female NSG mice until they reached a volume of approximately 175 mm. Mice were then randomized and dosed on day 33. Tumor volumes were measured twice weekly. NIP228-SG3932 (abbreviated as "Nip228") and AZD5305 (abbreviated as "PARPSel") alone demonstrated very weak or no antitumor activity. Moderate tumor growth inhibition (TGI) was observed with 1.25 mg / kg AZD5335 as a single agent, but the combination of AZD5335 with 1.25 mg / kg + 1 mg / kg AZD5305 resulted in better additive efficacy. The NIP228-SG3932 isotype in combination with AZD5305 demonstrated antitumor activity early in the study but failed to demonstrate a sustained response. The low-dose cohort, AZD5305 1.25 mg / kg + AZD5305 0.1 mg / kg, demonstrated moderate tumor growth inhibition with additive effects. Values ​​are mean ± SEM tumor volumes for n:6 animals per group. Dotted lines indicate dosing days. [Figure 23]

[0049] Figure 1 shows a CTG3718 PDX model study using monotherapy with AZD5335 or AZD5305, or combination therapy with AZD5335 and AZD5305. CTG3718 PDX tumors were grown in female NSG mice until tumors reached a volume of approximately 180 mm3. Mice were then randomized and dosed on day 26. Tumor volumes were measured twice weekly. AZD5305 (also abbreviated "5305") was inactive. Moderate tumor growth inhibition (TGI) was observed with AZD5335 (also abbreviated "5335") as a single agent, but the combination of 3.5 mg / kg AZD5335 + AZD5305 resulted in significant tumor growth inhibition. The NIP228-SG3932 isotype ADC (also abbreviated as "NIP228") did not show TGI, but a low level of tumor growth inhibition was observed in the group combined with AZD5305. Values ​​are mean ± SEM tumor volumes for n: 4 animals per group. [Figure 24] This shows an OV2022F PDX model study using monotherapy with AZD5335 or AZD5305, or combination therapy with AZD5335 and AZD5305. OV2022F xenograft tumors were grown in female NSG mice until they reached a volume of approximately 180 mm. Mice were then randomized and dosed on day 34. Tumor volumes were measured twice weekly. NIP228-SG3932 (abbreviated as "Nip228") and AZD5305 ("PARPSel") alone demonstrated some antitumor activity. Moderate tumor growth inhibition (TGI) was observed with 1.25 mg / kg AZD5335 as a single agent, but the combination of AZD5335 with 1.25 mg / kg + 1 mg / kg AZD5305 resulted in better efficacy. The low dose cohort AZD5335 1.25mg / kg + AZD5305 0.1mg / kg also showed strong tumour growth inhibition in combination. Values ​​are mean±SEM tumour volumes for n:5 animals per group. DETAILED DESCRIPTION OF THE INVENTION

[0060] 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, 20th ed., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991), provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0061] Unless otherwise specified, nucleic acid sequences are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation.

[0062] It should 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, a reference to "an agent" includes a plurality of such agents, a 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.

[0063] "About" may generally refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values. In certain embodiments, the term "about" as used herein shall be understood as plus or minus (±) 5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1% of the numerical value of the number with which it is used. Embodiments provided herein as "comprising" one or more features may also be considered to disclose corresponding embodiments "consisting of" such features.

[0064] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." As used herein, the terms "protein" and "polypeptide" are used interchangeably. In this disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0065] Concentrations, amounts, volumes, percentages, and other numerical values ​​may be presented herein in a range format, with it being understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​expressly stated as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly stated.

[0066] Anti-FRα antibody The present inventors developed an exemplary array of anti-FRα antibodies with high affinity and specific binding to FRα on cancer cells (without specific binding to other FR family members, such as FRβ and FRγ). The inventors conducted a thorough evaluation of the antibody's developability, confirming the tendency for reversible self-association, internalization, nonspecific binding, and hydrophobicity, as well as the stability of the mAbs against thermal and light stressors. Furthermore, the inventors used in vivo mouse PK testing on a focused panel of mAbs to eliminate those that showed insufficient in vivo half-life and increased clearance. To reduce the possibility of generating an anti-drug antibody (ADA) response in humans, the inventors performed an in silico immunogenicity assessment to eliminate from consideration any antibodies with a predicted increased risk of ADA responses. Through the above screening strategy, the inventors identified a panel of six antibodies with similar beneficial properties. In some embodiments, these antibodies are included in the ADCs described in the present disclosure.

[0067] Antibody sequence The ADCs of the present disclosure encompass antibodies or antigen-binding fragments (reference antibodies) defined herein having the enumerated CDR sequences or variable heavy and variable light chain sequences, as well as functional variants thereof. Functional variants may bind to the same target antigen as the reference antibody and exhibit the same antigen cross-reactivity as the reference antibody. Functional variants may have different affinities for the target antigen compared to the reference antibody, or may have substantially the same affinity.

[0068] In some embodiments, functional variants of a reference antibody exhibit sequence variations in one or more CDRs when compared to the corresponding reference CDR sequence. Thus, functional antibody variants may include functional variants of CDRs. When the term "functional variant" is used in the context of a CDR sequence, it means that a CDR has at most two, or at most one, amino acid differences when compared to the corresponding reference CDR sequence, which, when combined with the remaining five CDRs (or variants thereof), enables the variant antibody to bind to the same target antigen as the reference antibody and, in certain embodiments, exhibits the same antigen cross-reactivity as the reference antibody. Functional variants may be referred to as "variant antibodies."

[0069] Tables 1-5 show the CDR sequences, VH and VL sequences, heavy and light chain sequences, FR sequences and constant domain sequences of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 and AB1370117, respectively. In case of any discrepancy, the order in the tables prevails.

[0070] In some embodiments, 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.

[0071] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1; Heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS), Heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY), Light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, Light chain CDR2 (KASGLES) of SEQ ID NO: 5, and comprising a light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT), Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0072] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1; Heavy chain CDR2 of SEQ ID NO: 2 (RTYYRSKWYNDYAVSVKS), Heavy chain CDR3 of SEQ ID NO: 3 (GVGSFDY), Light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, Light chain CDR2 (KASGLES) of SEQ ID NO: 5, and It contains the light chain CDR3 of SEQ ID NO: 6 (QQYNSYSQLT).

[0073] 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.

[0074] 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: 49, 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: 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.

[0075] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SYAMS) of SEQ ID NO: 7, Heavy chain CDR2 of SEQ ID NO: 8 (SISSGRSYIYYADSVKG), Heavy chain CDR3 of SEQ ID NO: 9 (EMQQLALDY), Light chain CDR1 (RASQGISNFLA) of SEQ ID NO: 10, Light chain CDR2 (AASSLQS) of SEQ ID NO: 11, and comprising a light chain CDR3 of SEQ ID NO: 12 (QQYNSYPFT), Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0076] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SYAMS) of SEQ ID NO: 7, Heavy chain CDR2 of SEQ ID NO: 8 (SISSGRSYIYYADSVKG), Heavy chain CDR3 of SEQ ID NO: 9 (EMQQLALDY), Light chain CDR1 (RASQGISNFLA) of SEQ ID NO: 10, Light chain CDR2 (AASSLQS) of SEQ ID NO: 11, and Contains the light chain CDR3 of SEQ ID NO: 12 (QQYNSYPFT).

[0077] 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.

[0078] 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.

[0079] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 of SEQ ID NO: 13 (SNSAAWN), Heavy chain CDR2 of SEQ ID NO: 14 (RTYYRSNWYNDYTLSVKS), 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 comprising a light chain CDR3 of SEQ ID NO: 18 (QEYKTYSIFT), Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0080] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 of SEQ ID NO: 13 (SNSAAWN), Heavy chain CDR2 of SEQ ID NO: 14 (RTYYRSNWYNDYTLSVKS), 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 Contains the light chain CDR3 (QEYKTYSIFT) of SEQ ID NO: 18.

[0081] 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.

[0082] 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: 53, 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: 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.

[0083] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, 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 (AASSLQS) of SEQ ID NO: 23, and comprising a light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT), Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0084] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, 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 (AASSLQS) of SEQ ID NO: 23, and Contains the light chain CDR3 of SEQ ID NO: 24 (QQYISYPLT).

[0085] 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.

[0086] 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: 55, 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: 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.

[0087] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 of SEQ ID NO: 25 (SYSMN), Heavy chain CDR2 of SEQ ID NO: 26 (SISSRSSYVYYADSVKG), Heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 27, Light chain CDR1 (RASQGISSFLA) of SEQ ID NO: 28, Light chain CDR2 (AASSLQS) of SEQ ID NO: 29, and comprising a light chain CDR3 of SEQ ID NO: 30 (QQYNSYPLT), Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0088] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 of SEQ ID NO: 25 (SYSMN), Heavy chain CDR2 of SEQ ID NO: 26 (SISSRSSYVYYADSVKG), Heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 27, Light chain CDR1 (RASQGISSFLA) of SEQ ID NO: 28, Light chain CDR2 (AASSLQS) of SEQ ID NO: 29, and Contains the light chain CDR3 (QQYNSYPLT) of SEQ ID NO: 30.

[0089] 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.

[0090] 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.

[0091] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SDSATWN) of SEQ ID NO: 31; Heavy chain CDR2 of SEQ ID NO: 32 (RTYYRSKWYSDYAVSVKS), Heavy chain CDR3 of SEQ ID NO: 33 (GGAPFDY), Light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 34, Light chain CDR2 (KASSLES) of SEQ ID NO: 35, and comprising a light chain CDR3 of SEQ ID NO: 36 (QQYNSYSMYT), Any one or more of the CDRs may contain 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

[0092] In some embodiments, the anti-FRα antibody or antigen-binding fragment Heavy chain CDR1 (SDSATWN) of SEQ ID NO: 31; Heavy chain CDR2 of SEQ ID NO: 32 (RTYYRSKWYSDYAVSVKS), Heavy chain CDR3 of SEQ ID NO: 33 (GGAPFDY), Light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 34, Light chain CDR2 (KASSLES) of SEQ ID NO: 35, and Contains the light chain CDR3 (QQYNSYSMYT) of SEQ ID NO: 36.

[0093] 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.

[0094] 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: 59, 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: 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.

[0095] 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 constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 described in Table 4, and the antibody or fragment can bind only to FRα (e.g., in the form of a single-chain antibody fragment).

[0096] 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 constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 described in Table 4, and the antibody or fragment can bind only to FRα (e.g., in the form of a single-chain antibody fragment).

[0097] In some embodiments of any of the aspects described herein, the anti-FRα antibody or antigen-binding fragment thereof (a) comprises a light chain VL FR1, VL FR2, VL FR3, and VL FR4 that are at least 80%, 85%, 90%, or 95% identical to, 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 described in Table 4; and (b) comprises a light chain VL FR1, VL FR2, VL FR3, and VL FR4 that are at least 80%, 85%, 90%, or 95% identical to, or identical to, the 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 described in Table 4. The present invention also includes heavy chain VH FR1, VH FR2, VH FR3 and VH FR4 that are at least 80%, 85%, 90% or 95% identical to, or identical to, VH FR1, VH FR2, VH FR3 and VH FR4, respectively.

[0098] 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.

[0099] In some embodiments of any of the aspects of the antigen-binding fragments 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.

[0100] [Table 1]

[0101] [Table 2]

[0102] [Table 3-1]

[0103] [Table 3-2]

[0104] [Table 4]

[0105] [Table 5]

[0106] Minor variations in the amino acid sequences of the antibodies in the ADCs of the disclosure are contemplated as encompassed by the present disclosure, provided that the variations in the amino acid sequence maintain at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to an antibody or antigen-binding fragment thereof of the disclosure defined anywhere herein. In certain embodiments, the variations in the amino acid sequence maintain at least 99% sequence identity to an antibody or antigen-binding fragment thereof of the disclosure defined anywhere herein.

[0107] The antibodies in the ADCs of the present disclosure may include variants in which an amino acid residue from one species is substituted with the corresponding residue in another species, either at a conserved or non-conserved position. In some embodiments, an amino acid residue at a non-conserved position is substituted with a conservative or non-conserved residue. Conservative amino acid substitutions are particularly contemplated.

[0108] A "conservative amino acid substitution" refers to an amino acid residue being replaced with an amino acid residue having a similar side chain. In the art, families of amino acid residues with similar side chains have been defined, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, 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 conservative. The inclusion of conservatively modified variants in the antibodies in the ADCs of the disclosure does not exclude other forms of variants, such as polymorphic variants, interspecies homologs, and alleles.

[0109] "Non-conservative amino acid substitutions" include (i) a residue having a positive side chain (e.g., Arg, His, or Lys) that is replaced with or is substituted by a negative residue (e.g., Glu, Asp); (ii) a hydrophilic residue (e.g., Ser, Thr) that is replaced with or is substituted by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) a cysteine ​​or proline that is replaced with or is substituted by another residue; or (iv) a residue that has a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) that is replaced with a residue having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly), or no side chain at all.

[0110] 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 of antibodies in the ADCs of the present disclosure. 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 in the ADCs of the present disclosure may also include unnatural amino acid residues.

[0111] Unnatural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods for incorporating unnatural amino acid residues into proteins are known in the art. 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 Escherichia coli S30 extracts and commercially available enzymes and other reagents. The protein is 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 a second method, translation is carried out in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (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), which is incorporated into the polypeptide in place of its natural counterpart.See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-natural species by in vitro chemical modification. 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).

[0112] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, unnatural amino acids, and non-naturally occurring amino acids may be substituted for amino acid residues of antibodies in the ADCs of the disclosure.

[0113] Essential amino acids in antibodies in the ADCs of the present disclosure can be identified according to 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 the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, for example, 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 (e.g., translocation or protease components) of antibodies in the ADCs of the present disclosure.

[0114] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those disclosed by 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 in which two or more positions in a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the range of permissible substitutions at each position. Other methods that can 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, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0115] 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, 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 and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparison and percent identity determination between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0116] Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global methods, local methods, and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine procedures within the skill of those in 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 imposing 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, 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 Walle et al., Align-MA: A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0117] Percent sequence identity can be determined by conventional methods. See, e.g., 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 "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as follows (amino acids are indicated in standard one-letter code):

[0118] In some embodiments, the variable domains of both the heavy and light chains of the antibodies or antigen-binding fragments thereof in the ADCs are modified by at least partial replacement of one or more CDRs and / or by partial framework region replacement and sequence changes. While the CDRs can be derived from antibodies of the same class or subclass as the antibody from which the framework regions are derived, it is contemplated that the CDRs will be 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 from the donor variable region. Rather, only those residues necessary to maintain the activity of the antigen-binding site need to be transferred. Given the explanations provided in U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762 (each of which is incorporated herein by reference), routine experimentation to obtain functional antibodies with reduced immunogenicity is well within the capabilities of one of ordinary skill in the art.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof in the ADC may comprise 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, e.g., a human IgG constant region, e.g., a human IgG1 constant region.

[0120] In some embodiments, residues are inserted into the heavy chain constant region of an antibody in an ADC for site-specific conjugation, e.g., for cytotoxin attachment. For example, a cysteine ​​residue can be inserted between amino acids S239 and V240 in the CH2 region of IgG1, which can be referred to as a "239 insertion" or "239i."

[0121] In some embodiments, antibodies in the ADCs disclosed herein may be modified to include alterations or modifications 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 in the ADCs include domain-deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the omitted constant region domain may be replaced by a short amino acid spacer (e.g., 10 residues) that typically provides some of the molecular flexibility conferred by the absent constant region. Deletion or inactivation of constant region domains (by point mutation or other means) can reduce Fc receptor binding of circulating modified antibodies. In other cases, constant region modifications can mitigate complement binding and therefore reduce serum half-life and nonspecific association of conjugated cytotoxins. Still other modifications of the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties, which allow for enhanced localization due to increased antigen specificity or antibody flexibility. In some embodiments, the antibody or antigen-binding fragment thereof in the ADC does not have antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity.

[0122] In some embodiments, antibodies or antigen-binding fragments thereof in ADCs can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibody or fragment thereof. In other constructs, a peptide spacer can be inserted between the hinge region and the modified CH2 and / or CH3 domain. For example, compatible constructs can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is attached to the hinge region with a 5-20 amino acid spacer. Such spacers can be added, for example, to ensure that regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. Amino acid spacers can, in some cases, prove immunogenic and elicit an undesirable immune response against the construct. In some embodiments, any spacers added to the construct can be relatively non-immunogenic or even omitted entirely to maintain the desired biochemical qualities of the modified antibody.

[0123] In addition to deleting entire constant region domains, antibodies or antigen-binding fragments thereof in the ADCs provided herein can be modified by partial deletion or substitution of several or even single amino acids in the constant region. For example, mutation of a single amino acid in a selected region of the CH2 domain can 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) can be completely or partially deleted. Such partial deletion of the constant region can improve selected characteristics (e.g., serum half-life) of the antibodies or antigen-binding fragments thereof in the ADCs while leaving other desirable functions associated with the constant region domains of interest intact. Furthermore, the constant regions of antibodies and antigen-binding fragments thereof in the ADCs can be modified by 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 immunogenic profile of the modified antibodies or antigen-binding fragments thereof in the ADCs. In some embodiments, there may be one or more amino acid additions to the constant region to enhance desirable characteristics, such as reduced or increased effector function, or to provide more cytotoxin or carbohydrate attachment. In some embodiments, it may be desirable to insert or duplicate specific sequences from selected constant region domains. In some embodiments, the heavy chain constant region or fragment thereof, e.g., a human IgG constant region or fragment thereof, can contain one or more amino acid substitutions relative to a wild-type IgG constant domain, such that the modified IgG has an increased half-life compared to the half-life of an IgG having a wild-type IgG constant domain. For example, the IgG constant domain can contain one or more amino acid substitutions at amino acid residues 251-257, 285-290, 308-314, 385-389, and 428-436, where the numbering of amino acid positions is according to the EU index as set forth in Kabat.In some embodiments, the IgG constant domain comprises 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). substitution of the amino acid at Kabat position 311 with serine (S); substitution of the amino acid at Kabat position 428 with threonine (T), leucine (L), phenylalanine (F), or serine (S); substitution of the amino acid at Kabat position 433 with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q); or substitution of 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 in the ADC comprises a YTE variant. The term "YTE" or "YTE variant" refers to a mutation in the IgG1 Fc that results in increased binding to human FcRn and improves the serum half-life of antibodies carrying the 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, U.S. 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 variant has been shown to increase the serum half-life of an antibody by approximately four-fold 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.This patent is incorporated herein by reference in its entirety.

[0124] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC L at the N-terminus of VH (e.g., position 1); an E at the N-terminus (e.g., position 1) of VH, or Contains Q at the N-terminus (eg, position 1) of VH.

[0125] Antibody polynucleotide sequences In some embodiments, a polynucleotide encoding an anti-FRα antibody or antigen-binding fragment thereof in an ADC of the present disclosure is provided. The polynucleotide encoding the anti-FRα antibody or antigen-binding fragment thereof in an ADC of the present disclosure can be any of the nucleotide sequences in Tables 6-7. In the event of any discrepancy, the order in the tables takes precedence.

[0126] In another aspect, a polynucleotide encoding an anti-FRα antibody or antigen-binding fragment thereof in an ADC of the present disclosure 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 constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 listed in Table 6, 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 constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 listed in Table 6.

[0127] In another aspect, the polynucleotide encoding the anti-FRα antibody in the ADC of the present disclosure 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 the constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 listed in Table 7, 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 the constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 listed in Table 7.

[0128] [Table 6-1]

[0129] [Table 6-2]

[0130] [Table 7-1]

[0131] [Table 7-2]

[0132] [Table 7-3]

[0133] [Table 7-4]

[0134] [Table 7-5]

[0135] [Table 7-6]

[0136] Polynucleotide sequences include sequences that have been removed from their naturally occurring environment, recombinant or cloned (e.g., DNA) isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0137] Polynucleotide sequences can be prepared by any means known in the art. For example, large amounts of the sequence 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) that can be introduced and replicated in prokaryotic or eukaryotic cells. Usually, DNA constructs are suitable for autonomous replication in unicellular hosts such as yeast or bacteria, but introduction into and integration into the genome of cultured bacteria, insects, mammals, plants, or other eukaryotic cell lines is also contemplated.

[0138] Polynucleotide sequences can also be produced by chemical synthesis, for example, by the phosphoramidite or triester method, which can be performed in commercially available automated oligonucleotide synthesizers. Double-stranded (e.g., DNA) fragments can be obtained from the single-stranded product 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.

[0139] Polynucleotide variants are also described herein. Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are generated by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be generated for a variety of reasons, such as to optimize codon expression for a particular host (e.g., to change codons in human mRNA to those preferred by a bacterial host, such as E. coli).

[0140] Definitions and Antibody Formats As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen.

[0141] The antibodies in the ADCs of the present disclosure are generally isolated or recombinant. As used herein, "isolated" refers to a polypeptide, e.g., an antibody, that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, 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 specificities. For example, an isolated antibody that specifically binds to FRα is substantially free of antibodies that specifically bind to antigens other than FRα.

[0142] 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 domain (abbreviated herein as "CL").

[0143] In some embodiments, the antibody in the ADC is a full-length antibody. As used herein, an "intact" or "full-length" antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0144] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions VH and VL can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) (or hypervariable regions) interspersed with more conserved regions called framework regions (FRs). In certain embodiments, 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 the antibody in the ADC can further comprise all or a portion of a heavy or light chain constant region.

[0145] 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 bind to (e.g., is bound by) an antibody or antigen-binding fragment in the ADC of the present disclosure. CDRs are the primary determinants of antigen specificity. At least two techniques exist for determining CDRs: (1) an approach based on interspecies sequence variability (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). Furthermore, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0146] The sequences of the CDRs can be identified by 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 8).

[0147] [Table 8]

[0148] The "constant domains" (or "constant regions") of the heavy and light chains are not directly involved in binding the antibody to its target, but exhibit various effector functions. The constant regions of the antibody may 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 (Clq) of the classical complement system.

[0149] There are five major classes of heavy chain constant regions: IgA, IgG, IgD, IgE, and IgM. Each has a characteristic effector function designated by its isotype. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγRs) specific for the IgG class of antibody: FcγRI, FcγRII, and FcγRIII. Binding of antibodies to Fc receptors on the cell surface elicits several important and diverse biological responses, including phagocytosis 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 transfer, and regulation of immunoglobulin production. Sequences important for IgG binding to FcγR receptors have been reported to be located in the CH2 and CH3 domains.

[0150] In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC is of the IgG isotype. The anti-FRα antibody or antigen-binding fragment thereof in the ADC can be of any IgG subclass, for example, IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC is based on the IgG1 isotype. The use of wild-type human IgG1 molecules, which are closer to natural IgG, may reduce developability and other risks. For example, the inventors have designed an ADC using a human IgG1 mAb structure, which, without being bound by theory, is believed to be less immunogenic than other anti-FRα ADCs in development, such as IMGN151.

[0151] For the heavy chain constant region amino acid positions discussed in the antibodies in the ADCs of the present disclosure, numbering follows the EU index first 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). Hence, the terms "EU index as described in Kabat" or "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).

[0152] 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" contains a constant domain CH2 and a constant domain CH3. Each Fc chain may also contain a hinge region. Native Fc regions are homodimers. In some embodiments, Fc regions may contain modifications to force Fc heterodimerization and thus may be heterodimers. The Fc region contains carbohydrate moieties and binding sites for complement and Fc receptors (including the FcRn receptor) and does not have antigen binding activity. Fc can refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in several Fc domain sites, including, but not limited to, EU270, 272, 312, 315, 356, and 358, resulting in minor variations between the sequences described in this application and those known in the art. Consequently, all naturally occurring IgG Fc regions are referred to as "wild-type IgG Fc domains" or "WT IgG Fc domains" (i.e., any allele). Human IgG1, IgG2, IgG3, and IgG4 heavy chain sequences can be found in various sequence databases, including the UniProt database (www.uniprot.org), under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN), respectively.

[0153] In some embodiments, the anti-FRα antibody in the ADCs of the present disclosure is a monoclonal antibody. A "monoclonal antibody" (mAb) refers to a homogeneous antibody population that is involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses not only full-length monoclonal antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody moieties, and other modified immunoglobulin molecules containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to antibodies produced by various methods, including hybridoma, phage selection, recombinant expression, and transgenic animals. In certain embodiments, the anti-FRα antibody in the ADCs of the present disclosure is an isolated monoclonal antibody. In more specific embodiments, the antibody in the ADC is a fully human monoclonal antibody. In alternative embodiments, the methods of the present disclosure can use ADCs comprising polyclonal antibodies.

[0154] The anti-FRα antibodies and antigen-binding fragments thereof in the ADCs of the present disclosure can be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments can be derived from mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in human administration, non-human-derived antibodies or antigen-binding fragments in the ADCs can be genetically or structurally modified to reduce immunogenicity when administered to human patients. Particularly preferred are human or humanized antibodies, particularly recombinant human or humanized antibodies.

[0155] The term "human antibody" refers to an antibody generated in a human or an antibody having an amino acid sequence corresponding to an antibody generated in a human, produced using any technique 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 (e.g., an antibody comprising a murine light chain and a human heavy chain polypeptide). Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement, or mutations introduced by somatic mutation in vivo). Human antibodies can be produced in human cells (via recombinant expression), non-human animals, or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and light chain) genes. Linker peptides not found in native human antibodies can be included in single-chain human antibodies. For example, an Fv may have a linker peptide (e.g., 2 to about 8 glycine or other amino acid residues) connecting the heavy chain variable region and the light chain variable region. These linker peptides are believed to be of human origin. Human antibodies can be produced using a variety of techniques, including phage display techniques 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 produce human antibodies (see, e.g., PCT Publication Nos. WO 1998 / 24893, WO 1992 / 01047, WO 1996 / 34096, WO 1996 / 33735, U.S. 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, either immunized in vitro or isolated from immunized individuals, can be generated that produce antibodies against target antigens. See, e.g., 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.

[0156] The term "humanized antibody" refers to an antibody in which the framework or CDRs have been modified to comprise CDRs from 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, for example, 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 an antibody in which the constant region has been further modified or altered from the constant region of the original antibody to generate desired properties.

[0157] Humanized antibodies can optionally be prepared by a process of analysis of the 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 that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen, e.g., FRα. In this way, FR residues can be selected and combined from the consensus and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0158] Humanized antibodies can be further modified by substitution of additional residues within the Fv framework regions and / or within the replaced non-human residues to improve and optimize the antibody's specificity, affinity, and / or potency. Generally, a humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are consensus sequence regions of a human immunoglobulin. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641, each of which is incorporated herein by reference.

[0159] The term "chimeric antibody" refers to an antibody comprising 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, typically prepared by recombinant DNA technology. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present disclosure are those in which the constant region has been modified or changed from that of the original antibody to produce desired properties. Such chimeric antibodies are also referred to as "class-switched antibodies." Chimeric antibodies are the product of expressed immunoglobulin genes comprising 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, for example, 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 which is incorporated herein by reference.

[0160] In some embodiments, the antibody in the ADC of the present disclosure is a full-length antibody as described above. Alternatively, the antibody may be an antigen-binding fragment. As used herein, the term "antigen-binding fragment" includes any naturally occurring or artificially constructed configuration of an antigen-binding polypeptide comprising one, two, or three light chain CDRs and / or one, two, or three heavy chain CDRs, wherein the polypeptide is capable of binding to an antigen.

[0161] In some embodiments, the antigen-binding fragment in an ADC of the disclosure is a Fab fragment. The antibody in an ADC according to the disclosure may also be a Fab', Fv, scFv, Fd, V NAR 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.

[0162] The terms "Fab fragment" and "Fab" are used interchangeably herein and comprise a single light chain (e.g., constant domains C L and V L ) and a single heavy chain (e.g., constant domains C H and V H ). The heavy chain of a Fab fragment cannot form disulfide bonds with another heavy chain.

[0163] A "Fab' fragment" contains a single light chain and a single heavy chain, but in addition to CH1 and VH, it also contains the region of the heavy chain between the CH1 and CH2 domains required for interchain disulfide bond formation. Thus, two "Fab' fragments" can associate via disulfide bond formation to form an F(ab')2 molecule.

[0164] 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.

[0165] An "Fv fragment" contains only the variable regions of the heavy and light chains. It does not contain the constant regions.

[0166] A "single domain antibody" is an antibody fragment containing a single antibody domain unit (eg, VH or VL).

[0167] A "single-chain Fv" ("scFv") is an antibody fragment comprising 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.

[0168] "Tandem scFvs," also known as TandAbs®, are single-chain Fv molecules formed by covalently linking two scFvs in a tandem orientation with a flexible peptide linker.

[0169] "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.

[0170] "Diabodies" are small, bivalent, bispecific antibody fragments that contain a heavy-chain variable domain (VH) connected 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, promoting the assembly of a dimeric molecule with two functional antigen-binding sites.

[0171] "DARPins" are bispecific ankyrin repeat molecules. DARPins are derived from the natural ankyrin protein, one of the most abundant types of binding proteins found in the human genome. DARPin library modules are defined by the natural ankyrin repeat protein sequence, with 229 ankyrin repeats used for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin library. Library modules resemble the human genome sequence. DARPins are composed of 4-6 modules. Each module is approximately 3.5 kDa, so the average DARPin size is 16-21 kDa. Binder selection is performed by ribosome display, a completely cell-free method, as described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof in the ADC 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 antibody or antigen-binding fragment thereof in the ADC. The moieties may also reduce or eliminate any undesirable side effects of the antibody or antigen-binding fragment thereof in the ADC. A summary of these moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0173] FRα binding In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADCs of the present disclosure specifically binds to FRα. The term "specifically binds to FRα" refers to an antibody that can bind to a specified 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 in the ADCs of the present disclosure specifically binds to human FRα (UniProt ID: P15328) and / or cynomolgus monkey FRα (UniProt ID: A0A2K5U044). In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADCs of the present disclosure specifically binds to human FRα. In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADCs of the present disclosure specifically binds to human FRα and cynomolgus monkey FRα.

[0174] In some embodiments of any aspect of the disclosure, FRα has the sequence of SEQ ID NO: 112 or SEQ ID NO: 113. In particular embodiments, FRα has the sequence of SEQ ID NO: 112.

[0175] SEQ ID NO: 112: Human FRα protein (predicted mature secreted polypeptide) RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERV LNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMS

[0176] SEQ ID NO: 113: Cynomolgus monkey FRα protein (predicted mature secreted polypeptide) RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMS

[0177] In some embodiments, the antibody or antigen-binding fragment thereof in the ADC 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.

[0178] The term "does not bind" means that an antibody or antigen-binding fragment thereof in an ADC of the disclosure does not substantially bind to one or more of the 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 in a cell culture expressing one or more of the molecules are bound by (when contacted with) an antibody or antigen-binding fragment thereof in an ADC of the disclosure. Suitably, as used herein in the context of binding, the term "substantially free" can mean that such cells do not bind.

[0179] binding affinity "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate, whereas high-affinity antibodies generally bind antigens quickly and tend to remain bound for a long time.

[0180] Suitably, the antibody or antigen-binding fragment in the ADC of the present disclosure binds to the FRα molecule with sufficient affinity such that, when the antibody targets FRα, it is useful as a therapeutic agent or diagnostic reagent.

[0181] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC 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.

[0182] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC 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.

[0183] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC 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.

[0184] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC is at a concentration of 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.

[0185] The affinity or avidity of an antibody or antigen-binding fragment thereof for an antigen can be determined experimentally using any suitable method 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 as well as competitive binding assay formats can readily be 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.)

[0186] The binding affinity of an anti-FRα antibody or antigen-binding fragment thereof in an ADC of the present disclosure can be determined using the FRα binding affinity assay described herein. In some embodiments, the binding affinity of an anti-FRα antibody or antigen-binding fragment thereof in an ADC of the present disclosure is determined by Biacore, e.g., Biacore T200, at 25°C. For example, the affinity of recombinant human FRα ECD for an anti-FRα antibody or antigen-binding fragment thereof can be measured using a Biacore T200 at 25°C, e.g., using the following protocol: Protein A is covalently immobilized to a 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 at 10 μl / min in HBS-EP+ buffer, pH 7.4, to allow FRα ECD binding. Serial dilutions of FRαECD (0.4 nM to 100 nM human FRαECD, 0.8 nM to 200 nM cynomolgus monkey FRαECD, 30 nM to 4000 nM mouse FRαECD, and rat FRαECD in HBS-EP+ buffer, pH 7.4) were flowed over the chip at 50 μl / min and allowed to associate for 2 minutes and dissociate for 8 minutes. The chip surface was then fully regenerated with a pulse of 3 M MgCl2 to remove the captured antibody or its antigen-binding fragment, along with any bound FRαECD. Multiple injections of buffer alone were performed under the same conditions to allow for double reference subtraction of the final sensorgram set and analyzed using Biacore T200 evaluation software.

[0187] Alternatively, the binding affinity of the anti-FRα antibody or antigen-binding fragment thereof in the ADC of the present disclosure can be determined by Octet, e.g., Octet red. For example, the binding affinity of the anti-FRα antibody can be assayed by Octet red at 25°C, for example, using the following protocol. Binding assays are performed on Octet RED384 (ForteBio) in an assay buffer containing PBS, 0.1% v / v BSA (Sigma, A9576), 0.01% v / v Tween-20 (Sigma, P9416) (pH 7.4) at 25°C using a slanted-bottom black 384-well plate (ForteBio, 18-5076). Assays are set up according to the manufacturer's instructions using either Protein A or an anti-human capture biosensor (AHC) (ForteBio, 18-5089). 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 an anti-human capture biosensor (AHC) (ForteBio, 18-5089) for 180 seconds. Association was measured by incubating the loaded biosensor with 500 nM human FRα (autologous) or 500 nM rat FRα (Sino Biological, 81073-R08H). Dissociation was measured after transfer to assay buffer. Data were analyzed using Octet data analysis software version 7.0.

[0188] antibody preparation The antibodies or antigen-binding fragments thereof in the ADCs of the present disclosure can be produced by transfecting host cells with one or more vectors comprising polynucleotides encoding the respective antibodies or fragments, culturing the host cells under conditions that allow the synthesis of the antibody or antigen-binding fragment molecules, and recovering the antibody or antigen-binding fragment molecules from the culture.

[0189] The antibodies or antigen-binding fragments thereof (e.g., as monoclonal antibodies) in the ADCs can be produced using recombinant DNA methods, such as those described in U.S. Patent 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 antibody heavy and light chains, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into appropriate expression vectors, which, when transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, result in the production of monoclonal antibodies by the host cells. Alternatively, recombinant monoclonal antibodies or antigen-binding fragments thereof of a 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).

[0190] Affinity maturation and chain shuffling strategies are known in the art and can be used 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.

[0191] Various techniques are known for producing antibody fragments. Traditionally, these fragments are produced by proteolytic digestion of intact antibodies, as described, for example, in Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In some embodiments, the anti-FRα antibody fragments in the ADCs are recombinantly produced. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus enabling the production of large amounts of these fragments. Such anti-FRα antibody fragments can also be isolated from the antibody phage libraries described above. The anti-FRα antibody fragments in the ADCs can also be linear antibodies, as described in U.S. Patent No. 5,641,870, incorporated herein by reference. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0192] Techniques can be adapted for the production of single-chain antibodies specific for FRα (see U.S. Pat. No. 4,946,778). Furthermore, methods can be adapted for the construction of Fab expression libraries to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for FRα, or derivatives, fragments, analogs, or homologs thereof. See, e.g., Huse et al., Science 246:1275-1281 (1989). Antibody fragments can be produced by techniques known in the art, including, but not limited to, F(ab')2 fragments produced by pepsin digestion of antibody molecules, Fab fragments produced by reducing the disulfide bridges of F(ab')2 fragments, Fab fragments produced by treatment of antibody molecules with papain and a reducing agent, or Fv fragments.

[0193] Antibody-drug conjugates (ADCs) An "antibody-drug conjugate" (ADC) of the present disclosure comprises an anti-FRα antibody or antigen-binding fragment thereof described herein, wherein the anti-FRα antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0194] cytotoxin A cytotoxin (also called a cytotoxic drug) can be any molecule known in the art that inhibits or prevents the function of a cell, and / or causes destruction of a cell (cell death), and / or exerts an anti-tumor / anti-proliferative effect. Many classes of cytotoxic drugs are known to have potential utility in ADC molecules. Cytotoxic drugs suitable for the present disclosure include, but are not limited to, topoisomerase I inhibitors (TOPOi), amanitin, auristatins, daunomycin, doxorubicin, duocarmycin, dolastatins, enediynes, lexitropsins, taxanes, puromycin, maytansinoids, vinca alkaloids, tubulysins, and pyrrolobenzodiazepines (PBDs). 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, combretastatin, calicheamicin, maytansine, DM-1, vinblastine, methotrexate, and netropsin, as well as derivatives and analogs thereof. Further disclosure regarding cytotoxins suitable for use in ADCs can be found, for example, in International Patent Application Publication Nos. WO 2015 / 155345 and WO 2015 / 157592, which are incorporated herein by reference in their entireties.

[0195] Cytotoxic agents are typically linked to or "loaded onto" an antibody or antigen-binding fragment. Drug loading (p) is the average number of agents per antibody or antigen-binding fragment. Those skilled in the art will understand that more than one such agent (e.g., TOPOi) can be attached to an antibody or antigen-binding fragment thereof.

[0196] In some embodiments, the average number of cytotoxic agents per antibody (or antigen-binding fragment thereof) ranges from about 1 to 20. In some embodiments, this 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 agent per antibody (or antigen-binding fragment thereof). In some embodiments, the number of agents per antibody (or antigen-binding fragment thereof) can be expressed as a ratio of agents (i.e., drugs) to antibodies. This ratio is referred to as the Drug-to-Antibody Ratio (DAR). The DAR is the average number of agents (i.e., cytotoxic agents) conjugated to each antibody. In some embodiments of the present disclosure, the DAR ranges from about 1 to 20. In some embodiments, 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. In certain embodiments, the DAR is about 4 (e.g., 3.8 to 4.2) or about 8 (e.g., 7.6 to 8.4). In certain embodiments, the DAR is about 8 (e.g., 7.6 to 8.4).

[0197] In some embodiments, the antibody or antigen-binding fragment thereof in the ADC of the present disclosure 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 may be 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 provided below), or a combination thereof. In certain embodiments, the antibody or antigen-binding fragment thereof may be conjugated to a topoisomerase I inhibitor, such as the topoisomerase I inhibitor SG3932.

[0198] In certain embodiments, the antibody or antigen-binding fragment thereof in the ADC of the present disclosure is not conjugated to a microtubule inhibitor, such as a tubulin inhibitor (e.g., maytansinoid, auristatin), or the anti-FRα ADC of the present disclosure does not comprise a microtubule inhibitor. The microtubule inhibitor class of molecules has dosing-limiting and potentially difficult-to-treat toxicities.

[0199] Topoisomerase I inhibitors This disclosure uses in vivo and in vitro models to demonstrate the stability and efficacy of anti-FRα mAbs when conjugated to TOPOi payloads and deployed as ADCs.

[0200] In some embodiments, an anti-FRα ADC of the disclosure comprises a topoisomerase I inhibitor.

[0201] 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 the normal cell cycle. Topoisomerase I inhibitors are advantageous because they mediate highly effective tumor cell killing with less toxicity to patients. In particular, alternative payloads, such as microtubule inhibitors, which have been commonly used to date for the development of 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 (e.g., TOPOi) with less potent payloads promotes bystander killing in heterogeneous tumors. Bystander activity can be achieved by increasing potency and / or improving payload permeability through increased hydrophobicity, but this can result in increased toxicity due to nonspecific uptake.

[0202] General examples of suitable topoisomerase I inhibitors are represented by the following compounds:

[0203] [ka] The compound in question is A * and may be referred to herein as a "drug unit."

[0204] Compounds (e.g., A * ) may be provided with a linker for connecting (preferably, attaching) to an antibody or antigen-binding fragment in an ADC of the disclosure. In certain embodiments, the linker is cleavably attached (e.g., conjugated) to an amino acid residue of an amino acid of an antibody or antigen-binding fragment in an ADC of the disclosure, for example.

[0205] More specifically, examples of suitable topoisomerase I inhibitors are represented by the following compounds having formula "I" and salts and solvates thereof:

[0206] [ka] In the formula, R L is defined above.

[0207] Thus, general formula IV: L-(D L ) p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L and p are defined above and D L is a topoisomerase I inhibitor having a linker (e.g., drug linker unit) of Formula III: and salts and solvates thereof.

[0208] [ka] In the formula, R LL is defined above.

[0209] In some embodiments, the compound of formula I has formula I P :

[0210] [ka] and salts and solvates thereof, wherein R LP is a linker for connecting to an antibody or antigen-binding fragment thereof in an ADC of the disclosure, the linker comprising:

[0211] [ka] (In the formula, Q P teeth,

[0212] [ka] and Q XP Q P is an amino acid residue, a dipeptide residue, or a tripeptide residue, X P teeth,

[0213] [ka] and aP=0 to 5, bP=0 to 16, cP=0 or 1, dP=0 to 5, G L is defined above,

[0214] [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.

[0215] 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.

[0216] 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 or 12. In some of these embodiments, b is 0-8, and may be 0, 2, 4, or 8.

[0217] cP can be 0 or 1.

[0218] dP can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is 0-3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

[0219] X P In some embodiments, aP can be 0, cP can be 1, dP can be 2, and bP can be 0 to 8. In some of these embodiments, bP can be 0, 4, or 8.

[0220] Q above for compounds of formula I X The preference for Q XP may be applied (e.g., where appropriate).

[0221] G above for compounds of formula I L , R L1 , R L2 and the preference for e is given by formula I P The present invention can be applied to the compounds of the present invention.

[0222] In some embodiments, the ADC of Formula IV is P : L-(D LP ) p (IV P ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof;LP is represented by formula III P :

[0223] [ka] and a topoisomerase I inhibitor (e.g., a drug linker unit) which is R LLP is a linker connected to the antibody or antigen-binding fragment thereof, the linker comprising:

[0224] [ka] (In the formula, Q P , X P and G LL is as defined above), and

[0225] [ka] (In the formula, R L1 and R L2 is as defined above), p is an integer from 1 to 20.

[0226] In some embodiments, the compound of formula I has formula I P2 ,

[0227] [ka] and salts and solvates thereof, wherein R LP2 is a linker for connecting to an antibody or antigen-binding fragment thereof in an ADC of the disclosure, the linker comprising:

[0228] [ka] (In the formula, Q is

[0229] [ka] and Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, X P2 teeth,

[0230] [ka] and 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), GL is a linker for connecting to the antibody or antigen-binding fragment thereof in the ADC of the disclosure;

[0231] [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.

[0232] 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.

[0233] 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 may be 0, 2, 3, 4, 5, or 8.

[0234] 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 may be 0, 2, 3, 4, 5, or 8.

[0235] Preferably, only one of b1P2 and b2P2 may be non-zero.

[0236] cP2 may be 0 or 1.

[0237] 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.

[0238] 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. X 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. X 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. X P2In some embodiments, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The range for 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.

[0239] Q above for compounds of formula I X The preference for formula Ia P2 Q in X may be applied (e.g., where appropriate).

[0240] G above for compounds of formula I L , R L1 , R L2 and the preference for e is given by formula I P2 The present invention can be applied to the compounds of the present invention.

[0241] In some embodiments, the ADC of Formula IV is P2 : L-(D LP2 ) p (IV P2 ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof; LP2 is represented by formula III P2 :

[0242] [ka] and a topoisomerase I inhibitor (e.g., a drug linker unit) which is R LLP2 is a linker connected to the antibody or antigen-binding fragment thereof, the linker comprising:

[0243] [ka] (Wherein, Q and X P2 is as defined above, and G LLis a linker connected to the antibody or antigen-binding fragment thereof), and

[0244] [ka] (In the formula, R L1 and R L2 is as defined above), p is an integer from 1 to 20.

[0245] Particularly suitable topoisomerase I inhibitors include those having the formula:

[0246] [ka] is.

[0247] In certain embodiments, the antibody or antigen-binding fragment thereof in the ADC of the disclosure is conjugated to a topoisomerase I inhibitor having the following formula:

[0248] [ka]

[0249] Synthetic methods for making topoisomerase I inhibitors are described, for example, in WO 2020 / 200880, which is incorporated herein by reference.

[0250] While topoisomerase I inhibitors are preferred, as outlined above, it should be noted that any suitable agent (e.g., drug / cytotoxin) can be linked to an antibody or antigen-binding fragment thereof in the ADCs of the disclosure. Examples of other suitable agents are outlined below.

[0251] Tubulysins and pyrrolobenzodiazepines In some embodiments, the cytotoxin is a tubulysin or a tubulysin derivative. In some embodiments, the cytotoxin is tubulysin A, which has the following chemical structure:

[0252] [ka] is.

[0253] Tubulysin is a member of a class of natural products isolated from myxobacterial species. As cytoskeleton interactors, tubulysin is a mitotic poison that inhibits tubulin polymerization, leading to cell cycle arrest and apoptosis. As used herein, the term "tubulysin" refers collectively and individually to naturally occurring tubulysins and tubulysin analogs and derivatives. Illustrative examples of tubulysins are disclosed, for example, in WO 2004005326(A2), WO 2012019123(A1), WO 2009134279(A1), WO 2009055562(A1), WO 2004005327(A1), U.S. Patent Publication No. 7776841, U.S. Patent Publication No. 7754885, U.S. Patent Publication No. 20100240701, U.S. Patent Publication No. 7816377, U.S. Patent Publication No. 20110021568, and U.S. Patent Publication No. 20110263650, which are incorporated herein by reference. It should be understood that such derivatives include tubulysin prodrugs or tubulysins that include, for example, one or more protecting or protecting groups, one or more linking moieties.

[0254] In another embodiment, the cytotoxin can be a pyrrolobenzodiazepine (PBD) or a PBD derivative. PBDs translocate to the nucleus, where they crosslink DNA, preventing replication during mitosis and damaging DNA by inducing single-strand breaks, which subsequently leads to apoptosis. Some PBDs have the ability to recognize and bind to specific sequences in DNA. A preferred sequence is PuGPu. PBDs have the general structure:

[0255] [ka] is.

[0256] PBDs vary in the number, type, and position of substituents on both the aromatic A ring and the pyrrolo C ring, and in the degree of saturation of the C ring. The B ring contains either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, the electrophilic center involved in DNA alkylation. All known natural products possess the (S)-configuration at the chiral C11a position, which provides them with a right-handed twist when viewed from the C ring toward the A ring. This gives them the appropriate three-dimensional shape for isochimerism with the minor groove of B-form DNA, resulting in a snug fit at the binding site. Their ability to form additives in the minor groove allows them to interfere with DNA processing, thus enabling their use as antitumor agents.

[0257] The first PBD antitumor antibiotic, anthramycin, was discovered in 1965. Since then, numerous naturally occurring PBDs have been reported, and more than 10 synthetic routes have been developed for various analogs. Family members include abutamicin, ticamycin, DC-81, mazethramycin, neothramycin A and B, polothramycin, prothracarcin, sibanomycin (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.

[0258] Linker In the ADCs of the present disclosure, the antibody or antigen-binding fragment may be attached to a cytotoxin by a linker.

[0259] As used herein, the term "linker" or "spacer" refers to a bivalent chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody or antigen-binding fragment thereof to a cytotoxin to form an ADC. In some embodiments, the linker or spacer is a peptide spacer. In some embodiments, the linker or spacer is a non-peptide (e.g., chemical) spacer. A suitable linker has two reactive ends, one for antibody attachment and the other for cytotoxin attachment. For bond formation between the linker and / or cytotoxin and between the linker and / or antibody or antigen-binding fragment thereof, one or both of the reactive ends is absent or incomplete (e.g., only the carbonyl of a carboxylic acid). These attachment reactions are discussed in more detail below.

[0260] In certain embodiments, the linker is cleavably attached (e.g., conjugated) to an amino residue, such as an amino acid, of an antibody or antigen-binding fragment in an ADC described herein.

[0261] In some embodiments, the linker is cleavable in the intracellular environment, allowing the Drug Unit to be released from the antibody in the intracellular environment.

[0262] Alternatively, the linker unit may not be cleavable.In such an embodiment, the drug is released, for example, by antibody degradation.However, non-cleavable payload requires complete mAb digestion in lysosome, and the resulting drug-containing product may be too polar to achieve, for example, bystander effect.

[0263] ADCs should preferably be stable and intact, i.e., the antibody conjugated to the drug moiety, before being transported or delivered intracellularly. Outside the target cell, the linker is stable, but inside the cell, the linker can be cleaved at a high rate. An effective linker (i) maintains the specific binding properties of the antibody; (ii) allows intracellular delivery of the conjugate or drug moiety; (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 cytostatic 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 ADCs.

[0264] 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).

[0265] Examples of linkers that can be hydrolyzed under acidic conditions include hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc. (See, for example, U.S. Patent Nos. 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Examples of linkers that can be cleaved under reducing conditions include disulfides. A variety of disulfide linkers are known in the art, including those that can be formed using, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(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)).

[0266] In certain embodiments, the linker is susceptible to enzymatic hydrolysis. Such linkers may be advantageous over pH-sensitive cleavable linkers, which may not be sufficiently stable and may be prematurely cleaved before reaching the target cell, thus potentially resulting in off-target toxicity. Enzymatically cleavable linkers may be peptide-containing linkers that are cleaved by intracellular peptidases or protease enzymes, including, but not limited to, lysosomal or endosomal proteases. One advantage of using intracellular proteolytic release of therapeutic agents is that the drug is typically attenuated when conjugated, and the serum stability of the conjugate is typically 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 unnatural amino acid analogs such as citrulline are all examples of amino acid residues that can make up the amino acid linker moiety. Exemplary dipeptides include valine-citrulline (VC or Val-Cit) and alanine-phenylalanine (AF or Ala-Phe). Exemplary 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.

[0267] 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 within the target cancer cells. The low hydrophobicity of the linkers described herein may enable high drug loading onto antibodies or antigen-binding fragments (e.g., DAR 8) without aggregation, which is significantly higher than that of mirvetuximab soravance (DAR 3-4) or its derivatives, such as IMGN151 (DAR 3.5). This may enable the ADC to deliver significantly higher concentrations of cytotoxic payload to target cancer cells via binding to FRα on the cancer cells.

[0268] In some embodiments, the linker comprises a maleimide. The use of a maleimide in the linker can enable the production of DAR8 and DAR4 ADCs by taking advantage of the natural interchain disulfides in the antibody. This is advantageous over conjugation of surface amines from lysine residues, which can lead to mixtures of DAR species and batch-to-batch variability. If the conjugation site interferes with antigen binding, there can also be reproducibility issues that affect ADC efficacy. Furthermore, other conjugation methods, including engineered antibodies, such as azide-alkyne click chemistry, may not easily achieve a DAR greater than 4.

[0269] In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC of the disclosure is linked to a linker R chosen from: L linked to the cytotoxin via:

[0270] [ka] (In the formula, Q is

[0271] [ka] and Q Xis such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, X is

[0272] [ka] and a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and 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), G L is a linker for connecting to an antibody or antigen-binding fragment thereof in an ADC of the disclosure), and

[0273] [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, and e is 0 or 1; or

[0274] [ka] (In the formula, R L1 and R L2 is as defined above).

[0275] For example, G L , X, Q X Preferred embodiments of linkers Ia (e.g., those in linker Ia above) and Ib are outlined below.

[0276] The following preferences may apply to all aspects of the disclosure described herein or may relate to a single aspect. Preferences may be combined in any combination.

[0277] Various definitions associated with specific terms in this section are provided under the heading "Chemical Definitions" provided below.

[0278] G L G L may be selected from the following:

[0279] [Table 9-1]

[0280] [Table 9-2] In the formula, Ar represents a C5-6 arylene group (for example, phenylene), and X represents a C1-4 alkyl.

[0281] In some embodiments, G L is G L1-1 and G L1-2 In some of these embodiments, G L is G L1-1 is.

[0282] X X is preferably:

[0283] [ka] and a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5, at least b1 or b2=0 and at least c1 or c2=0.

[0284] 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.

[0285] 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 may be 0, 2, 3, 4, 5, or 8.

[0286] 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 may be 0, 2, 3, 4, 5, or 8. Preferably, only one of b1 and b2 may not be 0.

[0287] c1 may be 0 or 1. c2 may be 0 or 1. Preferably, only one of c1 and c2 may not be 0.

[0288] 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.

[0289] In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, and d is 2, and b2 can be from 0 to 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, and d is 0, and b1 can be from 0 to 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, and d is 1, and b2 can be from 0 to 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, and c2 is 0, and one of a and d is 0. The other of a and d is 1 to 5. In some of these embodiments, the other of a and d is 1. In other 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 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0290] Q X In some embodiments, Q is an amino acid residue. The amino acid can be a natural amino acid or an unnatural amino acid. For example, Q can be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0291] In some embodiments, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and unnatural amino acids. In some embodiments, the dipeptide comprises natural amino acids. When the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide is then the recognition site for the cathepsin.

[0292] 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 is selected from Cit is citrulline.

[0293] 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.

[0294] More preferably, Q is NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0295] 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 Includes:

[0296] Other dipeptide combinations can be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

[0297] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and unnatural amino acids. In some embodiments, the tripeptide comprises natural amino acids. When the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide is then 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 is.

[0298] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and unnatural 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. The tetrapeptide is then the recognition site for cathepsin. Tetrapeptide linkers of particular interest are as follows: NH -Gly-Gly-Phe-Gly C=O , and NH -Gly-Phe-Gly-Gly C=O is a tetrapeptide residue selected from:

[0299] In some embodiments, the tetrapeptide is NH -Gly-Gly-Phe-Gly C=O is.

[0300] In the above representation of peptide residues, NH - represents the N-terminus of the residue, C=O represents the C-terminus of the residue. The C-terminus is the NH of a "Drug unit" (e.g., a * )

[0301] Glu represents a glutamic acid residue, i.e.,

[0302] [ka]

[0303] αGlu represents the residue of glutamic acid when attached via the α chain, i.e.

[0304] [ka]

[0305] In some embodiments, amino acid side chains are chemically protected where appropriate. The side chain protecting groups can be those discussed above. The protected amino acid sequence can be cleaved by enzymes. For example, a dipeptide sequence containing a Boc-side-protected Lys residue can be cleaved by cathepsin.

[0306] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem catalogue and as described above.

[0307] Linker Ib R L1 and R L2 are independently selected from H and methyl, or may be taken together with the carbon atom to which they are attached to form a cyclopropylene or cyclobutylene group.

[0308] 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 of the groups are methyl.

[0309] In some embodiments, R L1 and R L2 together with the carbon atoms 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.

[0310] In Group Ib, in some embodiments, e is 0. In other embodiments, e is 1, and the nitro group can be present at any available position on the ring. In some of these embodiments, it is in the ortho position. In other of these embodiments, it is in the para position.

[0311] R L In some embodiments, R L is selected from the following:

[0312] [Table 10-1]

[0313] [Table 10-2]

[0314] Preferably, R L teeth

[0315] [ka] is.

[0316] For example, antibody-drug conjugates of the present disclosure may be represented by the general formula IV: L-(D L ) p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof in the ADC of the disclosure, and D L is the linker R connected to the antibody or antigen-binding fragment thereof in the ADC of the disclosure. LL wherein the linker is preferably

[0317] [ka] wherein Q and X are as defined above, and G LL is a linker connected to the antibody or antigen-binding fragment thereof in the ADC of the disclosure), and

[0318] [ka] (In the formula, R L1 and R L2 is as defined above), p is an integer from 1 to 20.

[0319] Drug loading is represented by p, which is the number of "drug units" (e.g., a cytotoxin such as TOPOi) per antibody or antigen-binding fragment thereof. Drug loading can range from 1 to 20 drug units (D) per antibody or antigen-binding fragment thereof. For compositions, 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. Preferably, p is 8.

[0320] G LL G LL may be selected from the following:

[0321] [Table 11] where Ar is C 5~6 represents an arylene group (e.g., phenylene), and X is C 1~4 Represents alkyl.

[0322] In some embodiments, G LL is G LL1-1 and G LL1-2 In some of these embodiments, G LL is G LL1-1 is.

[0323] In some embodiments, R LL is the above R L It is a group derived from the group.

[0324] It will be appreciated by those skilled 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.

[0325] In some embodiments, the compounds described herein are provided as single enantiomers or enantiomerically enriched forms, the enantiomerically enriched forms have 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, 97:3, or 99:1.

[0326] Specific ADC Embodiments In some embodiments, an anti-FRα ADC of the present disclosure comprises an anti-FRα antibody or antigen-binding fragment thereof described herein linked to a topoisomerase I inhibitor represented by the following compound having formula "I":

[0327] [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. In certain embodiments, the DAR is about 8.

[0328] In some embodiments, an anti-FRα ADC of the present disclosure comprises an anti-FRα antibody or antigen-binding fragment thereof described herein linked to a topoisomerase I inhibitor represented by the following compound having formula "I":

[0329] [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. In certain embodiments, the DAR is about 4.

[0330] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG3932.

[0331] [ka] and containing 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. In certain embodiments, the DAR is about 8.

[0332] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG3932.

[0333] [ka] and containing 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. In certain embodiments, the DAR is about 4.

[0334] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG4010.

[0335] [ka] and containing 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. In certain embodiments, the DAR is about 8.

[0336] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG4010.

[0337] [ka] and containing 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. In certain embodiments, the DAR is about 4.

[0338] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG4057.

[0339] [ka] and containing 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. In certain embodiments, the DAR is about 8.

[0340] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG4057.

[0341] [ka] and containing 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. In certain embodiments, the DAR is about 4.

[0342] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG4052.

[0343] [ka] and containing 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. In certain embodiments, the DAR is about 8.

[0344] In some embodiments, the anti-FRα ADCs of the disclosure comprise an anti-FRα antibody or antigen-binding fragment thereof described herein in combination with the topoisomerase I inhibitor SG4052.

[0345] [ka] and containing 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. In certain embodiments, the DAR is about 4.

[0346] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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":

[0347] [ka] In the formula, R L is defined above, (iii) 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. In certain embodiments, the DAR is about 8.

[0348] In some embodiments, (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, and optionally, the anti-FRα antibody or antigen-binding fragment thereof is sequenced with a VH having 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: 37. and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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":

[0349] [ka] In the formula, R L is defined above, (iii) 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. In certain embodiments, the DAR is about 4.

[0350] In some embodiments, (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, and optionally, the anti-FRα antibody or antigen-binding fragment thereof is sequenced with a VH having 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: 37. and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0351] [ka] and (iii) 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. In certain embodiments, the DAR is about 8.

[0352] In some embodiments, (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, and optionally, the anti-FRα antibody or antigen-binding fragment thereof is sequenced with a VH having 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: 37. and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0353] [ka] and (iii) 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. In certain embodiments, the DAR is about 4.

[0354] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG4010

[0355] [ka] and (iii) 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. In certain embodiments, the DAR is about 8.

[0356] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG4010

[0357] [ka] and (iii) 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. In certain embodiments, the DAR is about 4.

[0358] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG4057

[0359] [ka] and (iii) 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. In certain embodiments, the DAR is about 8.

[0360] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG4057

[0361] [ka] and (iii) 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. In certain embodiments, the DAR is about 4.

[0362] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG4052

[0363] [ka] and (iii) 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. In certain embodiments, the DAR is about 8.

[0364] In some embodiments, (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; and optionally, the anti-FRα antibody or antigen-binding fragment thereof comprises a VH having 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: 37; and a VL having 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: 38; optionally, the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain 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 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) Cytotoxicity is the topoisomerase I inhibitor SG4052

[0365] [ka] and (iii) 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. In certain embodiments, the DAR is about 4.

[0366] internalization Internalization can be a useful property of ADCs. For example, internalization enables delivery of a payload into cells. The inventors have shown that the antibodies and ADCs described herein demonstrate rapid internalization and lysosomal trafficking.

[0367] In some embodiments, the anti-FRα ADCs of the present disclosure bind to FRα on the surface of a cell and are internalized within the cell. In some embodiments, internalization of the anti-FRα ADCs of the present disclosure into FRα-expressing cells saturates 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.

[0368] cytotoxicity In some embodiments, the anti-FRα ADCs of the disclosure inhibit or suppress growth (e.g., of tumors) 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% (in certain embodiments, at least 40%) compared to the level of inhibition or suppression in the absence of the 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).

[0369] In some embodiments, the anti-FRα ADCs of the present disclosure exhibit cytotoxicity against 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.

[0370] In some embodiments, anti-FRα ADCs of the present disclosure exhibit cytotoxicity against 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.

[0371] In some embodiments, the anti-FRα ADCs of the present disclosure inhibit or suppress the proliferation of cell populations with heterogeneous expression of FRα and / or low expression of FRα. In some embodiments, the anti-FRα ADCs of the present disclosure inhibit or suppress the proliferation of cell populations with intermediate expression of FRα (e.g., Jeg-3, OVCAR-3 cell lines, or cells with similar or equivalent levels of FRα expression), cell populations with intermediate to high expression of FRα (e.g., Igrov-1 cell line, or cells with similar or equivalent levels of FRα expression), or cell populations with high expression of FRα (e.g., KB cell line, or cells with similar or equivalent levels of FRα expression).

[0372] Preparation of ADCs The ADCs of the present disclosure can be made in a variety of ways using known organic chemistry reactions, conditions, and reagents, such as: (1) reacting a reactive substituent on an antibody or antigen-binding fragment with a bivalent linker reagent, followed by reaction with a cytotoxin such as a topoisomerase I inhibitor, or (2) reacting a reactive substituent on a cytotoxin, such as a topoisomerase I inhibitor, with a bivalent linker reagent, followed by reaction with a reactive substituent on an antibody or antigen-binding fragment thereof.

[0373] Reactive substituents that may be present in the antibodies or antigen-binding fragments thereof disclosed herein include, but are not limited to, nucleophilic groups, such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups if 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 comprise amine or thiol moieties. Certain antibodies have cysteine ​​bridges that are reducible interchain disulfides. Antibodies can be made reactive for conjugation with linker reagents 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 the amine to a thiol. Reactive thiol groups can be inserted into antibodies (or fragments thereof) using one, two, three, four, or more cysteine ​​residues (e.g., preparing mutant antibodies 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, incorporated herein by reference.

[0374] 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, and an ADC is then formed by conjugation through the sulfhydryl groups' sulfur atoms.

[0375] 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 results in the formation of an ADC. Further protocols for modifying proteins for the attachment or association of cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002). Methods for conjugating linker-drug moieties to cell-targeting proteins, such as antibodies, immunoglobulins, or fragments thereof, are described, 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 (each of which is incorporated herein by reference).

[0376] Conventional conjugation strategies for antibodies or antigen-binding fragments thereof rely on randomly or stochastically attaching a payload to the antibody or fragment via lysine or cysteine. In some embodiments, antibodies or antigen-binding fragments thereof are stochastically conjugated to a cytotoxin, such as a topoisomerase I inhibitor, for example, by partial reduction of the antibody or fragment followed by reaction with the desired drug, with or without a linker moiety attached. The antibody or fragment can be reduced using DTT or other reducing agents to perform a similar reduction, e.g., TCEP. The drug, 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 any unreacted drug. 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.

[0377] In some embodiments, the cytotoxin is attached to the antibody or antigen-binding fragment thereof by site-specific conjugation. In some embodiments, site-specific attachment of a therapeutic moiety to an antibody using reactive amino acid residues at specific positions results in homogenous preparations of ADCs with uniform stoichiometry.

[0378] Site-specific conjugation may be via a cysteine ​​residue or a non-natural amino acid. In certain embodiments, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof via at least one cysteine ​​residue. The cysteine ​​amino acid can be engineered into the reactive site of the antibody (or antigen-binding fragment thereof) and preferably does not form intrachain or intermolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; U.S. Patent No. 7,521,541; U.S. Patent No. 7,723,485; WO 2009 / 052249). In some embodiments, the cytotoxin is attached to the antibody or antigen-binding fragment thereof via a cysteine ​​substitution at 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, where numbering corresponds to the EU index of Kabat. In some embodiments, the particular Kabat positions are 239, 442, or both. In some embodiments, the specific position is Kabat position 442, an amino acid insertion between Kabat positions 239 and 240, or both. In some embodiments, the cytotoxin is attached to the antibody or antigen-binding fragment thereof via a thiol-maleimide bond. In some aspects, the amino acid side chain is a sulfhydryl side chain.

[0379] When one or more nucleophilic or electrophilic groups on an antibody or antigen-binding fragment thereof reacts with a cytotoxic drug, the resulting product can be a mixture of ADCs containing a distribution of drug units (e.g., 1, 2, 3, etc.) attached to the antibody. Liquid chromatography methods, such as hydrophobic interaction (HIC), can separate compounds within a mixture by drug loading. Preparations of ADCs with a single drug loading (p) can be isolated.

[0380] The average number of cytotoxic agents per antibody (or antigen-binding fragment) in a preparation of ADCs from a conjugation reaction can be characterized by conventional means, such as UV, reverse-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 ADC (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070, Sanderson et al. (2005) Clin. Cancer Res. 11:843-852). In some instances, isolation, purification, and characterization of homogeneous ADCs (where p is a specific value from an antibody bearing another agent) can be achieved by means of reverse-phase HPLC, electrophoresis, TFF, SEC-FPLC, CHT, spin filter centrifugation, and the like. Such techniques are also applicable to other types of conjugation.

[0381] chemical definition The following definitions particularly relate to the description of the above topoisomerase I inhibitors.

[0382] C 5~6 Arylene: As used herein, "C 5~6 The term "arylene" refers to a divalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of an aromatic compound.

[0383] 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.

[0384] The ring atoms may be all carbon atoms, such as in a "carbarylene group," in which case the group is phenylene (C6).

[0385] Alternatively, the ring atoms may contain one or more heteroatoms, as in a "heteroarylene group." 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: As used herein, the term "C 1~4 "Alkyl" 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, fully unsaturated). As used herein, the term "C 1~n "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to n carbon atoms, and may be aliphatic or alicyclic, saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes subclasses such as alkenyl, alkynyl, and cycloalkyl, discussed below.

[0386] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), and butyl (C4).

[0387] Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4).

[0388] Examples of saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), sec-butyl (C4), and tert-butyl (C4) groups.

[0389] C 2~4 Alkenyl group: As used herein, "C 2~4 The term "alkenyl group" relates to an alkyl group having one or more carbon-carbon double bonds.

[0390] Examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1-methylvinyl, -C(CH3)=CH2), butenyl (C4), and the like.

[0391] C 2~4 Alkynyl: As used herein, the term "C 2~4 "Alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds.

[0392] Examples of unsaturated alkynyl groups include:

[0393] [ka] These include, but are not limited to:

[0394] C 3~4 Cycloalkyl: As used herein, "C 3~4 The term "cycloalkyl" refers to an alkyl group that is also a cyclyl group, i.e., a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has 3 to 7 carbon atoms, including 3 to 7 ring atoms.

[0395] 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: Cyclobutene (C3) and cyclobutene (C4).

[0396] Connection signs: (where

[0397] [ka] Superscript signs 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).

[0398] salt It may be convenient or desirable to prepare, purify, and / or handle the corresponding salt of the active compound / drug, e.g., a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are described in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0399] For example, if the compound is anionic or has a functional group that can be anionic (e.g., -COOH becomes -COO - (It may be a salt of 1,000 or more cations, such as 1,000 or more cations), salts may be formed with suitable cations. Examples of suitable inorganic cations include Na + and K. + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as +3 Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH +) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are substituted ammonium ions, but are not limited to those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + is.

[0400] If the compound is cationic or has a functional group that can be cationic (e.g., -NH2 is -NH3 + (It may be) 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: citrate, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0401] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, gluceptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, 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.

[0402] 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 a solute (e.g., an active compound, a salt of an active compound) and a solvent. When the solvent is water, the solvate may conveniently be referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, etc.

[0403] Isomers Certain compounds / agents of the present disclosure may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropisomer, stereoisomeric, tautomeric, conformational, 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-forms, meso-forms, D- and L-forms, d- and l-forms, (+)- and (−)-forms; keto-, enol-, and enolate-forms, synthon- and anti-forms, synclinal- and anticlinal-forms, α- and β-forms, axial and equatorial, boat-, chair-, twist-, envelope-, and half-chair-forms, and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

[0404] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures (wholly or partially). Methods for preparing (e.g., asymmetric synthesis) and separating (e.g., fractional crystallization and chromatographic means) such isomers are either known in the art or readily obtained by adapting the methods taught herein or known methods in a known manner.

[0405] PARP1 inhibitors The use of PARP inhibitors in BRCA-mutated ovarian cancer was the first clinical example of successfully using synthetic lethality to target tumor suppressor gene loss (Ledermann, Harter et al. (2014) Lancet Oncol 15(8):852-861). The basis for this finding is that PARP and both BRCA1 and BRCA2 are components of efficient DNA repair.

[0406] In some embodiments of any aspect of the disclosure, the inhibitor of PARP1 (poly(ADP-ribose) polymerase 1) is AZD5305. The terms "inhibit," "inhibition," or "inhibiting" include a decrease in the baseline activity of a biological activity or process.

[0407] The term "AZD5305" refers to the compound having the chemical name 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide and the structure shown below:

[0408] [ka]

[0409] The preparation of AZD5305 is disclosed in U.S. Patent Application Publication No. 2021 / 0040084 A1, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the PARP1 inhibitor is the free base of AZD5305. In some embodiments, the PARP1 inhibitor is a pharmaceutically acceptable salt of AZD5305. In some embodiments, the PARP1 inhibitor is crystalline AZD5305. In some embodiments, the PARP1 inhibitor is crystalline form A AZD5305.

[0410] In some embodiments of any aspect of the disclosure, PARP1 inhibitor also refers to a pharmaceutically acceptable salt thereof.

[0411] Pharmaceutical Composition An ADC and / or a PARP1 inhibitor of the present disclosure can be administered to a subject as a pharmaceutical composition. Accordingly, in one aspect, the present disclosure provides a pharmaceutical composition comprising (i) an ADC of the present disclosure, (ii) a PARP1 inhibitor of the present disclosure, and (iii) a pharmaceutically acceptable excipient.

[0412] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterilized and may contain a pharmaceutically acceptable carrier (e.g., physiological 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.

[0413] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0414] In some embodiments, pharmaceutical compositions according to and for use in accordance with the present disclosure may contain, in addition to the active ingredient (the ADC and PARP1 inhibitor of the present disclosure), a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other material known to those of skill 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 material will depend on the route of administration, which may be oral, or by injection (e.g., cutaneous, subcutaneous, or intravenous).

[0415] In some embodiments, (i) the anti-FRα antibody or antigen-binding fragment thereof of the ADC 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; and optionally, 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0416] [ka] and (iii) the ADC has a DAR of about 8; and (iv) The PARP1 inhibitor has the following formula:

[0417] [ka] or AZD5305 having a pharmaceutically acceptable salt thereof.

[0418] In some embodiments, pharmaceutical compositions of the present disclosure may comprise a pharmaceutically acceptable, non-toxic, sterile carrier 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).

[0419] Examples of suitable excipients may include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition.

[0420] One of skill in the art will understand that the appropriate choice of excipient for use with the ADCs and / or PARP1 inhibitors of the disclosure will depend on the desired properties of the pharmaceutical composition.

[0421] In some embodiments, the pharmaceutical compositions of the present disclosure 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 before use to make a suspension or solution, or a combination thereof.

[0422] In some embodiments, the pharmaceutical composition comprises one or more types of ADC and / or PARP1 inhibitor of the disclosure.

[0423] In some embodiments, the pharmaceutical composition may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like.

[0424] The pharmaceutical compositions disclosed herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, preventing, treating, managing, or ameliorating a disorder or one or more symptoms thereof, and / or in research. The pharmaceutical compositions disclosed herein may be suitable for veterinary use or for pharmaceutical use in humans.

[0425] The pharmaceutical compositions of the present disclosure 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.

[0426] The pharmaceutical compositions of the present disclosure 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, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most appropriate route for 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.

[0427] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. 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 solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, may be included. Capsules may contain a solid carrier such as gelatin.

[0428] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient is in the form of a pyrogen-free parenterally acceptable aqueous solution having appropriate pH, isotonicity and stability. Those skilled in the art are well able to prepare appropriate solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0429] In some embodiments, an ADC of the present disclosure and a PARP1 inhibitor of the present disclosure are formulated into two separate pharmaceutical compositions. Thus, in some embodiments, an ADC of the present disclosure and a PARP1 inhibitor of the present disclosure are administered separately or sequentially.

[0430] Kits, therapeutic combinations, articles of manufacture, and methods of manufacturing pharmaceuticals In one aspect, a kit is provided comprising an ADC of the disclosure and a PARP1 inhibitor of the disclosure. Further encompassed is the use of the kit in a method of the disclosure.

[0431] In one aspect, provided herein is a therapeutic combination for treating cancer in a human subject in need thereof, the therapeutic combination comprising an ADC of the disclosure and a PARP1 inhibitor of the disclosure.

[0432] In one aspect, provided herein is a method for treating a pulmonary arthritis, comprising: (i) an ADC of the disclosure; (ii) a PARP1 inhibitor of the present disclosure; (iii) a pharmaceutically acceptable excipient, and (iv) A product comprising instructions for administering the ADC and the PARP1 inhibitor in combination to a human subject in need of treatment for cancer.

[0433] In one aspect, provided herein is a method for treating a pulmonary arthritis, comprising: (i) a pharmaceutical composition of the present disclosure, and (ii) An article of manufacture comprising instructions for administering a combination of an ADC of the disclosure and a PARP1 inhibitor to a human subject in need of treatment for cancer.

[0434] In one aspect, provided herein is a method for manufacturing a medicament, comprising: (a) using an ADC of the disclosure and a PARP1 inhibitor of the disclosure; and (b) combining the ADC with a PARP1 inhibitor in a pharmaceutically acceptable carrier.

[0435] In some embodiments of any aspect of the present disclosure, the ADC can comprise any antibody or antigen-binding fragment thereof described herein. In some embodiments of any aspect of the present disclosure, the ADC can comprise any linker described herein. In some embodiments of any aspect of the present disclosure, the ADC can comprise any cytotoxic agent described herein.

[0436] In some embodiments of any aspect of the present disclosure, (i) the anti-FRα antibody or antigen-binding fragment thereof of the ADC 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; and optionally, 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0437] [ka] and (iii) the ADC has a DAR of about 8; and (iv) The PARP1 inhibitor has the following formula:

[0438] [ka] or AZD5305 having a pharmaceutically acceptable salt thereof.

[0439] In some embodiments, the kit comprises an isolated (e.g., purified) ADC described herein. In some embodiments, the kit comprises one or more containers. In some embodiments, the kit includes all of the components necessary and / or sufficient to administer a combination of an ADC and a PARP1 inhibitor to a subject. In some embodiments, the kit includes all of the instructions necessary to administer a combination of an ADC and a PARP1 inhibitor to a subject.

[0440] In some embodiments, the kit includes one or more containers filled with one or more of the ADCs and / or PARP1 inhibitors of the present disclosure. Such containers may optionally be accompanied by notices in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. Instructions may also be included, or made available to patients or healthcare providers, on how to use the provided pharmaceutical compositions in the treatment of cancers such as, for example, ovarian cancer, lung cancer (e.g., lung adenocarcinoma or 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.

[0441] In some embodiments, the kit may provide the antigen or antigen-binding fragment and a cytotoxin that is not conjugated to the antibody or antigen-binding fragment but is in a form suitable for binding thereto. Optionally, the kit further comprises instructions and / or reagents for conjugating the cytotoxin to the antibody or antigen-binding fragment. In some embodiments, the kit contains all of the components necessary and / or sufficient to perform the detection assay, including all controls, instructions for performing the assay, and any necessary software for analyzing and presenting results.

[0442] treatment The present disclosure encompasses therapies comprising administering to a subject an anti-FRα antibody-drug conjugate (ADC) of the present disclosure in combination with a PARP1 inhibitor, a pharmaceutical composition comprising an anti-FRα ADC of the present disclosure and a PARP1 inhibitor, or a therapeutic combination comprising an anti-FRα ADC of the present disclosure and a PARP1 inhibitor, to prevent, treat, or ameliorate symptoms associated with a disease, disorder, or infection.

[0443] "Treating" refers to a therapeutic measure that cures, slows, alleviates the symptoms, and / or halts the progression of a diagnosed condition or disorder. Thus, a person in need of treatment includes a person who already has the disorder. In some embodiments, a subject is successfully "treated" for a disease or disorder (preferably, cancer) according to the methods provided herein when the patient exhibits, for example, total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder (preferably, cancer).

[0444] "Preventing" refers to prophylactic or preventative measures that prevent and / or delay the onset of the targeted condition or disorder. Thus, those in need of prevention include those prone to having or susceptible to the disorder. In some embodiments, a disease or disorder (preferably cancer) is successfully prevented in accordance with the methods provided herein when a patient experiences, e.g., less severe or less severe symptoms associated with the disease or disorder, or a later onset of symptoms associated with the disease or disorder, either transiently or permanently, than patients not subjected to the methods of the present disclosure.

[0445] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammalian subject. In some embodiments, a "subject" is a human, livestock, farm animal, sport animal, or zoo animal, such as a human, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cow, etc. In some embodiments, the subject is a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the subject is a human. In the methods of the present disclosure, 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 be asymptomatic for cancer. The subject may also have cancer or be at risk for developing cancer. In some embodiments, the subject has previously undergone cancer treatment.

[0446] In one aspect, a method of treating a disease or disorder (e.g., cancer) is provided, comprising administering to a subject a therapeutically effective amount of an anti-FRα ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure, a pharmaceutical composition of the disclosure, or a therapeutic combination of the disclosure.

[0447] In one aspect, there is provided an anti-FRα ADC of the present disclosure in combination with a PARP1 inhibitor of the present disclosure, a pharmaceutical composition of the present disclosure, or a therapeutic combination of the present disclosure for use in therapy, e.g., to treat a disease or disorder (e.g., cancer).

[0448] In one aspect, an anti-FRα ADC of the disclosure is provided for use in therapy, e.g., for treating a disease or disorder (e.g., cancer), the treatment comprising administering to a subject an anti-FRα ADC and a PARP1 inhibitor of the disclosure.

[0449] In one aspect, a PARP1 inhibitor of the present disclosure is provided for use in therapy, e.g., for treating a disease or disorder (e.g., cancer), the treatment comprising administering to a subject an anti-FRα ADC of the present disclosure and a PARP1 inhibitor.

[0450] In one aspect, provided is a method for preventing the onset of a disease or disorder (e.g., cancer), comprising administering to a subject a therapeutically effective amount of an anti-FRα ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure, a pharmaceutical composition of the disclosure, or a therapeutic combination of the disclosure.

[0451] In one aspect, provided is an anti-FRα ADC of the present disclosure in combination with a PARP1 inhibitor of the present disclosure, a pharmaceutical composition of the present disclosure, or a therapeutic combination of the present disclosure for use in a method for preventing the onset of a disease or disorder (e.g., cancer).

[0452] In one aspect, an anti-FRα ADC of the present disclosure is also provided for use in a method for preventing the onset of a disease or disorder (e.g., cancer), the method comprising administering to a subject an anti-FRα ADC and a PARP1 inhibitor of the present disclosure.

[0453] Also provided in one aspect is a PARP1 inhibitor of the present disclosure for use in a method for preventing the onset of a disease or disorder (e.g., cancer), the method comprising administering to a subject an anti-FRα ADC of the present disclosure and a PARP1 inhibitor.

[0454] The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit can be at least an amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, e.g., determining dosage, is the responsibility of the general practitioner or other physician.

[0455] In one aspect, provided is a method for treating cancer comprising administering an anti-FRα ADC of the disclosure in combination with a PARP1 inhibitor of the disclosure, a pharmaceutical composition of the disclosure, or a therapeutic combination of the disclosure.

[0456] In one aspect, provided is an anti-FRα ADC of the present disclosure, a pharmaceutical composition of the present disclosure, or a therapeutic combination of the present disclosure in combination with a PARP1 inhibitor of the present disclosure.

[0457] Also provided in one aspect is an anti-FRα ADC of the present disclosure for use in treating cancer, the treatment comprising administering to a subject an anti-FRα ADC and a PARP1 inhibitor of the present disclosure.

[0458] Also provided in one aspect is a PARP1 inhibitor of the present disclosure for use in treating cancer, the treatment comprising administering to a subject an anti-FRα ADC of the present disclosure and a PARP1 inhibitor.

[0459] In some embodiments, the subject is a human subject in need thereof.

[0460] In some embodiments, the cancer is associated with FRα expression. In other words, the cancers referred to herein can include cancer cells that express FRα. The cancer cells can be contained within a tumor. In some embodiments, the cancer is a tumor or other malignant cell mass that includes cancer cells that express FRα. In some embodiments, the cancer includes cancer cells with heterogeneous expression of FRα and / or low expression of FRα. In another aspect, the FRα molecule is expressed in the cancer cells at a level similar to that in non-cancerous cells. In another aspect, the FRα molecule is expressed in the cancer cells at a level lower than that in non-cancerous cells. In some embodiments, the cancer further includes cancer cells that do not express FRα.

[0461] In certain embodiments, 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. In certain embodiments, the cancer is ovarian cancer or lung cancer. In some embodiments, the cancer is one or more non-small cell lung cancers (NSCLC), which in certain embodiments may be selected from squamous cell NSCLC, adenocarcinoma NSCLC, or a combination thereof.

[0462] Further examples of cancer include benign, pre-malignant, and malignant cell growths, including, but not limited to, neoplasias and tumors (e.g., histocytoma, glioma, astrocytoma, osteoma), cancers (e.g., ovarian cancer, lung cancer, non-small cell lung cancer (squamous cell carcinoma or adenocarcinoma), endometrial cancer, pancreatic cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma, 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, ovarian, renal, and pancreatic.

[0463] In some embodiments, the cancer is a homologous recombination deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. A mutation in an HRD gene may also be referred to as a mutation in a gene involved in homologous recombination repair (HRRm+). In some embodiments, the mutated HRD gene is selected from BRCA1, BRCA2, and ATM. In some embodiments, the mutated HRD gene is BRCA1. In some embodiments, the mutated HRD gene is BRCA2. In some embodiments, the mutated HRD gene is ATM.

[0464] In one aspect, a method for depleting a population of FRα-positive cells in a subject is provided, comprising administering a therapeutically effective amount of an anti-FRα ADC of the present disclosure in combination with a PARP1 inhibitor of the present disclosure, a pharmaceutical composition of the present disclosure, or a therapeutic combination of the present disclosure.

[0465] In one aspect, provided is an anti-FRα ADC of the present disclosure in combination with a PARP1 inhibitor of the present disclosure, a pharmaceutical composition of the present disclosure, or a therapeutic combination of the present disclosure for use in a method of depleting a population of FRα-positive cells in a subject.

[0466] In one aspect, an anti-FRα ADC of the present disclosure is also provided for use in a method for depleting a population of FRα-positive cells in a subject, the method comprising administering to the subject an anti-FRα ADC and a PARP1 inhibitor of the present disclosure.

[0467] In one aspect, a PARP1 inhibitor of the present disclosure is also provided for use in a method for depleting a population of FRα-positive cells in a subject, the method comprising administering to the subject an anti-FRα ADC of the present disclosure and a PARP1 inhibitor.

[0468] In some embodiments, FRα-positive cells have heterogeneous expression of FRα and / or low expression of FRα.

[0469] In some embodiments of any aspect of the disclosure, the ADC and the PARP1 inhibitor are administered separately or sequentially. In some embodiments of any aspect of the disclosure, the ADC and the PARP1 inhibitor are administered together.

[0470] In embodiments of any of the aspects of the present disclosure, (i) the anti-FRα antibody or antigen-binding fragment thereof of the ADC 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; and optionally, 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) Cytotoxicity is the topoisomerase I inhibitor SG3932

[0471] [ka] and (iii) the ADC has a DAR of about 8; and (iv) The PARP1 inhibitor has the following formula:

[0472] [ka] or AZD5305 having a pharmaceutically acceptable salt thereof.

[0473] In some embodiments of any aspect of the disclosure, the combination of an anti-FRα ADC and a PARP1 inhibitor has a synergistic effect, for example, in treating cancer.

[0474] 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.

[0475] The above-described embodiments should be understood as illustrative examples. Further embodiments are contemplated. It should be understood that features described in connection with any embodiment can be used alone or in combination with other described features, or in combination with one or more features of other embodiments or aspects, or in combination with any combination of other embodiments or aspects. Moreover, equivalents and modifications not described above may be employed without departing from the scope of the present disclosure, as defined in the appended claims.

[0476] Other examples and variations of the antibodies and methods described herein will be apparent to one of skill in the art in the context of this disclosure. Other examples and variations are within the scope of this disclosure, as set forth in the appended claims.

[0477] All documents cited herein are each incorporated by reference in their entirety, including all data, tables, figures, and text presented in the cited documents. [Example]

[0478] Example 1 - Generation of anti-FRα antibodies Aiming A hybridoma campaign was conducted using humanized transgenic mice to obtain a high-affinity, fully human antibody that binds to folate receptor alpha (FRα).

[0479] 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 together 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.

[0480] Human FRα protein was expressed and purified using standard methods. Briefly, plasmid DNA was prepared and transfected into 4 × 10 cells. 6 An autologous suspension-adapted CHO cell line was transfected using PEI-mediated delivery with cells at a density of 1000 cells / ml. Cells were cultured for 7 days at 34°C, 5% CO2, 140 rpm, and 70% humidity. Conditioned medium was collected and purified using a 5 ml HisTrap excel column (Cytiva) for affinity capture, followed by polishing on a HiLoad Superdex 75 16 / 600 pg column (Cytiva) equilibrated in DPBS. Fractions were analyzed for purity by SDS-PAGE, pooled, their concentrations determined by UV absorbance, snap-frozen in liquid nitrogen, and stored at -80°C. To site-specifically biotinylate the protein on its Avi tag, the protein was incubated with recombinant BirA enzyme, ATP, and biotin, followed by size-exclusion chromatography purification as described above. Antibodies were expressed as described for the target antigen and purified using protein A chromatography.

[0481] immunization A repeated immunization multiple site (RIMMS) strategy was employed to immunize Del-1 humanized mice and CD1 wild-type mice as follows. - Minus 4 days: Pre-blood draw - Day 0: Prime immunization - 7th day: 2nd booster - 13th: First blood draw - 15th: 3rd booster - 20th: Second blood draw - 22nd: 4th booster - 24th: 5th booster - 28th day: Final blood collection and fusion of spleen (SP) and lymph node (LN)

[0482] For immunization, six humanized Del-1 mice and four wild-type CD1 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. For the three subsequent injections, the immunogen was emulsified in incomplete Freund's adjuvant, and injections were performed as described above. A final boost was performed on day 24 by intraperitoneally injecting the recombinant protein in PBS.

[0483] Mice were bled via tail vein before immunization, 13 days after the first immunization, and 20 days after the second immunization. IgG titers against human FRα were determined by serum ELISA.

[0484] Evaluation of mouse immune response 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 an HRP-conjugated polyclonal goat anti-mouse IgG-specific secondary antibody (Jackson Immunolabs), and the assay was developed using TMB substrate (Sigma), followed by quenching with 0.5 M sulfuric acid. Plates were then read using a PerkinElmer EnVision 2103 multilabel plate reader.

[0485] Serum titration curves for human FRα and the negative protein control were plotted, and the area under the curve (AUC) for each was calculated.

[0486] Monoclonal mouse IgG isolation (hybridoma generation) Four days after the final booster immunization, 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 fusions were mixed with methylcellulose-based semi-solid medium and seeded onto 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 period, clonal colonies formed from single precursor hybridoma cells. These colonies secreted IgG, which was captured in the vicinity of the colony by FITC-conjugated anti-IgG present in the semi-solid medium. The resulting immune complex formation could be observed as a fluorescent "halo" around the cells when visualized using a ClonePix FL colony picker (Molecular Devices). These halo-forming colonies were then picked into 96-well microtiter plates. After 3–5 days of culture, supernatants from the harvested colonies were collected and screened for human FRα binding.

[0487] 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. PCR amplicons were sequenced by Sanger sequencing.

[0488] Mouse IgG Phynexus Purified Cells were grown in 24-well plates, and after 10 days, the supernatant was transferred to a 96-well master block. Mouse IgG of all subclasses (IgG1, IgG2a, IgG2b, and IgG3) was 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.

[0489] Reformatting Mouse IgG Mouse hybridoma IgG clones were molecularly reformatted to generate constructs expressing mouse VH and VL domains and the relevant mouse IgG constant domains for each hybridoma, essentially as described in Persic et al., Gene 187:9-18, 1997. The VH domains were cloned into the relevant vectors containing mouse heavy chain constant domains and regulatory elements to express the entire IgG1 heavy chain in mammalian cells. Similarly, the VL domains were cloned into vectors for expression of the appropriate mouse light chain (lambda or kappa) constant domains and regulatory elements to express the entire IgG light chain in mammalian cells. To obtain IgG, mammalian suspension CHO cells were transiently transfected with the heavy and light chain IgG vectors. IgG was expressed and secreted into the culture medium. The purification system included a MabSelect SuRe chromatography column (GE Healthcare Lifesciences catalog number: 11003493 for the 1 ml column and 11003495 for the 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 number: 17085101). The concentration of IgG was determined spectrophotometrically using the extinction coefficient based on the amino acid sequence of IgG (Pace et al., Protein Sci. 4:2411-2423, 1995), and the purified IgG was analyzed for purity using SDS-PAGE and HP-SEC analysis.

[0490] result Serum anti-FRα IgG titers after immunization Several approaches are available for mAb discovery, including phage display, immunization, and the use of binding profiles (including identifying mAbs that compete with folate binding to FRα). Here, a dual approach was used to generate anti-FRα antibodies via immunization. The first route involved immunization of human transgenic mice (i.e., Del-1) containing fully human VH and VK domains in the Ig locus. This resulted in high diversity of mAbs, ensuring they were 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 requires humanization of the mAbs.

[0491] 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.

[0492] Generation of hybridomas A total of 10,510 hybridoma clones were generated, and 2,586 were identified as IgG-secreting colonies. IgG-secreting colonies were picked into 96-well microtiter plates. After 3-5 days of culture, supernatants from the picked colonies were collected and screened for human FRα binding. All lead antibodies generated (see Example 2) were obtained from Del-1 transgenic mice.

[0493] Example 2 - Species cross-reactivity of anti-FRα antibodies Aiming The antibodies generated were characterized for strength of binding to the target, ortholog and paralog specificity.

[0494] Materials and Methods Generation of antigen for assay Table 9 shows the species from which the proteins originated, the vector into which the constructs were cloned, the signal peptide, and the epitope tag fused to the protein. Table 10 further shows the sequence of each insert. In each case, sequences corresponding to the soluble region of each folate receptor were cloned together with an N-terminal CD33 leader and C-terminal Avi and His6 tags. The sequences involved in GPI anchoring were removed to obtain soluble constructs.

[0495] [Table 12]

[0496] [Table 13]

[0497] All proteins in Table 10 were expressed and purified using the methods discussed in Example 1 for human FRα.

[0498] HTRF assay HTRF assays were performed in 384-well white shallow-well non-binding plates (Corning, 4513) in an 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 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:

[0499]

number

[0500] Negative control ratios were obtained from nonspecific binding (NSB) control wells. Curves were analyzed using GraphPad Prism software with a four-parameter logistic curve fitting equation.

[0501] HTRF antigen binding assay Mouse IgG-containing supernatants from hybridoma cells were incubated with 0.5 nM streptavidin-cryptate (CisBio, 610 SAKLB), 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% to give a final assay volume of 10 μl, and fluorescence was measured after 4 hours of incubation at room temperature.

[0502] Antigen binding assay of rat FRα Species binding assays were performed on Octet RED384 (ForteBio) plates at 25°C in a black, slanted-bottom 384-well plate (ForteBio, 18-5076) in assay buffer containing PBS, 0.1% v / v BSA (Sigma, A9576), and 0.01% v / v Tween-20 (Sigma, P9416), pH 7.4. Assays were set up according to the manufacturer's instructions using either Protein A or anti-human capture biosensors (AHC) (ForteBio, 18-5089). 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. Association was measured by incubating the loaded biosensor with 500 nM human FRα (autologous) or 500 nM rat FRα (Sino Biological, 81073-R08H). Dissociation was measured after transfer to assay buffer. Data were analyzed using Octet data analysis software version 7.0.

[0503] result 2586 hybridoma supernatants were tested for binding to human FRα, cynomolgus monkey FRα, mouse FRα, human FRβ, or human FRγ in an HTRF assay format. This experiment was performed to isolate antibodies cross-reactive with cynomolgus monkey FRα while ensuring that no binding was observed to paralogs: FRβ and FRγ. To determine the sequence similarity of the human folate receptor to its paralogs and orthologs, multiple sequence alignments were performed using the Clustal Omega v1.2.2 algorithm. The sequence identities of human FRα to its paralogs (Table 11) and orthologs (Table 12) are shown below.

[0504] [Table 14] N / A=Not Applicable

[0505] [Table 15] N / A = Not Applicable;

[0506] Binders were defined as IgGs with an assay signal of >30% ΔF. A total of 129 IgGs showed binding to only human and cynomolgus FRα, 9 IgGs showed binding to only human, cynomolgus, and mouse FRα, and an additional 30 IgGs showed binding to only human, cyno, or mouse FRα. Data for the 6 lead IgGs are shown in Table 13.

[0507] [Table 16]

[0508] 168 hybridoma IgGs identified in the hybridoma supernatant screening were produced as Phynexus-purified IgGs. These were tested in antigen binding assays at four dilutions of IgG. 115 IgGs were identified as IgGs that cross-react with human and cynomolgus monkey FRα but do not bind to human FRβ or FRγ. Data for the six lead IgGs are shown in Figures 1A-1F.

[0509] Furthermore, for the rat FRα antigen binding assay, the six lead IgGs also showed binding to human FRα, but not to rat FRα.

[0510] Overall, it has been demonstrated that a variety of different anti-FRα IgG antibodies have been generated through extensive, high-throughput antibody generation and screening campaigns.

[0511] Example 3 - High-throughput internalization assay Aiming The antibodies generated were characterized for their internalization rates.

[0512] Materials and Methods PhyNexus purified IgG samples from hybridoma output were evaluated in an internalization assay. IgG samples were added after overnight plating of frozen stocks of KB cells. The assay was performed using a fixative acquisition method with a 2.5-hour incubation period before fixation. The assay was performed using pHrodo Green-labeled detection reagent and Cellmask Red (Thermo Fisher Scientific).

[0513] Day 1 Frozen vials of KB cells were thawed, diluted in 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% CO in a humidified incubator.

[0514] 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) diluted 1:5000 was added to obtain a 3.7% formaldehyde fixative and a 1:10,000 dilution of nuclear stain. The plates were incubated for 20 minutes at room temperature and then washed with 1x HBSS (Thermo Fisher Scientific), after which 30 μl / well of Cellmask Red (1:5000 dilution in 0.1% Triton X-100 (Merck)) was added. The plates were incubated in the dark for 20 minutes at room temperature, washed twice with HBSS, and imaged on an Opera High Content Imaging system (see Table 14 for Opera Acquisition parameters).

[0515] [Table 17]

[0516] result A high-throughput internalization assay was performed to assess the rate of antibody internalization into KB cells. Phynexus purified antibodies were labeled and added to KB cells, and cells were fixed after a 2.5-hour time course. Image analysis was performed using Columbus, and the output was analyzed within Spotfire's High Content Profiler, allowing for multiparametric analysis of acquired image data and the development of a "hit list" containing mAbs that exhibited enhanced uptake rates (Figure 2). All six lead antibodies demonstrated internalization above the cutoff determined by positive control samples.

[0517] Example 4 - Epitope binning of generated anti-FRα antibodies Aiming The purpose of this experiment was to epitope bin the anti-FRα antibodies generated, specifically the six exemplary antibodies.

[0518] 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 the general setup of the HTRF assay).

[0519] HTRF epitope competition assays were set up in a 10 μl assay volume using a titration 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. Assay plates were incubated at room temperature for 3-4 hours before fluorescence measurements.

[0520] result One hundred fifteen human / cynomolgus folate receptor-specific hybridoma IgGs were profiled in three HTRF epitope competition assays to determine epitope diversity: 8 IgGs inhibited in all assays, 41 in comparative antibody 1 and comparative antibody 2 assays, 39 in comparative antibody 2 and comparative antibody 3 assays, 12 in comparative antibody 1 and comparative antibody 3 assays, 4 in comparative antibody 1 assay only, 6 in comparative antibody 3 assay only, and 5 IgGs did not inhibit in any assay.

[0521] Six exemplary antibodies were reformatted, expressed, purified as human IgG1, and profiled in HTRF epitope competition assays to confirm potency. Notably, all lead exemplary antibodies were able to inhibit in the Comparative Antibody 2 competition assay. AB1370117 and AB1370035 inhibited in all three epitope competition assays. AB1370026, AB1370083, and AB1370095 inhibited only in the Comparative Antibody 1 and Comparative Antibody 2 epitope competition assays. AB1370049 inhibited only in the Comparative Antibody 2 and Comparative Antibody 3 competition assays (see Table 15 and Figure 3).

[0522] [Table 18]

[0523] conclusion Six antibodies were profiled by epitope competition assays. A range of behaviors and epitope diversity was 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, weakest competition with comparative antibody 2, but did compete with comparative antibody 3.

[0524] Example 5 - Antigen binding affinity of generated anti-FRα antibodies Aiming To determine the binding kinetics and equilibrium dissociation constants for the generated antibodies, particularly a panel of six exemplary antibodies, against human and cynomolgus monkey FRα using surface plasmon resonance analysis.

[0525] Materials and Methods Antibody affinities for human and cynomolgus monkey FRα proteins were measured at 25°C using a Biacore T200 surface plasmon resonance system (Cytiva). Protein A was covalently immobilized to a CM5 chip surface at a concentration of 50 μg / ml in 10 mM sodium acetate (pH 4.0) using standard amine coupling techniques. Antibodies were captured onto the Protein A surface at 10 μl / min in HBS-EP+ buffer, pH 7.4, to allow FRαECD binding. Serial dilutions of FRαECD in HBS-EP+ buffer, pH 7.4 (0.4 nM to 100 nM human FRαECD, 0.8 nM to 200 nM cynomolgus monkey FRαECD, and 30 nM to 4000 nM mouse and rat FRαECD) were flowed over the chip at 50 μl / min, allowing for 2 minutes of association and 8 minutes of dissociation. The chip surface was thoroughly regenerated with a pulse of 3 M MgCl2 to remove the captured antibody along with any bound FRαECD. To allow for double-reference subtraction of the final sensorgram set, multiple buffer-only injections were performed under the same conditions and analyzed using Biacore T200 evaluation software to derive the equilibrium dissociation constant.

[0526] result The antibody affinities of a panel of antibodies to human and cynomolgus monkey FRα proteins were measured by SPR. All antibodies bound to both human and cynomolgus monkey proteins. The affinities to human FRα ranged from 1 to 16 nM, and to cynomolgus monkey FRα ranged from 1 to 37 nM. The kinetic binding parameters of six exemplary antibodies are summarized in Table 16 below.

[0527] [Table 19]

[0528] conclusion All six antibodies had dissociation constants in the low nM range, and in all cases, the affinity for binding to cynomolgus monkey FRα was within 2.5-fold of the affinity for binding to human FRα. Thus, all six exemplary antibodies appear to be suitable for further development as therapeutics from an affinity standpoint.

[0529] Example 6 - Physicochemical properties of generated anti-FRα antibodies Aiming The generation of therapeutic antibodies that are compatible with development and manufacturing requirements requires a thorough evaluation of the physicochemical properties of the antibody. In this study, a panel of generated antibodies was evaluated for their expression titer, stability, and tendency for reversible self-association.

[0530] 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 a species is, the later it elutes and therefore the longer its retention time.

[0531] 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 hour at room temperature. The nanoparticles were blocked by incubation with 100 nM PEG 2000 (Merck) for 1 hour at room temperature and then concentrated 10-fold. The analytical antibody was prepared in a 96-well plate 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 in a total volume of 120 μl. Samples were incubated at room temperature for 20 minutes, and then 50 μl aliquots were transferred in duplicate to a 384-well polystyrene plate (Nunc 384-well clear polystyrene plate, Thermo Scientific). The absorbance of the samples was measured on a plate reader, and the wavelength red shift was determined for each sample compared to that of the buffer-only control wells. A shift of more than 5 nm flagged as a risk of self-association.

[0532] Baculovirus ELISA Antibodies were assayed for nonspecific binding to baculovirus particles by ELISA as described by Hotzel et al. (Hotzel et al., 2012 mAbs 4:6, 753-760). Preparations of each antibody were made at either 100 nM or 10 nM in PBS (Gibco 14190-086) + 0.5% BSA (Sigma A9576) and used in duplicate in ELISA assays on 96-well Nunc Maxisorp F plates coated overnight at 4°C with 50 μl / well of either 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 either PBS + 0.5% BSA (background) or test antibody dilutions were added and incubated for 1 hour at room temperature. After washing three times with PBS, 50 μl / well of detection antibody (anti-human Fc-specific HRP Sigma A0170) diluted 1:5000 in PBS + 0.5% BSA was added. 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. After a 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 absorbance at 450 nm at 10 nM and 100 nM concentrations for each antibody sample and then dividing by the value for the secondary-only control sample. A BV score >5 may indicate a risk of increased clearance due to nonspecific binding.

[0533] Accelerated thermostability assay IgG was diluted to 1 mg / ml in PBS and then incubated at 4°C or 45°C for 2 weeks before filtering 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 column at a flow rate of 1 ml / min using 0.1 M anhydrous disodium phosphate and 0.1 M sodium sulfate, pH 6.8, as the isocratic running buffer. A 5 μm, 7.8 mm x 300 mm column was used. 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, the antibodies were profiled for changes in potency using an HTRF epitope competition assay.

[0534] 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 nonspecific uptake. Furthermore, more hydrophobic mAbs may result in more hydrophobic ADCs, which may lead to aggregation during binding, instability as ADCs, and potentially more nonspecific uptake into normal tissues and therefore toxicity.

[0535] The panel of antibodies generated exhibited retention times by HPLC-HIC ranging from approximately 2.0 to 2.8 minutes. Notably, six exemplary antibodies exhibited acceptably low retention times compared to the panel of antibodies tested (see Table 17).

[0536] [Table 20]

[0537] Affinity capture self-interacting nanoparticle spectroscopy (AC-SINS) Antibodies were also tested for their tendency to self-interact by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS). This behavior underlies or is associated with undesirable properties such as reversible self-association, aggregation, viscosity, opalescence, and phase separation. By monitoring the wavelength of peak absorbance (plasmon wavelength) of antibody-coated gold particles, AC-SINS indirectly measures the tendency of proteins to self-interact in an environment mimicking high protein concentration. The red shift of the exemplary antibodies indicated 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 that the risk of self-association is low for all six exemplary antibodies tested.

[0538] Baculovirus ELISA To test for nonspecific binding, which may indicate an increased clearance rate in vivo, the antibodies were assayed for their level of binding to baculovirus particles in an ELISA format. In all cases, the antibodies showed negligible levels of nonspecific binding, all below the assay cutoff threshold of 5. Advantageously, this suggests a low risk of insufficient clearance in vivo.

[0539] Accelerated thermostability assay A panel of six exemplary antibodies was evaluated for their thermal stability assayed by both HP-SEC and parallel HTRF epitope competition assays for changes in potency (Table 18). In all cases, any change in potency was <2-fold and the loss of monomer was <3%, indicating that all antibodies were thermally stable under the conditions tested.

[0540] [Table 21]

[0541] conclusion All six lead mAbs exhibit good thermal stability, low self-association risk, negligible nonspecific binding, and low hydrophobicity, and therefore all lead mAbs exhibit acceptable developability parameters.

[0542] Example 7 - Pharmacokinetics of generated anti-FRα antibodies in SCID mice Aiming To examine the pharmacokinetics of the generated anti-FRα antibody in wild-type SCID mice after a single intravenous administration.

[0543] Materials and Methods Wild-type SCID mice (n=3 per group) were intravenously (bolus) administered an exemplary anti-FRα antibody (5 mg / kg). Blood samples were collected 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 Phoenix 64 software (Certara).

[0544] Plasma antibody concentrations were determined by a universal ELISA IgG assay.

[0545] result Although the screening process based on the above examples appears exemplary, there was a range of PK profiles observed within this broader panel in mice. Through PK screening, leads with undesirably high clearance and short half-lives were removed.

[0546] The panel of antibodies tested exhibited clearances ranging from approximately 5 to 25 ml / kg / day and half-lives ranging from approximately 4 to 20 days. Among the antibodies tested, six exemplary anti-FRα antibodies exhibited relatively low clearance and long half-lives. Pharmacokinetic parameters of the six exemplary anti-FRα antibodies in mice are shown in Table 19.

[0547] [Table 22] C max = large plasma concentration AUC last = area under the plasma concentration versus time curve up to the last measurable time point CL = plasma clearance V ss = volume of distribution at steady state t 1 / 2 = elimination half-life

[0548] 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.

[0549] Example 8 - Pharmacokinetics of AB1370049 in hFcRn Tg32 mice Aiming To study the pharmacokinetics of AB1370049 in hFcRn Tg32 mice after a single intravenous administration.

[0550] Materials and Methods Human FcRn Tg32 mice (n=3 per group) were intravenously (bolus) administered AB1370049 (5 mg / kg). Blood samples were collected 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 Phoenix 64 software (Certara).

[0551] 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 sample was 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. The digestion was quenched by the addition of acid. An aliquot of the trypsin-digested liquid contents was then transferred to an injection plate for antibody analysis by LC-MS / MS.

[0552] A signature tryptic peptide on the human antibody Fc region (VVSVLTVLHQDWLNGK) was used to calculate the concentration of total antigen in the selected matrix. Tryptic digests from immunoaffinity-enriched samples were separated using reversed-phase chromatography (RPLC) and subsequently detected using multiple reaction monitoring (MRM) for the signature peptide. 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 generated by spiking the ADC reference material in the desired matrix.

[0553] Standard curves and QCs were prepared by spiking different levels of the target compound (ADC reference material) into the same matrix as the sample matrix. The quantification range covered 100 ng / ml to 12,000 ng / ml, and diluted QCs covered up to 50-fold dilutions. The standard curves were fitted with linear regression using a weighting factor of 1 / x2.

[0554] result The pharmacokinetic parameters of AB1370049 in hFcRn Tg32 mice are shown in Table 20.

[0555] [Table 23] C max = maximum observed plasma concentration AUC last = area under the plasma concentration versus time curve up to the last measurable time point CL = plasma clearance V ss = volume of distribution at steady state t 1 / 2 = elimination half-life

[0556] conclusion AB1370049 showed similar pharmacokinetic properties in hFcRn Tg32 and wild-type SCID mice. hFcRn Tg32 mice are a transgenic mouse model in which mouse FcRn is knocked out and human FcRn is knocked in. Therefore, this represents a particularly suitable model for predicting the human pharmacokinetics of AB1370049. Based on the pharmacokinetics in hFcRn Tg32 mice, it is expected that AB1370049 will likely have low human clearance and a half-life similar to that of other clinically used antibodies, thereby enabling convenient dosing.

[0557] Example 9 - Internalization / Lysosomal Trafficking of Exemplary Anti-FRα Antibodies in SCID Mice Aiming To evaluate the internalization of the lead FRα mAb into FRα-positive KB and JEG-3 cells.

[0558] Materials and Methods KB and Jeg-3 cells were stained with cell tracing violet (Thermo Scientific), seeded into 96-well plates, and incubated overnight. Monoclonal antibodies were incubated with 50 nM Fab-pHast human (ATS Bio, Carlsbad, CA) at room temperature before being added to the plated cells. Cell images (20x) were captured and analyzed automatically for fluorescence area every 30 minutes for up to 48 hours on a CellInsight CX7 High-Content Screening Platform (Thermo Scientific).

[0559] result All of the lead antibodies tested were rapidly internalized into FRα-expressing cell lines, saturating in approximately 6-7 hours in JEG-3 cells (Figure 5A) and 5 hours in KB cells (Figure 5B).

[0560] conclusion Despite low FRα expression on the surface of JEG-3 (choriocarcinoma) cells (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.

[0561] Example 10 - ADC generation in DAR8 format Aiming To generate DAR8 ADC for lead mAb with SG3932 payload for in vitro and in vivo evaluation of activity for lead selection.

[0562] Materials and Methods A solution of 50 mM tris(2-carboxyethyl)phosphine (TCEP, Pierce) in phosphate-buffered saline, pH 7.4 (PBS, Gibco) was added (50 molar equivalents / antibody, 16.7 micromoles, 333 μl) 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) to 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) with gentle shaking (60 rpm) in an incubated orbital shaker. 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 as a DMSO solution (12–24 molar equivalents / antibody, 4.8–9.0 μmol in 2.0–2.3 ml DMSO) at 2.0–2.5 mg / ml for a final DMSO concentration of 10% (v / v) to 18–19 ml of this reduced antibody solution (37.0–46.9 mg, 247–313 nmoles). The solutions were mixed at room temperature for 1–18 h, then binding was quenched by the addition of N-acetylcysteine ​​(22.2–45.2 micromolar, 222–452 μl at 100 mM) for >30 min 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.

[0563] result UHPLC-RP analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm × 2.1 mm column eluting with a gradient of water and acetonitrile for the reduced sample of the ADC at 214 nm and 330 nm (SG3932 specific) showed a mixture of unbound light chain (L0), light chain bound to a single molecule of SG3932 (L1), unbound heavy chain (H0), and heavy chains bound to up to three molecules of SG3932 (H1, H2, H3), where the major species were L1 and H3, and was consistent with a drug / antibody ratio (DAR) of 7.58 - 7.94 molecules of SG3932 per antibody as calculated by the following equation using 214 nm chromatogram peak integration. The free NAC-conjugated payload was <LOD / LOQ. Representative UHPLC-RP chromatograms of AB1370049-SG3932 DAR8 are shown in Figures 6A - 6B, which showed a DAR of 7.82.

[0564] [Number]

[0565] UHPLC-SEC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 × 150 mm column (equipped with a 4 μm 3.0 × 20 mm guard column) eluting with a 0.3 ml / min sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v) showed 98.5 - 99.5% monomer purity for the sample of the ADC at 280 nm. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR8 is shown in Figure 6C.

[0566] UHPLC-HIC analysis of the ADC samples at 214 nm on a Shimadzu Prominence system using a Sepax Proteomix HIC Butyl-NP5 5 μm nonporous 4.6 × 35 mm column and eluting with a gradient of 1.5 M to 0 M ammonium sulfate in 25 mM sodium phosphate pH 7.40 and 0 to 20% acetonitrile showed a complex single peak corresponding to the DAR8 species with retention times of 2.98 to 3.11 minutes. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR8 is shown in Figure 6D.

[0567] UV (Nanodrop, Thermo) analysis showed final ADC concentrations of 0.92–3.10 mg / ml in 5.0–6.0 ml, yielding masses of 11.0–37.0 mg of ADC (23–74% yield).

[0568] conclusion All lead antibodies were suitable for DAR8 binding, exhibiting high binding efficiencies of DAR>7.5, no loss of DAR during purification, no aggregation / fragmentation during manufacturing, and >98% monomer purity. Thus, the ADCs of the present disclosure can specifically deliver significantly higher concentrations of cytotoxic payloads to target cancer cells via binding to FRα on cancer cells. The DAR8 ADCs are also homogeneous, offering the advantages of reproducibility and limited batch-to-batch variability during manufacturing. This allows for the delivery of greater amounts of less potent drugs (e.g., TOPOi) to target cancer cells while maintaining tolerability.

[0569] Furthermore, the relatively low hydrophobicity of the resulting ADCs may result in less nonspecific uptake by normal tissues, thus potentially resulting in improved tolerability compared to comparable ADCs delivering more hydrophobic drugs such as mirvetuximab soravancencin or IMGN151.

[0570] 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).

[0571] Example 11 - ADC generation in DAR4 format Aiming To generate a DAR4 ADC for a lead mAb with an SG3932 payload, compared to the DAR8 ADC for in vitro and in vivo assessment of activity and for lead selection.

[0572] Materials and Methods A solution of 5 mM tris(2-carboxyethyl)phosphine (TCEP, Pierce) in phosphate-buffered saline, pH 7.4 (PBS, Gibco) was added (2.7–2.9 molar equivalents / antibody, 416–588 nmoles, 83.2–117.7 μl) to 9.2–12 ml of a solution of antibody (lead AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117, 23–30 mg each, 153–200 nmoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA, Molekula) to a final antibody concentration of 2.5 mg / ml. The reduction mixture was heated at 37°C for 3 h with gentle shaking (60 rpm) in an incubating orbital shaker. After cooling to room temperature, SG3932 was added as a DMSO solution (7 molar equivalents / antibody, 2.91–4.12 μmol, in 1.02–1.33 ml of DMSO) to the reduced antibody solution (23–30 mg, 153–200 nmol) to a final DMSO concentration of 10% (v / v). The solution was mixed for 17 hours at room temperature, and then the binding was quenched by the addition of N-acetylcysteine ​​(5.4–7.1 μmol, 54–71 μl at 100 mM) over 30 minutes at room temperature. The mixture was sterile filtered and purified on an AKTA™ Start FPLC using a GE Healthcare HiLoad™ 26 / 600 column packed with Superdex 200 PG and eluted with PBS at 2.6 ml / min. Fractions corresponding to the ADC monomer peak were pooled, concentrated using a 15 ml Amicon Ultracell 30 kDa MWCO spin filter, sterile filtered, and analyzed.

[0573] result UHPLC-RP analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm × 2.1 mm column eluting with a gradient of water and acetonitrile on the reduced sample of the ADC at 214 nm and 330 nm (SG3932-specific) showed a mixture of unbound light chain (L0), light chain bound to a single molecule of SG3932 (L1), unbound heavy chain (H0), and heavy chains bound to up to three molecules of SG3932 (H1, H2, H3), consistent with a drug / antibody ratio (DAR) of 4.13 - 4.27 molecules of SG3932 per antibody calculated as in Example 10. The free NAC quenched payload was <LOD. Representative UHPLC-RP chromatograms of AB1370049-SG3932 DAR4 are shown in Figures 7A - 7B, which showed a DAR of 4.24.

[0574] UHPLC-SEC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6 × 150 mm column (equipped with a 4 μm 3.0 × 20 mm guard column) eluting with a 0.3 ml / min sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v / v) showed >99.5% monomer purity for the ADC sample at 280 nm. Representative UHPLC-SEC chromatograms of AB1370049-SG3932 DAR4 are shown in Figure 7C.

[0575] UHPLC-HIC analysis at 214 nm on a Shimadzu Prominence system using a Sepax Proteomix HIC Butyl-NP5 5 μm non-porous 4.6 × 35 mm column eluting with a gradient of 1.5 M - 0 M ammonium sulfate and 0 - 20% acetonitrile in 25 mM sodium phosphate pH 7.40 on the ADC sample showed a mixture of DAR species corresponding to an average DAR of approximately 4. Representative UHPLC-SEC chromatograms of AB1370049-SG3932 DAR4 are shown in Figure 7D.

[0576] UV (Stunner, Unchained Labs) analysis indicated a final ADC concentration of 3.59–4.06 mg / ml in 5.0–6.0 ml, yielding an ADC mass of 18.0–24.0 mg (68–79% yield).

[0577] conclusion All lead antibodies were suitable for adjusting the TCEP equivalent for stochastic DAR4 binding, showed no DAR loss during purification, no aggregation / fragmentation during production, >99% monomer purity, and high yields.

[0578] Exemplary lead ADCs were then further evaluated for their in vivo mouse PK, as well as in vivo efficacy (see Examples below).

[0579] Example 12 - In vitro serum stability / deconjugation of exemplary ADCs Aiming To determine the stability of AB1370049-SG3932 DAR8 and other lead DAR8 ADCs across a range of serum.

[0580] 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 with 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).

[0581] Incubation of ADC in serum ADC (1.00 mg / ml, diluted as needed with PBS) was spiked into serum (mouse (ab7486, abcam), rat (C13SDZ, BioRad), IgG-depleted human (DHP-2001, Access Biologicals), and cynomolgus monkey (custom-made, BioIVT), 20-fold dilution of ADC in serum). The resulting mixture (1.8 ml) was aliquoted (200 μl) into 96-well plates (Sterile Nunc™). The 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 (0, 1, 3, and 7 days), and samples were stored at -80°C prior to analysis.

[0582] Preparation of capture resin PureProteome™ Streptavidin Magnetic Beads (4.90 ml) were washed five times in PBS, and CaptureSelect™ Human IgG-Fc PK Biotin-Conjugated (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 end-over-end (3 hours, 40 rpm). The remaining CaptureSelect was removed, and the beads were washed five times in PBS, brought to the original volume with PBS, and stored at 2–8°C until further use.

[0583] 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 hours. Meanwhile, on a KingFisher Flex magnetic bead handling system, capture resin (50 μl per sample) was washed sequentially with SN1 buffer (1× TBS pH 7.4, 0.05% TWEEN 20, 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). ADC capture was performed by incubating the sample with the prepared resin aliquot (15 min). The resin was then washed with PBS (3 x 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).

[0584] Samples were reduced (17 μl per sample) 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, mixed in a ratio of 15:20:6:10) for 15 min at 37° C. Quenching by addition of 2% formic acid in 50% v / v acetonitrile in water (40 μl per sample) provided samples prepared in their final state prior to LC-MS analysis.

[0585] LC-MS analysis Samples were analyzed using 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 using a segmented gradient (acetonitrile in water with 0.03% trifluoroacetic acid, 5–25% over 30 s, then 25–60% over 2 min). Analytes were detected at 280 nm on the VWD and on the EMR mass spectrometer (MS) in Full MS mode. MS tune file parameters were: 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 °C. MS method parameters: polarity: positive, in-source CID: 20.0 eV, microscans: 10, resolution 17500, AGC target: 3e6, max IT: automatic, scan range: 1000-4000 m / z.

[0586] MS data analysis Mass spectra were deconvoluted using Thermo BioPharma Finder. Peaks in the deconvoluted spectra that were deemed chemically significant were extracted by their percentage height values, allowing the DAR and percentage of significant chemical modification for each sample to be determined. Comparison of the DAR values ​​of a given ADC in a given serum over time allowed the percentage of deconjugation for each sample to be determined.

[0587] result All ADCs exhibited low payload deconjugation in all serum samples (Figure 8A). This was partially supported by all ADCs exhibiting significant levels of maleimide hydrolysis, at least in mouse, rat, and cynomolgus monkey serum (Figure 8B). Maleimide hydrolysis chemically stabilizes conjugated payloads and prevents them from deconjugating from the ADC's antibody. However, in IgG-depleted human serum, the prevalence of maleimide hydrolysis was lower. Thus, the reduced protective effect conferred by maleimide hydrolysis here demonstrates the inherent stability of these ADCs against deconjugation.

[0588] conclusion All ADCs showed good overall stability against all sera.

[0589] Example 13 - In vivo stability / deconjugation of AB1370049-SG3932 DAR8 Aiming To evaluate the integrity and warhead debinding of AB1370049-SG3932 DAR8 under in vivo conditions across the cynomolgus monkey DRF study timeframe.

[0590] Materials and Methods Male cynomolgus monkeys (N=3) were intravenously administered two single doses of AB1370049-SG3932 DAR8 (each 3 weeks apart, i.e., on days 1 and 22).

[0591] Blood samples were collected by venipuncture and centrifuged to obtain plasma. Total antibody and ADC assays were performed using an LBA-LCMS method. AB1370049-SG3932 DAR8 was immunocaptured with a biotinylated anti-human antibody bound to magnetic beads, which were then 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 the ADC assay. The injected samples were separated using reversed-phase chromatography (RPLC) and then detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte relative to the internal standard was used to calculate against a standard curve generated by spiking the ADC reference material in the desired matrix.

[0592] For the total antigen assay, two signature tryptic peptides on the AB1370049-SG3932 DAR8 antigen were used to calculate the concentration of total antigen in the selected matrix: heavy chain (VVSVLTVLHQDWLNGK) and light chain (DSTYSLSSTLTLSK). For the ADC assay, a papain release warhead was used to calculate the concentration. Isotopically labeled peptides or warheads were used as internal standards.

[0593] result The ratio between ADC and total antibody was approximately 1, indicating that there was no significant warhead debinding in vivo.

[0594] conclusion Under in vivo conditions, the AB1370049-SG3932 DAR8 ADC remained stable and no significant degradation was observed.

[0595] Example 14 - Pharmacokinetics of exemplary DAR8 ADCs in SCID mice Aiming To examine the pharmacokinetics of six exemplary anti-FRαDAR8 ADCs in wild-type SCID mice after a single intravenous administration.

[0596] Materials and Methods Wild-type SCID mice (n=3 per group) were intravenously (bolus) administered exemplary anti-FRα ADCs (5 mg / kg). Blood samples were collected 15 minutes, 4 hours, 1, 2, 3, 6, 10, 14, and 21 days after administration, centrifuged to obtain plasma, and analyzed for total ADC. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara).

[0597] 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 sample was then diluted in TBS and incubated with the 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 overnight enzymatic digestion with papain in the presence of an internal standard. The digested sample was quenched by the addition of acid before analysis by LC-MS / MS.

[0598] The concentration of the ADC was calculated using the papain-released warhead. The internal standard used in this experiment was an isotopically labeled warhead. Papain digests from the immunoaffinity-enhanced samples were separated using reversed-phase chromatography (RPLC) followed by detection of the released 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 generated by spiking the ADC reference material in the desired matrix.

[0599] Standard curves and QCs were prepared by spiking different levels of the target compound (ADC reference material) into the same matrix as the sample matrix. For rodent studies (FcRn and Tg32), the quantification range covered 100 ng / ml to 12,000 ng / ml, and diluted QCs covered up to 50-fold dilutions. The standard curves were fitted with linear regression using a weighting factor of 1 / x2.

[0600] result Table 21 shows the pharmacokinetic parameters of exemplary anti-FRαDAR8 ADCs in SCID mice.

[0601] [Table 24] C max = maximum observed plasma concentration AUC last = area under the plasma concentration versus time curve up to the last measurable time point CL = plasma clearance V ss = volume of distribution at steady state t 1 / 2 = elimination half-life

[0602] conclusion The exemplary anti-FRα ADCs exhibited various pharmacokinetic properties in wild-type SCID mice. The SCID mouse pharmacokinetics of all exemplary antibodies demonstrated low plasma clearance and long plasma half-lives in SCID mice.

[0603] Example 15 - Pharmacokinetics of DAR4 and DAR8 ADCs formed from AB1370049 and SG3932 in hFcRn Tg32 mice Aiming To study the pharmacokinetics of DAR4 and DAR8 ADCs formed from AB1370049 and SG3932 in hFcRn Tg32 mice after a single intravenous administration.

[0604] Materials and Methods Human FcRn Tg32 mice (n=3 per group) were intravenously (bolus) administered DAR4 or DAR8 ADCs (5 mg / kg) formed from AB1370049 and SG3932. Blood samples were collected 15 minutes, 4 hours, 1, 2, 3, 6, 10, 14, and 21 days after administration, centrifuged to obtain plasma, and analyzed for total ADC. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara).

[0605] The concentrations of AB1370049-SG3932 DAR4 or AB1370049-SG3932 DAR8 were measured using an immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μl of plasma sample was then diluted in TBS and incubated with the 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 overnight enzymatic digestion with papain in the presence of an internal standard. The digested samples were quenched by the addition of acid before analysis by LC-MS / MS.

[0606] Papain-released warheads were used to calculate the concentration of DAR4 or DAR8 ADC. The internal standard used in this experiment is an isotope-labeled warhead. Papain digests from immunoaffinity-enhanced samples were separated using reversed-phase chromatography (RPLC) followed by detection of the released warheads 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 DAR4 or DAR8 ADC reference material in the desired matrix.

[0607] Standard curves and QCs were prepared by spiking different levels of DAR4 or DAR8 ADC (ADC reference material) into the same matrix as the sample matrix. The quantification range covered 100 ng / ml to 12,000 ng / ml, and diluted QCs covered up to 50-fold dilutions. The standard curves were fitted by linear regression with a weighting factor of 1 / x2.

[0608] result Table 22 shows the pharmacokinetic parameters of DAR4 and DAR8 ADCs in hFcRn Tg32 mice.

[0609] [Table 25] C max = maximum observed plasma concentration AUC last= area under the plasma concentration versus time curve up to the last measurable time point CL = plasma clearance V ss = volume of distribution at steady state t 1 / 2 = elimination half-life

[0610] conclusion AB1370049-SG3932 DAR8 showed slightly higher clearance and a shorter half-life in hFcRn Tg32 mice than in wild-type SCID mice. hFcRn Tg32 mice are a transgenic mouse model in which mouse FcRn is knocked out and human FcRn is knocked in. Therefore, this represents an appropriate model for predicting the human pharmacokinetics of AB1370049-SG3932 DAR8. Based on the pharmacokinetics in hFcRn Tg32 mice, AB1370049-SG3932 DAR8 is likely to have low human clearance, and its half-life is consistent with other clinically used ADCs, which should allow for convenient dosing. This data is consistent with the pharmacokinetic data obtained with exemplary mAbs of the present disclosure. The DAR4 ADC showed a slightly longer half-life in hFcRn Tg32 mice, driven by slightly lower plasma clearance.

[0611] Example 16 - In vitro cell killing activity of exemplary DAR8 ADCs Aiming To evaluate the cytotoxic activity of exemplary anti-FRαDAR8 ADCs using cancer cell lines expressing different levels of FRα.

[0612] Materials and Methods Cancer cell lines with varying levels of FRα expression were obtained from the American Tissue Culture Collection (ATCC). 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 on 96-well plates. After 24 h of incubation, cells were treated with the lead ADC or the non-targeting ADC control NIP228 at concentrations ranging from 0 to 66.66 nM (0 to 10 μg / ml) for 6 days. 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 cell-free medium and subtracted from experimental values. IC50 values ​​were calculated using GraphPad Prism.

[0613] result Exemplary ADCs were tested in various cancer cell lines with different expression levels of FRα: KB (high), IGROV-1 (high to moderate), and JEG-3 (moderate). As shown in Figures 9A-9C, all exemplary ADCs had cytotoxic activity in the three tested FRα-positive cell lines. The ADC lead candidates were more potent in the high FRα-expressing cell lines KB (IC50 = 0.13-0.23 μg / ml) and JEG-3 (IC50 = 0.01-0.14 μg / ml).

[0614] conclusion All ADCs demonstrated high potency in these in vitro cell killing assays, demonstrating their potential efficacy in treating cancers that overexpress FRα. Notably, AB1370049-SG3932 DAR8 had the best cytotoxic activity across the three FRα-expressing cell lines.

[0615] Example 17 - Bystander killing of AB1370049-SG3932 DAR8 Aiming To evaluate the bystander activity of AB1370049-SG3932 DAR8 in co-culture experiments with FRα-positive and -negative KB cells.

[0616] 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 a GFP tag (KB FRα k / o GFP) was incubated in the presence of AB1370049-SG3932 DAR8. After 6 days of culture, the remaining cell population was harvested using trypsin and analyzed by flow cytometry. Viable antigen-positive and antigen-negative cells were identified, counted, and compared with untreated samples.

[0617] 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).

[0618] conclusion AB1370049-SG3932 DAR8 can induce apoptosis and ultimately cell death not only in tumors that uniformly express FRα, but also in tumors with heterogeneous expression, as shown in co-culture experiments in which 50% of the cells express FRα and 50% do not. The ADC is internalized by FRα-positive cells, releasing free warheads that kill the target cancer cells. The free warheads can also spread to neighboring cancer cells that do not express FRα and kill them as well.

[0619] Example 18 - In vivo anti-cancer activity of exemplary ADCs in a cell-derived xenograft model Aiming To evaluate the anti-tumor activity of exemplary ADCs, particularly AB1370049-SG3932 DAR8, in CDX models of various human cancers representing different target expression levels.

[0620] Materials and Methods For the KB xenograft model, 6 × 10 6Cells / mouse were subcutaneously inoculated into female CB17-SCID mice (Charles River Laboratories). Tumors were approximately 150-200 mm 3 When the IL-16 receptor agonist (IL-16) level reached 0.3125 mg / kg, mice were randomly assigned to groups. Each of the six lead DAR8 ADCs was administered intravenously as a single dose (day 7) at 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 ADC was administered intravenously 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 corresponding dose levels of the isotype control NIP228.

[0621] For the Caco-2 xenograft model, 5 × 10 cells in 50% Matrigel 6 Cells / mouse were inoculated subcutaneously into female athymic nude mice (Harlon Laboratories). Tumors grew to approximately 150 mm 3 Upon reaching 18 days, mice were randomly assigned to groups and administered intravenously as a single dose (day 17) of AB1370049-SG3932 DAR8 at 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg, the corresponding isotype control NIP228 at 1.25 mg / kg or 5 mg / kg, and the FRα-DM4 ADC ("mirvetuximab soravancencin biosimilar," DAR approximately 3) at 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg, the corresponding isotype control NIP228 at 1.25 mg / kg or 5 mg / kg.

[0622] 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 3At tumor volume ≥ 32 days, mice were randomly assigned to groups based on tumor volume. Control animals received a 100 μl intravenous dose of vehicle, while treated animals received a single intravenous dose of 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg of AB1370049-SG3932 DAR8, 1.25 mg / kg, 2.5 mg / kg, or 5 mg / kg of the corresponding isotype control NIP228, and 5 mg / kg of FRα-DM4 ADC ("mirvetuximab soravancencin biosimilar" with a DAR of approximately 3), along with 5 mg / kg of the corresponding isotype control NIP228, dosed at 4 ml / kg.

[0623] For 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, and FRα-DM4 ADC ("mirvetuximab soravancencin and biosimilar," DAR approximately 3) was administered at 1.25 mg / kg, 2.5 mg / kg, and 5 mg / kg.

[0624] Tumor volumes were measured twice weekly with calipers.

[0625] result Complete tumor regression was observed at 5 mg / kg (KB and OVCAR-3) and 1.25 mg / kg (OVCAR-3 only) for all DAR 8 ADCs tested (Figures 11-12). In IGROV-1 xenografts with high to moderate FRα expression, AB1370049-SG3932 DAR8 was active at 5 mg / kg, with partial tumor regression over 70 days before tumors began to regrow (Figure 13). Lead ADC AB1370049-SG3932 DAR8 was selected because it had more durable responses compared to other lead candidates, particularly in the KB and OVCAR-3 xenograft models.

[0626] The lead DAR4 ADC was also tested in the KB model (Figure 14). Complete tumor regression was achieved at 5 mg / kg for all of the ADCs tested, indicating that the DAR4 ADC also showed good efficacy.

[0627] AB1370049-SG3932 DAR8 was also tested against the FRα-DM4 ADC (mirvetuximab soravance, biosimilar, DAR approximately 3) in the xenograft models OVCAR-3 and CaCo-2, which have moderate to moderately low FRα expression (Figures 15A-B). AB1370049-SG3932 DAR8 was more effective at lower dose levels (1.25 and 2.5 mg / kg) than FRα-DM4 in these models, and had more durable antitumor activity.

[0628] conclusion All exemplary lead ADCs demonstrated strong antitumor activity at different doses in a variety of different xenograft models, which was similar to or better than the comparator molecule FRα-DM4.

[0629] Example 19 - In vivo anti-cancer activity of AB1370049-SG3932 DAR8 in Champions and autologous patient-derived xenograft models Aiming To evaluate the antitumor activity of AB1370049-SG3932 DAR8 in Low Passage Champions PDX and autologous PDX models of human non-small cell lung, ovarian, colorectal, and endometrial cancers representing a range of target expression levels.

[0630] 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-Foxn1nu stock mice. Enough stock animals were 1000-1500 mm 3 When tumors reached 150-300 mm, tumors were harvested and fragments were transplanted into pre-test animals. 3When tumors reached a mean tumor volume of 1000 mg / kg, 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 a single dose intravenously on day 0.

[0631] The animals were observed daily, and tumor size and body weight were measured and recorded twice a week. Tumor volume was measured using a digital caliper, and tumor volume was 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.

[0632] The study endpoint for all models was a mean tumor volume of 1200mm in the control group. 3 The tumor volume was increased to 1200 mm before day 28, or if the maximum tumor volume was not reached, it was increased to a maximum of 60 days. 3 Treatment groups were measured until day 28 when tumor volumes of approximately 400-600 mm were obtained. After randomization of animals for testing, tumor samples were collected using three animals per model. 3 and half of the tumors were processed for immunohistochemical analysis and the other half for genomic analysis.

[0633] Homemade model Autologous in vivo patient-derived xenograft (PDX) efficacy studies were conducted using different tumor models, including ovarian, colon, endometrial, and non-small cell lung cancer. Tumor fragments from these PDX models were subcutaneously implanted into female NOD-SCID mice (Envigo) using a trocar. Tumors were approximately 150–200 mm 3When the mice reached 100 mg / kg, they were randomly assigned to groups. AB1370049-SG3932 DAR8 was administered intravenously as a single dose at 2.5 mg / kg or 5 mg / kg, along with the isotype control NIP228 at the corresponding dose level. Tumor volume was 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 treatment tolerance.

[0634] Determination of antitumor response The response in terms of final tumor volume (FTV) from the initial tumor volume (ITV) was calculated as the time point that resulted in the greatest reduction from the initial tumor volume. The anti-tumor response was calculated for each tumor-bearing mouse using the following formula: Tumor growth (%) = [(FTV-ITV) / ITV] × 100 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.

[0635] Each treatment group was determined to have a response or no response to the study drug. Response criteria were based on RECIST 1.1, with a response being based on a 30% reduction in tumor volume from baseline tumor measurement.

[0636] result The median percent tumor growth resulting from a single dose of 5.0 mg / kg AB1370049-SG3932, DAR8, or NIP228-SG3932 in 51 PDX models is summarized in Table 23 and Figure 16A. 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 resulted in a 30% or greater reduction in tumor volume from baseline in 22% (11 of 49) of models tested. In the ovarian cancer PDX models tested, AB1370049-SG3932 DAR8 at 5.0 mg / kg resulted in a 30% or greater reduction in tumor volume from baseline in 78.3% (18 of 23; 95% CI 58%-91%) of models tested. NIP228-SG3932 at 5.0 mg / kg resulted in a 30% or greater reduction in tumor volume from baseline in 43.5% (10 of 23; 95% CI 26%-63%) of models tested.

[0637] [Table 26-1]

[0638] [Table 26-2]

[0639] The median percent tumor growth resulting from a single administration of 2.5 mg / kg of AB1370049-SG3932, DAR8, or NIP228-SG3932 in 39 PDX models is summarized in Table 24 and Figure 16B. Specific tumor growth inhibition was observed in 49% (19 of 39) of the PDX models tested after a single intravenous administration of 2.5 mg / kg of AB1370049-SG3932 DAR8, while 51% (20 of 39) of the models did not respond. Only two of the 37 models tested responded to the same dose of the nontargeted ADC NIP228-SG3932 DAR8. The activity of the nontargeted ADC is dose-dependent, as fewer models responded to NIP228-SG3932 DAR8 (5%) at lower dose concentrations.

[0640] [Table 27-1]

[0641] [Table 27-2]

[0642] AB1370049-SG3932 DAR8 demonstrated antitumor responses in several 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 cancer).

[0643] In ovarian cancer, the objective response rate was 80%, 30% in NSCLC, 20% in CRC, and 50% in endometrium. For AB1370049-SG3932 DAR8, there was a positive correlation between FRα expression and activity.

[0644] conclusion AB1370049-SG3932 DAR8 is highly effective in tumors with high, medium, and medium-low levels of FRα expression. In general, there is a positive correlation between the antitumor activity of AB1370049-SG3932 DAR8 and the expression level of FRα.

[0645] Example 20 - In vitro safety studies with exemplary ADCs Aiming 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.

[0646] 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 25 ng / ml SCF, 50 ng / ml TPO, and 50 ng / ml Flt3-L human recombinant protein (all from Peprotech)) in a humidified incubator at 37°C with 5% CO. The next day, cells were resuspended in Cell Expand medium, which can support erythroid (Preferred Cell Systems, SEC-BFU1-40H), myeloid (Preferred Cell Systems, SEC-GM1-40H), or megakaryocytic differentiation (Stem Cell Technologies, 09707) in the presence of drugs, at a concentration of 5,000 cells / ml for erythroid and myeloid cells or 15,000 cells / ml for megakaryocytic cells.

[0647] Cells (100 μl) were seeded 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.

[0648] The effect of exemplary ADCs on proliferating and differentiated cells was further evaluated. To do so, cells were seeded at a concentration of 5000 cells / ml for 5 days for erythroid differentiation, followed by seeding into 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 of exemplary ADCs (e.g., AB1370049-SG3932 DAR8), non-FRα-targeting NIP228 control ADC, or non-binding mAb (NIP228 and AB1370049, respectively). The cells were then seeded into triplicate white-walled, clear-bottom 96-well tissue culture plates for 5 days in a humidified incubator at 37°C with 5% CO2. For myeloid or megakaryocytic differentiation, cells were seeded at 10,000 and 15,000 cells / ml, respectively, for 5 days, then spun down and seeded in fresh medium at 10,000 and 30,000 cells / ml, respectively, for another 5 days, and then plated in myeloid differentiation medium or megakaryocytic at 20,000 and 30,000 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).

[0649] Viability was determined using CellTiter-Glo 2.0 (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 in GraphPad software (Prism), with control set to 100 and maximum cell death set to 0.

[0650] result 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 the 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 assess the toxicity induced by AB1370049-SG3932 DAR8 and its related non-FRα-targeting ADC control to assess any exacerbated toxicity of AB1370049-SG3932 DAR8.

[0651] AB1370049-SG3932 DAR8 exhibits similar toxicity levels to non-FRα-targeted ADC molecules with the same payload (Figures 18A-18F). This observation is consistent with the results of primary CD34 cells differentiated into any lineage, regardless of the level of differentiation. + This is seen in bone marrow cells. Similar results were observed with other exemplary ADCs of the present disclosure, including mAbs AB1370095, AB1370026, and AB1370117, when conjugated to the same TOPOi payload. When administered under culture conditions similar to those described for AB1370049-SG3932 DAR8, the 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 exhibited a similar level of toxicity to HSPCs treated with the non-FRα-targeted ADC DAR4.

[0652] conclusion The results suggest that there is no target-mediated toxicity or exacerbated toxicity driven by the lead antibody compared to non-FRα-targeted ADC molecules.

[0653] Example 21 - In vivo pharmacokinetic study using AB1370049 and AB1370049-SG3932 DAR8 Aiming Plasma pharmacokinetic (PK) analysis of AB1370049-SG3932 DAR8 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 for the lead ADC candidate and the non-conjugated antibody. Non-compartmental analysis was performed to estimate PK parameters.

[0654] Materials and Methods Administration of AB1370049 and AB1370049-SG3932 DAR8 to cynomolgus monkeys Male cynomolgus monkeys (N=3) were administered two single doses (3 weeks apart, i.e., on days 1 and 22) of 15 mg / kg AB1370049 or 15 and 25 mg / kg AB1370049-SG3932 DAR8 intravenously.

[0655] Blood samples were collected by venipuncture pre-dose, and 0.04, 0.25, 1, 3, 7, 14, and 21 days post-dose on days 1 and 22, centrifuged to obtain plasma, which was analyzed for total antibody, total ADC, and unbound payload. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara).

[0656] Whole 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 sample was 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. The digestion was quenched by the addition of acid. An aliquot of the trypsin-digested liquid contents was then transferred to an injection plate for total Ab analysis.

[0657] For the ADC assay, an aliquot of the trypsin-digested supernatant was subjected to overnight enzymatic digestion with papain in the presence of an internal standard. The digested samples were quenched by the addition of acid and then analyzed by LC-MS / MS. Concentrations were calculated using the papain-released warhead. The internal standard used in this experiment is an isotope-labeled warhead. Papain digests from the immunoaffinity-enhanced samples were separated using reversed-phase chromatography (RPLC) and subsequently detected using multiple reaction monitoring (MRM) for the released warhead. The peak area ratio of the analyte to the internal standard was used to calculate the concentration against a standard curve generated by spiking the ADC reference material in the desired matrix.

[0658] For the total Ab assay, a signature tryptic peptide on the human antibody Fc region (VVSVLTVLHQDWLNGK) was used to calculate the concentration of total antibody in the selected matrix. Tryptic digests from immunoaffinity-enriched samples were separated using reverse-phase chromatography (RPLC) and subsequently detected using multiple reaction monitoring (MRM) for the signature peptide. 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 generated by spiking the ADC reference material in the desired matrix.

[0659] 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 testing, the quantification range was 100-15,000 ng / mL with a dilution factor of up to 100x. The standard curve was fitted by linear regression using a 1 / x2 weighting. The accuracy and precision of the assay were within 20% for all levels except the LLOQ (25%).

[0660] Unbound warhead assay The unbound warhead assay was performed by precipitation. The internal standard used in this experiment was an isotopically 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 and reconstituted in an appropriate buffer for injection into the LCMS. The samples were then separated using reversed-phase chromatography (RPLC) followed by detection of the unbound 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 generated by spiking the ADC reference material in the desired matrix. 【066...

Claims

1. 1. A method of treating cancer in a human subject in need thereof, comprising administering to said human subject: (a) an antibody-drug conjugate (ADC) comprising an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin; and (b) a method comprising administering a PARP1 inhibitor.

2. 2. The method of claim 1, 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.

3. 3. The method of claim 1 or claim 2, wherein the cancer is selected from ovarian cancer, lung cancer (e.g., lung adenocarcinoma), 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, optionally wherein the cancer is selected from ovarian cancer and lung cancer.

4. 4. The method of claim 3, 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.

5. A kit comprising: (a) an ADC comprising an anti-FRα antibody or antigen-binding fragment thereof linked to a cytotoxin; and (b) A kit comprising a PARP1 inhibitor.

6. The PARP1 inhibitor has the following formula: 【Chemistry 1】 AZD5305 or a pharmaceutically acceptable salt thereof.

7. the anti-FRα antibody or antigen-binding fragment thereof, (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, 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, 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, light chain CDR3 (QEYKTYSIFT) of SEQ ID NO: 18; (d) heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, heavy chain CDR2 (SISSSGSSYIYYADSMKG) 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, 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, a light chain CDR3 (QQYNSYPLT) of SEQ ID NO: 30, or (f) The method of any one of claims 1 to 4 or 6, or the kit of claim 5 or 6, comprising 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.

8. The anti-FRα antibody or antigen-binding fragment thereof is (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.

9. the anti-FRα antibody or antigen-binding fragment thereof, (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 9. The method of claim 8, or the kit of claim 8, comprising (c) a Q at the N-terminus (e.g., position 1) of the VH.

10. The anti-FRα antibody or antigen-binding fragment thereof is (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) VH of SEQ ID NO: 41 and VL of SEQ ID NO: 42; (d) VH of SEQ ID NO: 43 and VL of SEQ ID NO: 44; (e) a VH of SEQ ID NO: 45 and a VL of SEQ ID NO: 46; or (f) the method of any one of claims 1 to 4 or 6 to 9, or the kit of any one of claims 5 to 9, comprising a VH of SEQ ID NO: 47 and a VL of SEQ ID NO:

48.

11. The method of any one of claims 1 to 4 or 6 to 10, or the kit of any one of claims 5 to 10, 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.

12. 12. The method of any one of claims 1 to 4 or 6 to 11, or the kit of any one of claims 5 to 11, wherein 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.

13. The anti-FRα antibody (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.

14. The anti-FRα antibody (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 method of any one of claims 1 to 4 or 6 to 13, or the kit of any one of claims 5 to 13, comprising a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO:

60.

15. The method of any one of claims 1 to 4 or 6 to 12, or the kit of any one of claims 5 to 12, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

16. The method of any one of claims 1 to 4 or 6 to 15, or the kit of any one of claims 5 to 15, wherein the anti-FRα antibody or antigen-binding fragment thereof is a fully human antibody.

17. The method of any one of claims 1 to 4 or 6 to 16, or the kit of any one of claims 5 to 16, wherein the anti-FRα antibody or antigen-binding fragment thereof is monoclonal, polyclonal, recombinant, or multispecific.

18. The method of any one of claims 1 to 4 or 6 to 17, or the kit of any one of claims 5 to 17, wherein the anti-FRα antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 type.

19. The method according to claim 18 or the kit according to claim 18, wherein the anti-FRα antibody or antigen-binding fragment thereof is of the IgG1 type.

20. The cytotoxin is 【Chemistry 2】 (Wherein Q is 【Transformation 3】 and Q Ｘ is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue; X is 【Chemistry 4】 and a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and 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), G Ｌ is a linker for connecting the anti-FRα antibody or antigen-binding fragment thereof, and 【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, and e is 0 or 1; or 【Transformation 6】 (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; Ｌ The method of any one of claims 1 to 4 or 6 to 19, or the kit of any one of claims 5 to 19, wherein the anti-FRα antibody or antigen-binding fragment thereof is linked to the anti-FRα antibody or antigen-binding fragment thereof via

21. G Ｌ but 【Transformation 7】 21. The method of claim 20, or the kit of claim 20, wherein

22. R Ｌ but 【Transformation 8】 22. The method of claim 20 or claim 21, or the kit of claim 20 or claim 21, wherein

23. 23. The method of any one of claims 1 to 4 or 6 to 22, or the kit of any one of claims 5 to 22, wherein the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof.

24. 24. The method of claim 23 or the kit of claim 23, wherein the cytotoxin is a topoisomerase I inhibitor.

25. 4. The method of claim 1, wherein the topoisomerase I inhibitor is 【Chemistry 9】 and salts and solvates thereof, In the formula, R Ｌ is defined according to any one of claims 20 to 22, or the method according to claim 23 or 24, or the kit according to claim 23 or 24.

26. The topoisomerase I inhibitor is 【Chemistry 10】 The method according to any one of claims 23 to 25, or the kit according to any one of claims 23 to 25,

27. The topoisomerase I inhibitor is 【Chemistry 11】 27. The method of claim 26 or the kit of claim 26, wherein

28. 28. The method of any one of claims 1 to 4 or 6 to 27, or the kit of any one of claims 5 to 27, wherein the drug-to-antibody ratio (DAR) of the ADC is in the range of about 1 to 20, optionally wherein 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.

29. 29. The method of claim 28 or the kit of claim 28, wherein the DAR is about 8 or about 4.

30. 30. The method of claim 29; or the kit of claim 29, wherein the DAR is about 8.

31. (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; and optionally, 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 12】 and (iii) The method of any one of claims 1 to 4 or 6 to 30, or the kit of any one of claims 5 to 30, wherein the DAR of the ADC is about 8.

32. (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; and optionally, 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 13】 and (iii) the DAR of the ADC is about 8; and 【Chemistry 14】 or a pharmaceutically acceptable salt thereof.

33. 33. The method of any one of claims 1 to 4 or 6 to 32, wherein the ADC and the PARP1 inhibitor are administered separately or sequentially.

34. 33. The method of any one of claims 1 to 4 or 6 to 32, wherein the ADC and the PARP1 inhibitor are administered together.

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