TW202448517A - Combination therapies for treatment of cancer with therapeutic binding molecules - Google Patents

Abstract

Provided herein are methods of treating cancer comprising combination therapies of (i) an ADC comprising an anti-FR[alpha] antibody, and (ii) a PARP1 inhibitor, and kits comprising the same.

Description

Combination therapy for cancer using therapeutic binding molecules

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 an antigen-binding fragment thereof linked to a cytotoxin; and (b) a PARP1 inhibitor. The present disclosure also relates to a kit comprising (a) an ADC comprising an anti-FRα antibody or an antigen-binding fragment thereof linked to a cytotoxin; and (b) a PARP1 inhibitor.

Despite years of research into the mechanisms of cancer and the development of many potential anti-cancer drugs, cancer remains one of the leading diseases worldwide. In particular, lung cancer and ovarian cancer rank third and fifth, respectively, among the most common cancers in women. Chemotherapy and radiation are the most common cancer treatments. However, these therapies are associated with a variety of negative side effects, including fatigue, nausea, and hair loss. These issues are compounded by the fact that chemotherapy treatments require frequent administration over long periods of time. Over the past few decades, a variety of antibody therapies have been developed and marketed against cancer, resulting in a reduction in the need for conventional chemotherapy forms for many cancer types. Although the availability of methods to produce antibodies (e.g., monoclonal antibodies) has greatly improved during this period, relatively few anticancer antibodies are clinically available, and even fewer antibodies are available to target multiple cancer types. In addition, there is a need to increase the potency of therapeutic antibodies, which is often limited by the ubiquity of target antigen expression and the subsequent effects of the antibody on cancer cells after binding. Conjugating monoclonal antibodies to cytotoxic molecules to generate antibody-drug conjugates (ADCs) is a new approach to deliver targeted therapies to malignant tumors. This approach has been successfully implemented against specific targets (HER2, CD30, and CD79b), resulting in the launch of ADCs such as fam-trastuzumab deruxtecan-nxki (breast and gastric cancer), brentuximab vedotin (Hodgkin’s lymphoma), and polatuzumab vedotin-piiq (non-Hodgkin’s lymphoma).

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

FRα is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that has a high affinity for the active form of folate, 5-methyltetrahydrofolate ( 5-MTF). FRα is expressed by the FOLR1 gene. Previous studies have shown that FRα plays a crucial role in embryogenesis (Kelemen, Int J Cancer. 2006;119(2):243-250). However, folate transport in adults is mainly driven by the ubiquitous expression of the reduced folate mediator and the proton-coupled folate transporter (Zhao et al. , Annu Rev Nutr . 2011;31:177-201). In adults, FRα expression is usually restricted to the apical surface of polarized epithelial cells, such as the vascular plexus, kidney, lung, and placenta.

Overexpression of FRα, also known as folate receptor 1 (FOLR1) or folate binding protein (FBP), is often observed in tumor cells such as ovarian cancer, lung cancer ( e.g., non-small cell lung cancer (NSCLC)), and breast cancer (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 an early ovarian cancer biomarker (Basal et al., PLoS One. 2009;4(7):e6292). Preclinical ovarian models also revealed that overexpression of FRα is associated with tumor progression and that folate binding to FRα can mediate activation of the oncogene STAT3 (Hansen et al., Cell Signal. 2015; 27(7):1356-1368).

Prior art antibodies against FRα have defects such as poor internalization, short half-life, and insufficient cytotoxicity. In addition, ADCs targeting FRα in the field use microtubule inhibitors, which have been associated with specific toxicities in clinical trials such as corneal inflammation (mirvetuximab soravtansine, which consists of the anti-FRα antibody M9346A fused to the maytansinoid warhead DM4 via a sulfo-SPBD linker) (Moore et al. (2017) Cancer 123:3080-7), interstitial lung disease (MORAb-202, which consists of the LK26-derived humanized antibody farletuzumab fused to the eribulin warhead) (Sato et al. (2020) ESMO abstract https://doi.org/10.1016/j.annonc.2020.01.026), and neuropathy and neutropenia (STRO-002, which consists of the anti-FRα antibody SP8166 conjugated to a 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 found that cancer cells may acquire resistance to microtubule inhibitors (Ganguly et al ., Biochim Biophys Acta. 2011 Dec ;1816(2):164–171).

There is a need to develop new cancer therapies based on ADCs targeting FRα.

The present disclosure provides, inter alia, methods for treating cancer comprising combination therapy comprising an ADC comprising an anti-FRα antibody and a PARP1 inhibitor, and kits comprising the same.

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

In some embodiments of any aspect of the disclosure, the cancer comprises cancer cells having heterogeneous expression of FRα and/or low expression of FRα; optionally, wherein the cancer cells have similar expression of FRα to the Igrov-1 cell line.

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 specific embodiments, the cancer can be selected from ovarian cancer and lung cancer.

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

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

In some embodiments of any aspect of the present disclosure, the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.

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

In some embodiments of any aspect of the present disclosure, the anti-FRα antibody or antigen-binding fragment thereof comprises: (a) a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 37 and a VL comprising an amino acid sequence 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 at least 90% identical to the amino acid sequence of SEQ ID NO: 45 and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 46; or (f) a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 47 and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 48.

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

In some embodiments of any aspect of the present disclosure, the anti-FRα antibody or antigen-binding fragment thereof comprises: (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) VH of SEQ ID NO: 45 and VL of SEQ ID NO: 46; or (f) VH of SEQ ID NO: 47 and VL of SEQ ID NO: 48.

In some embodiments of any aspect of the disclosure, the anti-FRα antibody contains 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.

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

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

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

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

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

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

In some embodiments of any aspect of the present disclosure, the anti-FRα antibody or antigen-binding fragment thereof is of IgG1, IgG2, IgG3, or IgG4 type. In a specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof is of IgG1 type.

In some embodiments of any aspect of the present disclosure, the cytotoxin is linked to the anti-FRα antibody or antigen-binding fragment thereof via a linker ^RL selected from the group consisting of: (Ia): , where Q is: , wherein Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; and X is: , wherein a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, wherein 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); ^GL is a linker for linking an anti-FRα antibody or an antigen-binding fragment thereof; (Ib): , wherein R ^L1 and R ^L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group; and e is 0 or 1; or (Ib'): wherein R ^L1 and R ^L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group.

In some implementations of any aspect of the present disclosure, ^GL is .

In some implementations of any aspect of the present disclosure, R ^L is .

In some embodiments of any aspect of the present disclosure, the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof. In a specific embodiment, the cytotoxin is a topoisomerase I inhibitor.

In some embodiments of any aspect of the present disclosure, the topoisomerase I inhibitor is represented by formula (I): and its salts and solvates; wherein ^RL is as defined above.

In some embodiments of any aspect of the present disclosure, the topoisomerase I inhibitor is: ; ; ; and/or .

In certain embodiments, the topoisomerase I inhibitor is: .

In some embodiments of any aspect of the present disclosure, the drug to antibody ratio (DAR) ranges from about 1 to 20, optionally wherein the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10.

In some embodiments of any aspect of the present disclosure, the DAR is about 8 or about 4. In a specific embodiment, the DAR is about 8.

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, optionally wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) the cytotoxin is the topoisomerase I inhibitor SG3932 ; and (iii) the DAR is approximately 8.

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

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

In another aspect, a method for treating cancer in a human subject in need thereof is provided, the method comprising administering to the human subject: (a) an antibody-drug conjugate (ADC) comprising an anti-FRα antibody or an antigen-binding fragment thereof linked to a cytotoxin, wherein: (i) the anti-FRα antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, a light chain CDR2 (KASGLES) of SEQ ID NO: 5, and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, optionally wherein the anti-FRα antibody or the antigen-binding fragment thereof has 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 NO: 37 VH and SEQ ID NO: 38 VL; (ii) the cytotoxin is topoisomerase I inhibitor SG3932 ; and (iii) the DAR is about 8; and (b) a PARP1 inhibitor, wherein the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.

In another aspect, a kit is provided, comprising: (a) an antibody-drug conjugate (ADC) comprising an anti-FRα antibody or an antigen-binding fragment thereof linked to a cytotoxin, wherein: (i) the anti-FRα antibody or an antigen-binding fragment thereof comprises a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, a light chain CDR2 (KASGLES) of SEQ ID NO: 5, and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, optionally wherein the anti-FRα antibody or an antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VH of SEQ ID NO: 38 VL; (ii) the cytotoxin is the topoisomerase I inhibitor SG3932 ; and (iii) the DAR is about 8; and (b) a PARP1 inhibitor, wherein the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof.

Various aspects and implementations of the present disclosure are set forth in the appended claims. These and other aspects and implementations of the present disclosure are also described herein.

Cross-references to related patent applications

This application claims the benefit of priority to U.S. Provisional Application No. 63/485,366, filed on February 16, 2023; U.S. Provisional Application No. 63/578,233, filed on August 23, 2023; and U.S. Provisional Application No. 63/619,055, filed on January 9, 2024. The above-referenced applications are incorporated herein by reference in their entirety for all purposes. General Definitions

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 the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ed., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in the present disclosure.

Unless otherwise indicated, any nucleic acid sequence is written from left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxyl orientation, respectively.

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

"About" can generally mean an acceptable degree of error in a measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value or range of values. In specific embodiments, the term "about" should be understood herein as plus or minus (±) 5%, ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1% of the numerical value of the digit used. Embodiments described herein as "comprising" one or more features may also be considered disclosures of corresponding embodiments "consisting of such features."

The name, three-letter abbreviation or single-letter abbreviation of amino acids are used herein to refer to amino acids. 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 cases, the term "amino acid sequence" is synonymous with the term "peptide". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and the scope of the patent application, conventional one-letter and three-letter codes for amino acid residues can be used. The 3-letter code for amino acids follows the definition of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

Concentrations, amounts, volumes, percentages and other numerical values may be presented in a range format. It should also be understood that such range format is used for convenience and brevity only and should be flexibly interpreted to include not only the numerical values explicitly listed as limits of the range but also all individual numerical values or sub-ranges contained within the range, as if each numerical value and sub-range were explicitly listed. Anti-FRα Antibody

The inventors developed a series of exemplary anti-FRα antibodies that have high affinity and specifically bind to FRα on cancer cells (e.g., do not specifically bind to other FR family members, such as FRβ and FRγ). The inventors conducted a comprehensive evaluation of the developability of the antibodies, examining the propensity for reversible self-association, internalization, non-specific binding and hydrophobicity, as well as the stability of the mAbs to heat and light stressors. In addition, the inventors used in vivo PK studies in mice for a panel of key mAbs to eliminate any antibodies that exhibited poor in vivo half-life and increased clearance. To reduce the potential for generating anti-drug antibody (ADA) responses in humans, an in silico immunological assessment was performed to exclude from consideration any antibodies with an expected risk of increasing ADA responses. Through the above screening strategy, the inventors identified a group of 6 antibodies with similar, beneficial properties. In some embodiments, the antibodies are included in the ADC described in the present disclosure. Antibody Sequence

The ADC disclosed herein encompasses antibodies or antigen-binding fragments (reference antibodies) as defined herein having the CDR sequences or variable heavy and variable light chain sequences and functional variants thereof. Functional variants bind to the same target antigen as the reference antibody and may exhibit the same antigen cross-reactivity as the reference antibody. When compared to the reference antibody, the functional variants may have different affinities for the target antigen, or may have substantially the same affinity.

In some embodiments, the functional variant of the reference antibody shows sequence variation at one or more CDRs when compared to the corresponding reference CDR sequence. Therefore, the functional antibody variant may comprise a functional variant of the CDR. When the term "functional variant" is used in the context of a CDR sequence, this means that the CDR has at most 2 or at most 1 amino acid difference when compared to the corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof), the variant antibody can bind to the same target antigen as the reference antibody, and in a specific embodiment, exhibits the same antigen cross-reactivity as the reference antibody. Functional variants may be referred to as "variant antibodies".

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. If there are any differences, the sequences in the table shall prevail.

In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises 6 CDRs of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 of Table 2 , wherein the CDRs are determined by Kabat, Chothia, or IMGT.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, light chain CDR2 (KASGLES) of SEQ ID NO: 5, and light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, wherein any one or more of the CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, light chain CDR2 (KASGLES) of SEQ ID NO: 5, and light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6.

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.

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: a heavy chain amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence of SEQ ID NO: 50.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SYAMS) of SEQ ID NO: 7, heavy chain CDR2 (SISSGRSYIYYADSVKG) of SEQ ID NO: 8, heavy chain CDR3 (EMQQLALDY) of SEQ ID NO: 9, light chain CDR1 (RASQGISNFLA) of SEQ ID NO: 10, light chain CDR2 (AASSLQS) of SEQ ID NO: 11, and light chain CDR3 (QQYNSYPFT) of SEQ ID NO: 12, wherein any one or more of the CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

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

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.

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: a heavy chain amino acid sequence of SEQ ID NO: 51 and a light chain amino acid sequence of SEQ ID NO: 52.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SNSAAWN) of SEQ ID NO: 13, heavy chain CDR2 (RTYYRSNWYNDYTLSVKS) of SEQ ID NO: 14, heavy chain CDR3 (GVGRFDS) of SEQ ID NO: 15, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 16, light chain CDR2 (KASSLES) of SEQ ID NO: 17, and light chain CDR3 (QEYKTYSIFT) of SEQ ID NO: 18, wherein any one or more of the CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SNSAAWN) of SEQ ID NO: 13, heavy chain CDR2 (RTYYRSNWYNDYTLSVKS) of SEQ ID NO: 14, heavy chain CDR3 (GVGRFDS) of SEQ ID NO: 15, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 16, light chain CDR2 (KASSLES) of SEQ ID NO: 17, and light chain CDR3 (QEYKTYSIFT) of SEQ ID NO: 18.

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.

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: a heavy chain amino acid sequence of SEQ ID NO: 53 and a light chain amino acid sequence of SEQ ID NO: 54.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, heavy chain CDR2 (SISSGSSYIYYADSMKG) of SEQ ID NO: 20, heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 21, light chain CDR1 (RASQGISTFLA) of SEQ ID NO: 22, light chain CDR2 (AASSLQS) of SEQ ID NO: 23, and light chain CDR3 (QQYISYPLT) of SEQ ID NO: 24, wherein any one or more of the CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SYNMN) of SEQ ID NO: 19, heavy chain CDR2 (SISSGSSYIYYADSMKG) of SEQ ID NO: 20, heavy chain CDR3 (GMTTLTFDY) of SEQ ID NO: 21, light chain CDR1 (RASQGISTFLA) of SEQ ID NO: 22, light chain CDR2 (AASSLQS) of SEQ ID NO: 23, and light chain CDR3 (QQYISYPLT) of SEQ ID NO: 24.

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.

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: a heavy chain amino acid sequence of SEQ ID NO: 55 and a light chain amino acid sequence of SEQ ID NO: 56.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SYSMN) of SEQ ID NO: 25, heavy chain CDR2 (SISSRSSYVYYADSVKG) of SEQ ID NO: 26, 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 light chain CDR3 (QQYNSYPLT) of SEQ ID NO: 30, wherein any one or more of the CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SYSMN) of SEQ ID NO: 25, heavy chain CDR2 (SISSRSSYVYYADSVKG) of SEQ ID NO: 26, 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 light chain CDR3 (QQYNSYPLT) of SEQ ID NO: 30.

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.

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: a heavy chain amino acid sequence of SEQ ID NO: 57 and a light chain amino acid sequence of SEQ ID NO: 58.

In some embodiments, the anti-FRα antibody or antigen-binding fragment comprises: heavy chain CDR1 (SDSATWN) of SEQ ID NO: 31, heavy chain CDR2 (RTYYRSKWYSDYAVSVKS) of SEQ ID NO: 32, heavy chain CDR3 (GGAPFDY) of SEQ ID NO: 33, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 34, light chain CDR2 (KASSLES) of SEQ ID NO: 35, and light chain CDR3 (QQYNSYSMYT) of SEQ ID NO: 36, wherein any one or more of the CDRs comprises 1, 2 or 3 conservative amino acid substitutions compared to the sequence.

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

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.

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: a heavy chain amino acid sequence of SEQ ID NO: 59 and a light chain amino acid sequence of SEQ ID NO: 60.

In some embodiments of any aspect 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 a reference heavy chain VH FR1, VH FR2, VH FR3 and/or VH FR4 of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117 described in Table 4, respectively, wherein the antibody or fragment is capable of binding to FRα alone (e.g., as a single-chain antibody fragment).

In some embodiments of any aspect 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 of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 described in Table 4, respectively, wherein the antibody or fragment is capable of binding to FRα alone (e.g., as a single-chain antibody fragment).

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

In some embodiments, 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 an 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 an amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-FRα antibody comprises: a constant heavy chain amino acid sequence of SEQ ID NO: 109 and a constant light chain amino acid sequence of SEQ ID NO: 110.

In some embodiments of any antigen-binding fragment described herein, the anti-FRα antigen-binding fragment comprises a constant heavy chain and a constant light chain, wherein the constant heavy chain comprises 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 the constant light chain comprises 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: a constant heavy chain amino acid sequence of SEQ ID NO: 111 and a constant light chain amino acid sequence of SEQ ID NO: 110. [ Table 1 ]: FRα Antibody CDR Sequences Construct VH-CDR1 VH-CDR2 VH-CDR3 VL-CDR1 VL-CDR2 VL-CDR3 AB1370049 SDSATWN (SEQ ID NO: 1) RTYYRSKWYNDYAVSVKS (SEQ ID NO: 2) GVGSFDY (SEQ ID NO: 3) RASQSISSWLA (SEQ ID NO: 4) KASGLES (SEQ ID NO: 5) QQYNSYSQLT (SEQ ID NO: 6) AB1370026 SYAMS (SEQ ID NO: 7) SISSGRSYIYYADSVKG (SEQ ID NO: 8) EMQQLALDY (SEQ ID NO: 9) RASQGISNFLA (SEQ ID NO: 10) AASSLQS (SEQ ID NO: 11) QQYNSYPFT (SEQ ID NO: 12) AB1370035 SNSAAWN (SEQ ID NO: 13) RTYYRSNWYNDYTLSVKS (SEQ ID NO: 14) GVGRFDS (SEQ ID NO: 15) RASQSISSWLA (SEQ ID NO: 16) KASSLES (SEQ ID NO: 17) QEYKTYSIFT (SEQ ID NO: 18) AB1370083 SYNMN (SEQ ID NO: 19) SISSGSSYIYYADSMKG (SEQ ID NO: 20) GMTTLTFDY (SEQ ID NO: 21) RASQGISTFLA (SEQ ID NO: 22) AASSLQS (SEQ ID NO: 23) QQYISYPLT (SEQ ID NO: 24) AB1370095 SYSMN (SEQ ID NO: 25) SISSRSSYVYYADSVKG (SEQ ID NO: 26) GMTTLTFDY (SEQ ID NO: 27) RASQGISSFLA (SEQ ID NO: 28) AASSLQS (SEQ ID NO: 29) QQYNSYPLT (SEQ ID NO: 30) AB1370117 SDSATWN (SEQ ID NO: 31) RTYYRSKWYSDYAVSVKS (SEQ ID NO: 32) GGAPFDY (SEQ ID NO: 33) RASQSISSWLA (SEQ ID NO: 34) KASSLES (SEQ ID NO: 35) QQYNSYSMYT (SEQ ID NO: 36) [ Table 2 ] : FRα antibody VH and VL sequences Construct VH V L AB1370049 LVQLQQSGPGLVKPSQTLSLTCAISGDSVSSDSATWNWIRQSPSRGLEWLGRTYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGVGSFDYWGQGTLVTVSS (SEQ ID NO: 37) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASGLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSQLTFGGGTKVEIK (SEQ ID NO: 38) AB1370026 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSGRSYIYYADSVKGRFTISSRDNAKNSLYLKMNSLRDEDTAVYYCAREMQQLALDYWGQGTLVTVSS (SEQ ID NO: 39) DIQMTQSPSSSLSASVGDRVTITCRASQGISNFLAWFQQAPGKAPKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPFTFGQGTKLEIK (SEQ ID NO: 40) AB1370035 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSNWYNDYTLSVKSRITVNPDTSKNQFSLQLNSVTPEDTAVYYCVRGVGRFDSWGQGTLVTVSS (SEQ ID NO: 41) DIQMTQSPSTLSASSVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTITSLQPDDFASYYCQEYKTYSIFTFGGPGTKVDIK (SEQ ID NO: 42) AB1370083 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSSISSGSSYIYYADSMKGRFTISSRDNAKNSLFLQMNSLRAEDTAVYYCARGMTTLTFDYWGQGTLVTVSS (SEQ ID NO: 43) DIQMTQSPSSSLSASVGDRVTITCRASQGISTFLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGSETDFTLTISSLQPEDFATYYCQQYISYPLTFGGGTKVEIK (SEQ ID NO: 44) AB1370095 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSRSSYVYYADSVKGRFTISSRDNAKNSLYLQMNSLRAEDTAVYYCARGMTTLTFDYWGQGTLVTVSS (SEQ ID NO: 45) DIQMTQSPSSSLSASVGDRVTITCRASQGISSFLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK (SEQ ID NO: 46) AB1370117 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSDSATWNWIRQSPSRGLEWLGRTYYRSKWYSDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYFCARGGAPFDYWGQGTLVTVSS (SEQ ID NO: 47) DIQMTQSPSTLSASSVGDRVTINCRASQSISSWLAWYQQKPGKAPNLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSMYTFGQGTKLEIK (SEQ ID NO: 48) [ Table 3 ] : FRα antibody heavy chain and light chain sequences Construct VH-CH VL-CL AB1370049 LVQLQQSGPGLVKPSQTLSLTCAISGDSVSSDSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGVGSFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 49) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASGLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSQLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50) AB1370026 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSGRSYIYYADSVKGRFTISRDNAKNSLYLKMNSLRDEDTAVYYCAREMQQLALDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51) DIQMTQSPSSSLSASVGDRVTITCRASQGISNFLAWFQQAPGKAPKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 52) AB1370035 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSNWYNDYTLSVKSRITVNPDTSKNQFSLQLNSVTPEDTAVYYCVRGVGRFDSWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) DIQMTQSPSTLSASSVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTITSLQPDDFASYYCQEYKTYSIFTFGPGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 54) AB1370083 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSSISSSGSSYIYYADSMKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARGMTTLTFDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55) DIQMTQSPSSSLSASVGDRVTITCRASQGISTFLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGSETDFTLTISSLQPEDFATYYCQQYISYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56) AB1370095 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSRSSYVYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGMTTLTFDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 57) DIQMTQSPSSSLSASVGDRVTITCRASQGISSFLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58) AB1370117 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSDSATWNWIRQSPSRGLEWLGRTYYRSKWYSDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYFCARGGAPFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 59) DIQMTQSPSTLSASSVGDRVTINCRASQSISSWLAWYQQKPGKAPNLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSMYTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 60) [ Table 4 ] : FRα antibody FR region Construct VH-FR1 VH-FR2 VH-FR3 VH-FR4 VL-FR1 VL-FR2 VL-FR3 VL-FR4 AB1370049 LVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 61) WIRQSPSRGLEWLG (SEQ ID NO: 62) RITINPDTSKNQFSLQLNSVTPEDTAVYYCAR (SEQ ID NO: 63) WGQGTLVTVSS (SEQ ID NO: 64) DIQMTQSPSTLSAVGDRVTITC (SEQ ID NO: 65) WYQQKPGKAPKLLIY (SEQ ID NO: 66) GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 67) FGGGTKVEIK (SEQ ID NO: 68) AB1370026 EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 69) WVRQAPGKGLEWVS (SEQ ID NO: 70) RFTISRDNAKNSLYLKMNSLRDEDTAVYYCAR (SEQ ID NO: 71) WGQGTLVTVSS (SEQ ID NO: 72) DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO: 73) WFQQAPGKAPKSLIY (SEQ ID NO: 74) GVPSKFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 75) FGQGTKLEIK (SEQ ID NO: 76) AB1370035 QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 77) WIRQSPSRGLEWLG (SEQ ID NO: 78) RITVNPDTSKNQFSLQLNSVTPEDTAVYYCVR (SEQ ID NO: 79) WGQGTLVTVSS (SEQ ID NO: 80) DIQMTQSPSTLSAVGDRVIITC (SEQ ID NO: 81) WYQQKPGKAPKLLIY (SEQ ID NO: 82) GVPSRFSGSGSGTEFTLTITSLQPDDFASYYC (SEQ ID NO: 83) FGPGTKVDIK (SEQ ID NO: 84) AB1370083 EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 85) WVRQAPGKGLEWVS (SEQ ID NO: 86) RFTISRDNAKNSLFLQMNSLRAEDTAVYYCAR (SEQ ID NO: 87) WGQGTLVTVSS (SEQ ID NO: 88) DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO: 89) WFQQKPGKAPKSLIY (SEQ ID NO: 90) GVPSKFSGSGSETDFTLTISSLQPEDFATYYC (SEQ ID NO: 91) FGGGTKVEIK (SEQ ID NO: 92) AB1370095 EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO: 93) WVRQAPGKGLEWVS (SEQ ID NO: 94) RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 95) WGQGTLVTVSS (SEQ ID NO: 96) DIQMTQSPSSSLSASVGDRVTITC (SEQ ID NO: 97) WFQQKPGKAPKSLIY (SEQ ID NO: 98) GVPSKFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 99) FGGGTKVEIK (SEQ ID NO: 100) AB1370117 QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 101) WIRQSPSRGLEWLG (SEQ ID NO: 102) RITINPDTSKNQFSLQLNSVTPEDTAVYFCAR (SEQ ID NO: 103) WGQGTLVTVSS (SEQ ID NO: 104) DIQMTQSPSTLSAVGDRVTINC (SEQ ID NO: 105) WYQQKPGKAPNLLIY (SEQ ID NO: 106) GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 107) FGQGTKLEIK (SEQ ID NO: 108) [ Table 5 ] : FRα antibody constant domain sequences Constant region sequence CH (CH1-hinge-CH2-CH3) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109) CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 110) CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 111)

It is expected that minor changes in the amino acid sequence of the antibody in the ADC of the present disclosure are included in the present disclosure, provided that the changes in one or more amino acid sequences maintain at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity with the antibody or antigen-binding fragment thereof of the present disclosure as defined anywhere herein. In a specific embodiment, the changes in one or more amino acid sequences maintain at least 99% sequence identity with the antibody or antigen-binding fragment thereof of the present disclosure as defined anywhere herein.

The antibodies in the disclosed ADCs may include variants in which amino acid residues from one species are replaced by corresponding residues in another species, either at conservative positions or at non-conservative positions. In some embodiments, amino acid residues at non-conservative positions are replaced by conservative or non-conservative residues. In particular, conservative amino acid substitutions are contemplated.

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, The side chains of the polypeptides described herein are amino acids, ...

"Non-conservative amino acid substitutions" include those in which (i) a residue with a cationic side chain (e.g., Arg, His, or Lys) replaces or is replaced by a cationic residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) replaces or is replaced by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) cysteine or proline replaces or is replaced by any other residue, or (iv) a residue with a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) replaces or is replaced by a residue with a smaller side chain (e.g., Ala or Ser) or a residue without a side chain (e.g., Gly).

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaleric acid and α-methylserine) can replace the amino acid residues of the antibody in the ADC disclosed herein. A limited number of non-conservative amino acids, amino acids not encoded by genetic codes and unnatural amino acids can replace amino acid residues. The antibodies in the ADC disclosed herein can also contain non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-oxomethylene-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine, nitro-glutamic acid, homoglutamic acid, hexahydronicotinic acid, tertiary leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be employed in which a chemically amine-acylated suppressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and amine-acylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing an E. coli S30 extract 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 approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and a chemically aminated suppressor tRNA (Turcatti et al ., J. Biol. Chem. 271:19991-8, 1996). In a third approach, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired one or more non-naturally occurring amino acids (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acids are incorporated into the polypeptide in place of their natural counterparts. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring 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).

A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can replace amino acid residues of antibodies in the ADCs of the present disclosure.

Essential amino acids of antibodies in the disclosed ADCs can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Biological interaction sites can also be determined by physical analysis of the structure, such as by nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, together with mutating putative contact site amino acids. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al. , FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from homology analysis with relevant components of the antibody in the ADC of the present disclosure, such as the translocation component or the protease component.

A variety of amino acid substitutions can be made and tested using known mutation 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 for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the induced polypeptides 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. Patent No. 5,223,409; Huse, WIPO Publication No. WO 92/06204) and localized mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

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, % 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 % 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 comparisons and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms familiar to those of skill, such as BLAST.

Any of a variety of sequence alignment methods can be used to determine the percent identity, including but not limited to global methods, local methods, and hybrid methods, such as, for example, segment methods. The protocol for determining percent identity is a routine procedure familiar to those skilled in the art. The global method aligns the sequence from the beginning to the end of the molecule and determines the best alignment by adding the scores of individual residual base pairs and applying gap penalties. Non-limiting methods include, e.g., 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 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); Align-M, see, e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences [Alin-M - A new algorithm for multiple alignment of highly diverse sequences], 20(9) Bioinformatics: 1428-1435 (2004).

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 using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) as shown below to optimize the alignment score (amino acids are represented by standard single letter codes).

In some embodiments, the variable domains in both the heavy and light chains of the antibody or antigen-binding fragment thereof in the ADC are altered by at least partially replacing one or more CDRs and/or by replacing and altering the sequence of a partial framework region. Although the CDRs may be derived from antibodies of the same class or even subclass as the antibody from which the framework region is derived, it is envisioned that the CDRs will be derived from antibodies of different classes and, in certain embodiments, from antibodies of different species. It is not necessary to replace all CDRs with complete CDRs from the donor variable region to transfer the antigen-binding ability of one variable domain to another. Instead, only those residues necessary to maintain the activity of the antigen-binding site need to be transferred. In view of the explanations set forth in U.S. Patent Nos. 5,585,089, 5,693,761 and 5,693,762, each of which is incorporated herein by reference, one skilled in the art is well able to obtain functional antibodies with reduced immunogenicity by performing routine experiments.

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

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

In some embodiments, the antibodies in the ADC disclosed herein can be modified to include changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, modified constant regions are contemplated in which one or more domains are partially or completely deleted. In some embodiments, the modified antibodies in the ADC will include a domain-deficient construct or variant in which the entire CH2 domain is removed (ΔCH2 construct). In some embodiments, the omitted constant region domain can be replaced by a short amino acid spacer (e.g., 10 residues) that provides a certain molecular flexibility that is usually conferred by the non-existent constant region. The deletion or inactivation of the constant region domain (via point mutation or other means) can reduce the Fc receptor binding of the circulating modified antibody. In other cases, constant region modifications can mitigate complement binding and thereby reduce the serum half-life and non-specific association of the cytotoxin yoke. Additional modifications of the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties, thereby allowing for enhanced localization due to increased antigen specificity or antibody flexibility. In some embodiments, the antibody or antigen-binding fragment thereof in the ADC has no antibody-dependent cellular cytotoxicity (ADCC) activity and/or has no complement-dependent cytotoxicity (CDC) activity.

In some embodiments, the antibody or antigen-binding fragment thereof in the ADC can be engineered to fuse the CH3 domain directly to the hinge region of the corresponding 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 domains. For example, a compatible construct can be presented in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is bound to the hinge region with a spacer of 5-20 amino acids. This spacer can be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible, or that the hinge region remains flexible. In some cases, the amino acid spacer can prove to be immunogenic and induce an undesirable immune response against the construct. In some embodiments, any spacer added to the construct may be relatively non-immunogenic, or even omitted entirely, in order to maintain the desired biochemical properties of the modified antibody.

In addition to the deletion of the entire constant region domain, the antibody or its antigen-binding fragment in the ADC provided herein can be modified by partial deletion or substitution of several or even single amino acids in the constant region. For example, the mutation of a single amino acid in a selected region in 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 deletions of the constant region can improve the selected characteristics (e.g., serum half-life) of the antibody or its antigen-binding fragment in the ADC, while keeping other desired functions associated with the subject constant region domain intact. In addition, the constant region of the antibody and its antigen-binding fragment in the ADC can be modified by mutation or substitution of one or more amino acids that enhance the properties of the resulting construct. In this regard, the activity provided by the conserved binding site (e.g., Fc binding) can be interfered with while substantially maintaining the conformation and immunological properties of the modified antibody or antigen-binding fragment thereof in the ADC. In some embodiments, one or more amino acids can be added to the constant region to enhance the desired characteristics, such as reducing or increasing effector function, or providing more cytotoxin or carbohydrate attachment. In some embodiments, it may be desirable to insert or replicate a specific sequence derived from a selected constant region domain. In some embodiments, a heavy chain constant region or fragment thereof (e.g., a human IgG constant region or fragment thereof) may include one or more amino acid substitutions relative to a wild-type IgG constant domain, wherein 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, an IgG constant domain may contain one or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein the amino acid positions are numbered according to the EU index as set forth in Kabat. In some embodiments, the IgG constant domain may contain one or more of the following substitutions: the amino acid at Kabat position 252 is substituted with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T), the amino acid at Kabat position 254 is substituted with threonine (T), the amino acid at Kabat position 256 is substituted with serine (S), arginine (R), glutamine (Q), glutamine (E), aspartic acid (D), or threonine (T), the amino acid at Kabat position 257 is substituted with leucine (L), the amino acid at Kabat position 309 is substituted with The amino acid at Kabat position 433 is substituted with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q), or the amino acid at Kabat position 434 is substituted 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 mutant . The term "YTE" or "YTE mutant" refers to a mutation in IgG1 Fc that results in increased binding to human FcRn and improves the serum half-life of the antibody with the mutation. The YTE mutant comprises a combination of three mutations M252Y/S254T/T256E introduced into the heavy chain of IgG1 (EU numbering, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC). See U.S. Patent No. 7,658,921, which is incorporated herein by reference. The YTE mutant was shown to increase the serum half-life of the antibody by approximately four-fold compared to the wild-type of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al. , (2013) Antimicrob. Agents Chemother. 57, 6147-6153). See also U.S. Patent No. 7,083,784, which is hereby incorporated by reference in its entirety.

In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC comprises: L at the N-terminus (e.g., position 1) of the VH; E at the N-terminus (e.g., position 1) of the VH; or Q at the N-terminus (e.g., position 1) of the VH. Polynucleotide sequence of the antibody

In some aspects, a polynucleotide encoding an anti-FRα antibody or an antigen-binding fragment thereof of an ADC disclosed herein is provided. The polynucleotide encoding an anti-FRα antibody or an antigen-binding fragment thereof in an ADC disclosed herein may be any one of the nucleotide sequences in Tables 6-7 . If there are any differences, the sequence in the table shall prevail.

In another aspect, the polynucleotide encoding the anti-FRα antibody or antigen-binding fragment thereof in the ADC disclosed herein comprises a sequence encoding: (a) a VL that is at least 80%, 85%, 90% or 95% identical or identical to a reference VL nucleotide sequence of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 as described in Table 6 ; and (b) a VH that is at least 80%, 85%, 90% or 95% identical or identical to a reference VH nucleotide sequence of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 as described in Table 6.

In another aspect, the polynucleotide encoding the anti-FRα antibody in the ADC disclosed herein comprises a sequence encoding: (a) a light chain that is at least 80%, 85%, 90% or 95% identical or identical to a reference light chain nucleotide sequence of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 as described in Table 7 ; and (b) a heavy chain that is at least 80%, 85%, 90% or 95% identical or identical to a reference heavy chain nucleotide sequence of any one of constructs AB1370049, AB1370026, AB1370035, AB1370083, AB1370095 or AB1370117 as described in Table 7. [ Table 6 ] : FRα antibody VH and VL nucleotide sequences Construct VH V L AB1370049 CTGGTACAGCTGCAGCAGTCTGGACCTGGACTGGTCAAGCCTTCTCAGACCCTGTCTCTGACCTGCGCCATCTCTGGCGACTCTGTGTCCTCTGATTCTGCCACCTGGAACTGGATCCGGCAGTCTCCATCTAGAGGCCTGGAATGGCTGGGCAGAACCTACTACCGGTCCAAGTGGT ACAACGACTACGCCGTGTCCGTGAAGTCCCGGATCACCATCAATCCCGACACCTCCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACCCTGAGGATACCGCCGTGTACTATTGTGCTAGAGGCGTGGGCTCCTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 114) GACATCCAGATGACCCAGTCTCCATCCACACTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCTCAGTCCATCTCTAGCTGGCTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAAGGCCTCTGGACTG GAATCCGGCGTGCCCTCTAGATTTTCTGGCTCTGGATCTGGCACCGAGTTCACCCTGACCATTTCTAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGCCAGCAGTACAACTCCTACAGCCAGCTGACCTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 115) AB1370026 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTGGTCGTAGTTACATATAC TACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGAAAATGAACAGCCTGAGAGACGAGGACACAGCTGTTTATTACTGTGCGAGAGAAATGCAGCAGCTGGCCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 116) GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAATTTTTTAGCCTGGTTTCAGCAGGCACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCACAGTATAATAGTTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 117) AB1370035 CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAATTGGT ATAATGATTATACATTATCTGTGAAAAGTCGAATAACCGTCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGTAAGAGGGGTGGGACGCTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 118) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCATCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTA GAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATTACCAGCCTTCAGCCTGATGATTTTGCAAGTTATTACTGCCAAGAGTATAAAACTTATTCTATATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO: 119) AB1370083 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAACCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAACATGAACTGGGTCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTGGTAGTAGTTACATATAC TACGCAGACTCAATGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGATGACTACATTAACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 120) GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCACTTTTTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGAGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCACAGTATATTAGTTACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 121) AB1370095 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAATTGGGTCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGGAGTAGTTACGTATAC TACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCTGTGTATTACTGTGCGAGAGGGATGACTACATTAACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 122) GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAGTTTTTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 123) AB1370117 CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCGACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGT ATAGTGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTTCTGTGCAAGAGGGGGAGCTCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 124) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCGAGTTTA GAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCCATGTATACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 125) [ Table 7 ] : FRα antibody heavy chain and light chain nucleotide sequences Construct VH-CH VL-CL AB1370049 CTGGTACAGCTGCAGCAGTCTGGACCTGGACTGGTCAAGCCTTCTCAGACCCTGTCTCTGACCTGCGCCATCTCTGGCGACTCTGTGTCCTCTGATTCTGCCACCTGGAACTGGATCCGGCAGTCTCCATCTAGAGGCCTGGAATGGCTGGGCAGAACCTACTACCGG TCCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCCGGATCACCATCAATCCCGACACCTCCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACCCCTGAGGATACCGCCGTGTACTATTGTGCTAGAGGCGTGGGCTCCTTCGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACAGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 126) GACATCCAGATGACCCAGTCTCCATCCACACTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCTCAGTCCATCTCTAGCTGGCTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACAAGGCCTCTGGACT GGAATCCGGCGTGCCCTCTAGATTTTCTGGCTCTGGATCTGGCACCGAGTTCACCCTGACCATTTCTAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGCCAGCAGTACAACTCCTACAGCCAGCTGACCTTCGGCGGAGGGACCAAGGTGGAGATCA AACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAG GAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC (SEQ ID NO: 127) AB1370026 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTGGTCGTAGT TACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGAAAATGAACAGCCTGAGAGACGAGGACACAGCTGTTTATTACTGTGCGAGAGAAATGCAGCAGCTGGCCCTTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCAAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTTCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 128) GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAATTTTTTAGCCTGGTTTCAGCAGGCACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCACAGTATAATAGTTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 129) AB1370035 CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGG TCCAATTGGTATAATGATTATACATTATCTGTGAAAAGTCGAATAACCGTCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTATTGTGTAAGAGGGGTGGGACGCTTTGACTCCTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC GTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCAAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTTCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA CAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 130) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCATCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTT AGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATTACCAGCCTTCAGCCTGATGATTTTGCAAGTTATTACTGCCAAGAGTATAAAACTTATTCTATATTCACTTTCGGCCCTGGGACCAAAGTGGATATCA AACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 131) AB1370083 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAACCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTGGTAGTAGT TACATATACTACGCAGACTCAATGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTTTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGATGACTACATTAACTTTTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCAAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTTCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 132) GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCACTTTTTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGAGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCACAGTATATTAGTTACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 133) AB1370095 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAATTGGGTCCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGGAGTAGT TACGTATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACAGCTGTGTATTACTGTGCGAGAGGGATGACTACATTAACTTTTGACTACTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCAAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTTCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTAC AAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 134) GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTCGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAGTTTTTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAAGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCACAGTATAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 135) AB1370117 CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCGACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGG TCCAAGTGGTATAGTGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTTCTGTGCAAGAGGGGGAGCTCCCTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC GTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCAAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTTCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACATGCGTGGTGGTGGATGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCAGCCGCGAGGAGATGACCCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA CAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 136) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCGAGTTT AGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCCATGTATACTTTTGGCCAGGGGACCAAGCTGGAGATCA AACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 137)

The polynucleotide sequence or 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.

One or more polynucleotide sequences can be prepared by any means known in the art. For example, large quantities of sequences can be produced by replication and/or expression in suitable host cells. Natural or synthetic DNA fragments encoding the desired fragments are usually incorporated into recombinant nucleic acid constructs, usually DNA constructs, which can be introduced into prokaryotic or eukaryotic cells and replicated therein. Typically, DNA constructs are suitable for autonomous replication in single-cell hosts (such as yeast or bacteria), but can also be used for introduction and integration into the genome of cultured bacteria, insects, mammals, plants or other eukaryotic cell lines.

One or more polynucleotide sequences can also be produced by chemical synthesis (e.g., by the phosphoramidite method or triester method to synthesize polynucleotides), and can be performed on commercial automated oligonucleotide synthesizers. Double-stranded (e.g., DNA) fragments can be obtained from single-stranded products of chemical synthesis by synthesizing complementary strands and annealing the strands together under appropriate conditions or by joining complementary strands using a DNA polymerase with appropriate primer sequences.

Variants of polynucleotides are also described herein. Polynucleotide variants can contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants comprise alterations that produce silent substitutions, additions, or deletions without altering the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon representation for a particular host (changing codons in human mRNA to those that are better for bacterial hosts such as E. coli). Definitions and Antibody Forms

As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or immunologically reacts with a specific antigen.

The antibodies in the ADCs disclosed herein are typically isolated or recombinant. "Isolated," when used herein to refer to a polypeptide (e.g., an antibody) that has been identified and separated and/or recovered from a cell or cell culture expressing it. Typically, an isolated antibody will be prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to FRα is substantially free of antibodies that specifically bind to antigens other than FRα.

Typically, antibodies contain at least two "light chains" (LC) and two "heavy chains" (HC). The light and heavy chains of such antibodies are polypeptides composed of several domains. Each heavy chain contains a heavy chain variable region (abbreviated herein as "VH") and a heavy chain constant region (abbreviated herein as "CH"). The heavy chain constant region contains heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD and IgG) and, if necessary, a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain contains a light chain variable domain (abbreviated herein as "VL") and a light chain constant domain (abbreviated herein as "CL").

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.

The "variable region" of an antibody refers to the variable region of the antibody light chain alone or the variable region of the antibody heavy chain or a combination thereof. The variable regions VH and VL can be further subdivided into hypervariable regions (also called hypervariable regions) called complementarity determining regions (CDRs), interposed with more conserved regions called framework regions (FRs). In a specific embodiment, each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of the antibody in the ADC may further include all or part of the heavy chain or light chain constant region.

The binding between an antibody and its target antigen or epitope is mediated by the CDRs. The term "epitope" refers to a region of a target protein (e.g., a polypeptide) that is capable of binding to (e.g., being bound by) an antibody or antigen-binding fragment in the ADC disclosed herein. CDRs are the primary determinant of antigen specificity. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda Md.); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol . 273:927-948). In addition, the art sometimes uses a combination of these two methods to determine CDRs.

The sequence of a CDR may be identified by reference to any numbering system known in the art, for example, 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 and 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, [IMGT unique numbers for variable domains of immunoglobulins and cell receptors and Ig superfamily V-like domains]" Dev. Comp . Immunol. [Developmental and Comparative Immunology] 27, 55-77 (2003)) ( see Table 8 ). [ Table 8 ] : CDR definition Kabat Chothia IMGT VH CDR1 31-35 26-32 27-38 VH CDR2 50-65 52-56 56-65 VH CDR3 95-102 95-102 105-117 VL CDR1 24-34 24-34 27-38 VL CDR2 50-56 50-56 56-65 VL CDR3 89-97 89-97 105-117

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

There are five major classes of heavy chain constant regions, classified as IgA, IgG, IgD, IgE, and IgM, each with characteristic effector functions designated by isotype. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγRs), namely FcγRI, FcγRII, and FcγRIII, which are specific for the IgG class of the antibody. Binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, cytolytic killing of antibody-coated target cells (termed antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. Sequences important for IgG binding to FcγR receptors have been reported to be located in the CH2 and CH3 domains.

In a specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof in the ADC is of IgG isotype. The anti-FRα antibody or antigen-binding fragment thereof in the ADC may be of any IgG subclass, such as IgG1, IgG2, IgG3 or IgG4 isotypes. In a specific embodiment, 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 that are close to natural IgG may reduce developability and other risks. For example, the inventors of the present invention designed an ADC using a human IgG1 mAb structure, which is considered to be not subject to theoretical constraints and its immunogenicity will be lower than other anti-FRα ADCs under development, such as IMGN151.

For the heavy chain constant region amino acid positions discussed in the antibodies of the ADC disclosed herein, the numbering is according to the EU index first described by Edelman, G.M. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 63 (1969) 78-85. Edelman's EU numbering is also described in Kabat et al. (1991) (supra). Therefore, the terms "EU index as described in Kabat", "EU index" in the context of the heavy chain. "Kabat's EU index" or "EU numbering" refers to the residue numbering system based on the human IgG1 EU antibody as described by Edelman et al. (1991). The numbering system used for the light chain constant region amino acid sequences is similarly described in Kabat et al. (supra). Thus, as used herein, "according to Kabat numbering" refers to the Kabat numbering system as set forth by Kabat et al. (supra).

The terms "Fc region", "Fc portion" and "Fc" are used interchangeably and refer to the portion of a native immunoglobulin formed by two Fc chains. Each "Fc chain" comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. The native Fc region is homodimeric. In some embodiments, the Fc region may be heterodimeric, as it may comprise modifications that implement Fc heterodimerization. The Fc region contains carbohydrate moieties and binding sites for complements and Fc receptors (including FcRn receptors), and has no antigen binding activity. Fc may refer to this region in isolation, or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in many Fc domain positions, including but not limited to EU positions 270, 272, 312, 315, 356 and 358, resulting in minor differences between the sequences described in this application and sequences known in the art. Therefore, each naturally occurring IgG Fc region is referred to as a "wild-type IgG Fc domain" or "WT IgG Fc domain" (i.e., any allele). Human IgG1, IgG2, IgG3 and IgG4 heavy chain sequences are available 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.

In some embodiments, the anti-FRα antibody in the ADC disclosed herein is a monoclonal antibody. "Monoclonal antibody" (mAb) refers to a homologous antibody group that is involved in the highly specific recognition and binding of a single epitope or epitopes. This is in contrast to polyclonal antibodies that typically include different antibodies against different epitopes. The term "monoclonal antibody" can cover full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal antibody" refers to such antibodies prepared in any number of ways, including but not limited to fusion tumors, phage selection, recombinant expression, and transgenic animals. In specific embodiments, the anti-FRα antibody in the ADC disclosed herein is an isolated monoclonal antibody. In a more specific embodiment, the antibody in the ADC is a fully human monoclonal antibody. In an alternative embodiment, the method of the present disclosure can use an ADC with multiple clones of antibodies.

The anti-FRα antibodies and antigen-binding fragments thereof in the ADC disclosed herein can be derived from any species by recombinant means. For example, the antibody or antigen-binding fragment can be mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human or a chimeric form thereof. For use in administration to humans, the non-human derived antibodies or antigen-binding fragments in the ADC can be genetically or structurally altered to have lower immunogenicity when administered to human patients. Particularly preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies.

The term "human antibody" means an antibody produced in the human body or an antibody having an amino acid sequence corresponding to an antibody produced in the human body prepared using any technique known in the art. Human antibodies include complete or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy chain and/or light chain polypeptide, such as antibodies comprising mouse light chain and human heavy chain polypeptides. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific induced mutations in vitro or during gene rearrangement, or by somatic cell mutations in vivo). Human antibodies may be prepared in human cells (expressed by recombination), non-human animals, or prokaryotic or eukaryotic cells that can express functionally rearranged human immunoglobulin (such as heavy and light chain) genes. Linker peptides not found in natural human antibodies may be included in single-chain human antibodies. For example, Fv may have a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the heavy chain variable region and the light chain variable region. Such 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 that cannot express functional inherent immunoglobulins but can express human immunoglobulin genes can also be used to prepare human antibodies (see, e.g., PCT Publication Nos. WO 1998/24893, WO 1992/01047, WO 1996/34096, WO 1996/33735; U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598, each of which is incorporated herein by reference). Human antibodies can also be prepared directly using various techniques known in the art. Immortalized human B lymphocytes can be generated that are immunized in vitro or isolated from an immunized individual that produces antibodies to a target antigen. See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77 (1985); Boemer et al. , J. Immunol. 147 (1): 86-95 (1991); U.S. Patent 5,750,373.

The term "humanized antibody" refers to an antibody in which the framework or CDR has been modified to contain CDRs of an immunoglobulin of different specificity compared to the parent immunoglobulin. For example, mouse CDRs can be grafted into the framework region of a human antibody to prepare a "humanized antibody". See, e.g., Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, M.S. et al., Nature 314 (1985) 268-270. In some embodiments, "humanized antibodies" are those in which the constant region has been additionally modified or altered from that of the original antibody to produce desired properties.

Humanized antibodies can be prepared as needed by using the three-dimensional model of the parent, engineering and humanized sequence to analyze the parent sequence and various conceptual humanization and engineering products. The three-dimensional immunoglobulin model is usually available and is familiar to those familiar with the technology. The computer program that illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence is available. Checking these displays allows the analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing the residues that affect the ability of the candidate immunoglobulin to bind its antigen such as FRα. In this way, FR residues can be selected and combined from the consensus sequence and the input sequence so that the desired antibody characteristics (such as increasing the affinity to one or more target antigens) are achieved.

Humanized antibodies can be further modified by substitution of additional residues within the Fv framework region and/or substituted non-human residues to improve and optimize antibody specificity, affinity and/or ability. Generally speaking, humanized antibodies will contain substantially all of at least one (and typically two or three) variable domains, which contain all or substantially all CDR regions corresponding to non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin consensus sequences. Humanized antibodies may also include at least a portion of an immunoglobulin constant region or domain (Fc), typically a constant region or domain of a human immunoglobulin. Examples of methods for producing humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641, each of which is incorporated herein by reference.

The term "chimeric antibody" refers to an antibody comprising a variable region (i.e., binding region) from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA technology. Chimeric antibodies comprising mouse variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" covered by the present disclosure are those in which the constant region has been modified or changed from that of the original antibody to produce the desired properties. Such chimeric antibodies are also referred to as "class-switched antibodies." Chimeric antibodies are the product of expressed immunoglobulin genes, which include a DNA fragment encoding an immunoglobulin variable region and a DNA fragment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies involving conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Patent Nos. 5,202,238 and 5,204,244, each of which is incorporated herein by reference.

In some embodiments, the antibody in the ADC disclosed herein is a full-length antibody as described above. Alternatively, the antibody may be an antigen-binding fragment. The term "antigen-binding fragment" as used herein 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.

In some embodiments, the antigen binding fragment in the ADC disclosed herein is a Fab fragment. The antibody in the ADC disclosed herein can also be Fab′, Fv, scFv, Fd, V NAR domain, IgNAR, intracellular antibody, IgG CH2, miniantibody, single domain antibody, Fcab, scFv-Fc, F(ab′)2, di-scFv, bispecific T cell engager ( ^BiTE® ), F(ab′)3, tetrabodies, triabodies, bibodies, DVD-Ig, (scFv)2, mAb2 or DARPin.

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

"Fab' fragments" contain a single light chain and a single heavy chain, but in addition to CH1 and VH, "Fab' fragments" also contain a heavy chain region between the CH1 and CH2 domains, which is necessary for the formation of interchain disulfide bonds. Therefore, two "Fab' fragments" can be combined by forming disulfide bonds to form a F(ab')2 molecule.

The "F(ab')2 fragment" contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for forming interchain disulfide bonds between the two heavy chains.

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

"Single-domain antibodies" are antibody fragments that contain a single antibody domain unit (e.g., VH or VL).

"Single-chain Fv" ("scFv") is an antibody fragment containing the VH and VL domains of an antibody, linked together to form a single chain. A polypeptide linker is usually used to connect the VH and VL domains of the scFv.

"TandAb ® ", also known as TandAb ^® , is a single-chain Fv molecule formed by covalently bonding two scFvs in a tandem orientation to a flexible peptide linker.

Bispecific T cell engagers (BiTE ^® ) are fusion proteins consisting of two single-chain variable fragments (scFv) on one peptide chain. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to tumor cell antigens.

"Diabodies" are small bivalent and bispecific antibody fragments that comprise: a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected 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 the complementary domain of the other chain and promotes the assembly of a dimeric molecule with two functional antigen-binding sites.

"DARPin" is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrins, which can be found in the human genome and are one of the most abundant types of binding proteins. The DARPin library module is defined by the natural ankyrin repeat protein sequence, with 229 ankyrin repeat sequences used for initial design and another 2200 for subsequent refinement. This module serves as a building block for the DARPin library. The library module is similar to the human genome sequence. DARPins consist of 4 to 6 modules. Since each module is approximately 3.5 kDa, the average DARPin size is 16-21 kDa. Selection of binders was accomplished by completely cell-free ribosome display and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007 Nov;35(Pt 5):962-5.

In some embodiments, the antibody or antigen-binding fragment thereof in the ADC can be further modified to contain additional chemical moieties that are not normally part of a protein. Those derivatized moieties can improve the solubility, biological half-life, or absorption of the antibody or antigen-binding fragment thereof in the ADC. Such moieties can also reduce or eliminate any desired side effects of the antibody or antigen-binding fragment thereof in the ADC. A review of those moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Lloyd V. Allen, Jr., ed., (2012). FRα Binding

In a specific embodiment, the anti-FRα antibody or its antigen-binding fragment in the ADC disclosed herein specifically binds to FRα. The term "specifically binds to FRα" refers to an antibody that is able to bind to a defined target with sufficient affinity so that the antibody can be used as a therapeutic agent targeting FRα. In some embodiments, the 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 its antigen-binding fragment in the ADC disclosed herein specifically binds to human FRα (UniProt ID: P15328) and/or cynomolgus monkey FRα (UniProt ID: A0A2K5U044). In a specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof in the ADC disclosed herein specifically binds to human FRα. In a specific embodiment, the anti-FRα antibody or antigen-binding fragment thereof in the ADC disclosed herein specifically binds to human FRα and cynomolgus monkey FRα.

In some embodiments of any aspect of the disclosure, FRα has a sequence of SEQ ID NO: 112 or SEQ ID NO: 113. In specific embodiments, FRα has a sequence of SEQ ID NO: 112. SEQ ID NO: 112 : Human FRα protein (predicted mature, secreted polypeptide) RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMS SEQ ID NO: 113 : Cynomolgus monkey FRα protein (predicted mature, secreted polypeptide) RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERV LNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMS

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.

The term "does not bind" means that the antibody or antigen-binding fragment thereof in the ADC disclosed herein 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). When used in the context of binding herein, the term "substantially free" may mean that less than 5%, 2%, 1%, 0.5% or 0.1% of the cells in the cell culture expressing one or more of the molecules are bound (upon contact) by the antibody or antigen-binding fragment thereof in the ADC disclosed herein. Suitably, when used in the context of binding herein, the term "substantially free" may mean that no such cells are bound. Binding Affinity

"Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed 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 antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer.

Suitably, the antibody or antigen-binding fragment in the ADC of the present disclosure binds to the FRα molecule with sufficient affinity such that the antibody can be used as a therapeutic or diagnostic agent targeting FRα.

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.

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.

In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC binds to cynomolgus monkey 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.

In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC is about 1 to about 100 nM, about 1 to about 80 nM, about 1 to about 60 nM, about 1 to about 40 nM, about 2 to about 100 nM, about 2 to about 80 nM, about 2 to about 60 nM, about 2 to about 40 nM, about 5 to about 100 nM, about 5 to about 80 nM, about 5 to about 60 nM, about 5 to about 40 nM, 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, nM, or a KD of about 30 to about 40 nM to bind to cynomolgus monkey FRα.

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

The binding affinity of the anti-FRα antibody or antigen-binding fragment thereof in the ADC disclosed herein can be determined using a FRα binding affinity assay as described herein. In some embodiments, the binding affinity of the anti-FRα antibody or antigen-binding fragment thereof in the ADC disclosed herein is determined by Biacore (e.g., Biacore T200) at 25°C. For example, the affinity of the recombinant human FRα ECD to the anti-FRα antibody or antigen-binding fragment thereof can be measured using Biacore T200 at 25°C, for example using the following protocol. Protein A at a concentration of 50 μg/ml in 10 mM sodium acetate (pH 4.0) is covalently immobilized on the surface of a CM5 chip using standard amine coupling techniques. The antibody or antigen-binding fragment thereof is captured to the protein A surface in HBS-EP+ buffer (pH 7.4) at 10 μl/min to ensure FRα ECD binding. FRα ECD was serially diluted (0.4 nM-100 nM human FRα ECD, 0.8 nM-200 nM cynomolgus FRα ECD, 30 nM-4000 nM mouse FRα ECD, and rat FRα ECD) in HBS-EP+ buffer (pH 7.4) and flowed over the chip at 50 µl/min, associated for 2 min, and dissociated for 8 min. The chip surface was fully regenerated with a pulse of ₃ M MgCl2 to remove captured antibodies or their antigen-binding fragments, as well as any bound FRα ECD. Multiple buffer-only injections were performed under the same conditions to allow double reference subtraction of the final sensorgrams analyzed using Biacore T200 Evaluation Software.

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 determined by Octet red at 25°C, for example, using the following protocol. The binding assay was performed on an Octet RED384 (ForteBio) at 25°C in an assay buffer containing PBS, 0.1% v/v BSA (Sigma, A9576), 0.01% v/v Tween-20 (Sigma, P9416) (pH 7.4), using a tilt bottom black 384-well plate (ForteBio, 18-5076). The assay was set up using either protein A or anti-human capture biosensors (AHC) (FoteBio, 18-5089) according to the manufacturer's instructions. 10 µg/ml of anti-rat FRα IgG (Sino Biological, 81073-RP01) was coated onto protein A biosensors (FoteBio, NC9490476), and 10 µg/ml of test human IgG was loaded onto anti-human capture biosensors (AHC) (FoteBio, 18-5089) for 180 seconds. Binding was measured by incubating the loaded biosensors with 500 nM human FRα (in-house) or 500 nM rat FRα (Sino Biological, 81073-R08H). Dissociation was measured after transfer into assay buffer. Data were analyzed using Octet data analysis software version 7.0. Antibody preparation

The antibodies or antigen-binding fragments thereof in the ADC disclosed herein can be produced by: transfecting host cells with one or more vectors comprising polynucleotides encoding the corresponding 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.

The antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies) in the ADC can be prepared using recombinant DNA methods as 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 fused tumor cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into appropriate expression vectors which, when transfected into non-immunoglobulin-producing host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, produce monoclonal antibodies by the host cells. In addition, recombinant monoclonal antibodies of the desired species or antigen-binding fragments thereof 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).

Affinity maturation strategies and linkage modification group principles 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), which is incorporated by reference in its entirety.

Various techniques for producing antibody fragments are known. Traditionally, such fragments are derived by proteolysis of intact antibodies, such as described by 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 ADC are recombinantly produced. All Fab, Fv and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, thereby allowing the production of large quantities of such fragments. Such anti-FRα antibody fragments can also be isolated from the antibody phage libraries discussed above. The anti-FRα antibody fragments in the ADC may also be linear antibodies as described in U.S. Patent No. 5,641,870, which is incorporated herein by reference. Other techniques for generating antibody fragments will be clear to the skilled practitioner.

The techniques can be adapted to generate single chain antibodies specific for FRα. (See, e.g., U.S. Patent No. 4,946,778). In addition, the methods can be adapted to construct Fab expression libraries to allow rapid and efficient identification of single cloned Fab fragments with desired specificity for FRα or its derivatives, fragments, analogs or homologs. 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 bonds of F(ab')2 fragments; Fab fragments produced by treating antibody molecules with papain and a reducing agent; or Fv fragments. Antibody-drug conjugate (ADC)

The "antibody-drug conjugate" (ADC) disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof as described herein, wherein the anti-FRα antibody or an antigen-binding fragment thereof is conjugated to a cytotoxin. Cytotoxin

A cytotoxin (also called a cytotoxic agent) can be any molecule known in the art that inhibits or prevents the function of a cell and/or causes cell destruction (cell death), and/or exerts an anti-tumor/anti-proliferative effect. Many classes of cytotoxic agents are known to have potential utility in ADC molecules. Suitable cytotoxic agents of the present disclosure include, but are not limited to, topoisomerase I inhibitors (TOPOi), muscimol, auristatin, daunorubicin, doxorubicin, duocarmixin, dolastatin, enediynes, lexitropsin, taxanes, puromycin, maytansinoids, vinblastine, tubulysin, and pyrrolobenzodiazepine (PBD). Examples of such cytotoxic agents are AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, thiabendazole, root mycin, cyanothiabendazole, dolastatin-10, echinocytin, compretin, chalicheamicin, maytansine, DM-1, vinblastine, methotrexate and cytotoxin, and derivatives and analogs thereof. Additional disclosures about 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 entirety.

The cytotoxic agent is typically linked to or "loaded onto" the antibody or antigen-binding fragment. The agent loading (p) is the average amount of agent per antibody or antigen-binding fragment. Those skilled in the art will appreciate that more than one of the agents (e.g., TOPOi) may be conjugated to the antibody or antigen-binding fragment thereof.

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, the range is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In some embodiments, there is one 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 agent (i.e., drug) to antibody. This ratio is referred to as the drug-antibody ratio (DAR). The DAR is the average number of drugs (i.e., cytotoxic agents) attached to each antibody. In some embodiments of the present disclosure, the DAR ranges from about 1 to 20. In some embodiments, the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In specific embodiments, the DAR is about 4 ( e.g., 3.8-4.2) or about 8 ( e.g., 7.6-8.4). In specific embodiments, the DAR is about 8 ( e.g., 7.6-8.4).

In some embodiments, the antibody or antigen-binding fragment thereof in the ADC disclosed herein is conjugated to one or more cytotoxins selected from the group consisting of topoisomerase I inhibitors, tubulysin derivatives, pyrrolobenzodiazepines, or combinations thereof. For example, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxins selected from the group consisting of topoisomerase I inhibitor SG3932 (also known as AZ14170133), SG4010, SG4057 or SG4052 (structures of which are provided below), or combinations thereof. In specific embodiments, the antibody or antigen-binding fragment thereof may be conjugated to a topoisomerase I inhibitor (e.g., topoisomerase I inhibitor SG3932).

In certain embodiments, the antibody or antigen-binding fragment thereof in the ADC disclosed herein is not conjugated to a microtubule inhibitor such as a microtubule inhibitor (e.g., maytansinoid, auristatin), or the anti-FRα ADC disclosed herein does not contain a microtubule inhibitor such as a microtubule inhibitor (e.g., maytansinoid, auristatin). Microtubule inhibitor molecules have potential for intractable toxicity that limits dose. Topoisomerase I inhibitors

Using in vivo and in vitro models, the present disclosure demonstrates the stability and efficacy of anti-FRα mAbs when harpoonated with TOPOi payloads and deployed as ADCs.

In some embodiments, the anti-FRα ADCs disclosed herein comprise a topoisomerase I inhibitor.

Topoisomerase inhibitors are chemical compounds that block the action of topoisomerases (topoisomerase I and II), a class of enzymes that control changes in DNA structure during normal cell cycle by catalyzing the breaking and rejoining of the phosphodiester backbone of the DNA chain. 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 that have been commonly used to develop anti-FRα ADCs to date are known to have toxicity issues (Hinrichs et al. AAPS J. 2015 Sep; 17(5): 1055-1064). In addition, the use of less hydrophobic linkers and less potent warheads (e.g., TOPOi) will facilitate bystander killing in heterogeneous tumors. Although bystander activity can be achieved by increasing potency by increasing hydrophobicity and/or improving warhead permeability, this may result in increased toxicity due to nonspecific uptake.

General examples of suitable topoisomerase I inhibitors are represented by the following compounds: Such compounds are denoted A* and may be referred to herein as "drug units."

The compound (e.g., A*) can provide a linker for attachment (preferably conjugation) to an antibody or antigen-binding fragment in the ADC of the present disclosure. In a specific embodiment, the linker is attached (e.g., conjugated) to an amino residue, such as an amino acid of an antibody or antigen-binding fragment in the ADC of the present disclosure in a cleavable manner.

More particularly, examples of suitable topoisomerase I inhibitors are the following compounds of formula "I": and its salts and solvates, wherein ^RL is as defined above.

Therefore, for the ADC of the present disclosure having the general formula IV: L-( ^DL ) _p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L and p are as defined above, ^DL is a topoisomerase I inhibitor with a linker (e.g., a drug linker unit) having the formula III: and its salts and solvates, wherein R ^LL is as defined above.

In some embodiments, the compound having Formula I has Formula I ^P : ; and salts and solvates thereof, wherein R ^LP is a linker for linking to an antibody or an antigen-binding fragment thereof in the ADC of the present disclosure, wherein the linker is selected from: (Ia ^P ): , where Q ^P is: , wherein Q ^XP makes Q ^P an amino acid residue, a dipeptide residue or a tripeptide residue; ^XP is: , wherein aP = 0 to 5, bP = 0 to 16, cP = 0 or 1, dP = 0 to 5; ^GL is as defined above; (Ib): , wherein ^RL1 and ^RL2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group; and e is 0 or 1.

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 other embodiments, aP is 0.

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

cP can be 0 or 1.

dP can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is 0 to 3. In some of these embodiments, dP is 1 or 2. In other embodiments, dP is 2.

In some embodiments of ^XP , aP is 0, cP is 1 and dP is 2, and bP can be from 0 to 8. In some of these embodiments, bP is 0, 4 or 8.

The above preferences for ^QX of compounds of Formula I may apply to ^QXP (e.g., where appropriate).

The above preferences for ^GL , ^RL1 , ^RL2 and e for compounds of Formula I may apply to compounds of Formula ^IP .

In some embodiments, the ADC having Formula IV has the formula IV ^P : L-(D ^LP ) _p ( ^IVP ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof, and D ^LP is a topoisomerase I inhibitor (e.g., a drug linker unit) having the formula III ^P : ; R ^LLP is linked to a linker of the antibody or antigen-binding fragment thereof, wherein the linker is selected from (Ia ^P '): , wherein Q ^P , ^XP and G ^LL are as defined above; and (Ib'): , wherein R ^L1 and R ^L2 are as defined above; and p is an integer from 1 to 20.

In some embodiments, the compound having Formula I has Formula I ^P2 : and its salts and solvents, wherein R ^LP2 is a linker for linking to the antibody or antigen-binding fragment thereof in the ADC of the present disclosure, wherein the linker is selected from: (Ia ^P2 ): , where Q is: , wherein Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X ^P2 is: , wherein aP2 = 0 to 5, b1P2 = 0 to 16, b2P2 = 0 to 16, cP2 = 0 or 1, dP2 = 0 to 5, wherein at least b1P2 or b2P2 = 0 (i.e., only one of b1 and b2 may not be 0); GL is a linker for linking to the antibody or antigen-binding fragment thereof in the ADC of the present disclosure; (Ib): , wherein ^RL1 and ^RL2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group; and e is 0 or 1.

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 other embodiments, aP2 is 0.

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

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

Preferably, only one of b1P2 and b2P2 can be non-zero.

cP2 can be 0 or 1.

dP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP2 is 0 to 3. In some of these embodiments, dP2 is 1 or 2. In other embodiments, dP2 is 2. In other embodiments, dP2 is 5.

In some embodiments of ^XP2 , aP2 is 0, b1P2 is 0, cP2 is 1 and dP2 is 2, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. In some embodiments of ^XP2 , aP2 is 1, b2P2 is 0, cP2 is 0 and dP2 is 0, and b1P2 may be from 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8. In some embodiments of ^XP2 , aP2 is 0, b1P2 is 0, cP2 is 0 and dP2 is 1, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. In some embodiments of ^XP2 , b1P2 is 0, b2P2 is 0, cP2 is 0 and one of aP2 and dP2 is 0. The other of aP2 and dP2 is from 1 to 5. In some of these embodiments, the other of aP2 and dP2 is 1. In other of these embodiments, the other of aP2 and dP2 is 5.

The above preferences for ^QX in compounds of Formula I may apply to ^QX in Formula Ia ^P2 (eg, where appropriate).

The above preferences for ^GL , ^RL1 , ^RL2 and e for compounds of Formula I may apply to compounds of Formula ^IP2 .

In some embodiments, the ADC having Formula IV has Formula IV ^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, and D ^LP2 is a topoisomerase I inhibitor (e.g., a drug linker unit) having Formula III ^P2 : ; R ^LLP2 is linked to a linker of the antibody or antigen-binding fragment thereof, wherein the linker is selected from (Ia ^P2 '): , wherein Q and ^XP2 are as defined above, and ^GLL is a linker linked to the antibody or antigen-binding fragment thereof; and (Ib'): , wherein R ^L1 and R ^L2 are as defined above; and p is an integer from 1 to 20.

Particularly suitable topoisomerase I inhibitors include those having the formula: ; ; ; ; and/or .

In a specific embodiment, the antibody or antigen-binding fragment thereof in the ADC of the present disclosure is conjugated to a topoisomerase I inhibitor having the following formula: .

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

Although topoisomerase I inhibitors are preferred as described above, it should be noted that any suitable agent (e.g., drug/ cytotoxin ) can be linked to the antibody or antigen-binding fragment thereof in the ADC of the present disclosure. Examples of other suitable agents are summarized below. Tubulolysin and pyrrolobenzodipiperidin

In some embodiments, the cytotoxin is tubulysin or a tubulysin derivative. In some embodiments, the cytotoxin is tubulysin A, which has the following chemical structure: .

Tubulysin is a member of a class of natural products isolated from myxobacterial species. As cytoskeletal interactors, tubulysin is a mitotic poison that inhibits tubulin polymerization and causes cell cycle arrest and apoptosis. As used herein, the term "tubulelysin" refers collectively and individually to naturally occurring tubulysin as well as analogs and derivatives of tubulysin. Illustrative examples of tubulysin are disclosed, for example, in WO 2004005326 A2, WO 2012019123 A1, WO 2009134279 A1, WO 2009055562 A1, WO 2004005327 A1, US 7776841, US 7754885, US 20100240701, US 7816377, US 20110021568 and US 20110263650, which are incorporated herein by reference. It will be understood that such derivatives include, for example, tubulysin prodrugs or tubulysin comprising one or more protection or protecting groups, one or more linking moieties.

In another embodiment, the cytotoxin may be a pyrrolobenzodiazepine (PBD) or a PBD derivative. PBDs are transported to the cell nucleus where they crosslink DNA, prevent replication during mitosis, damage the DNA by inducing single strand breaks, and subsequently cause apoptosis. Some PBDs have the ability to recognize and bind to specific sequences of DNA; a preferred sequence is PuGPu. PBDs have the following general structure: .

PBDs differ in the number, type and position of substituents, in both their aromatic A ring and pyrrolo C ring, and in the saturation of the C ring. In the B ring, there is an imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for alkylating the DNA. All known natural products have the (S)-configuration at the chiral C11a position, which provides them with a right-handed twist when viewed from the C ring to the A ring. This provides them with a suitable three-dimensional shape to have isohelicity with the minor groove of the B-form DNA, thereby fitting tightly at the binding site. Their ability to form adducts in the minor groove enables them to interfere with DNA processing, thus arguing for their use as antitumor agents.

The first PBD antitumor antibiotic, anisomycin, was discovered in 1965. Since then, many naturally occurring PBDs have been reported, and more than 10 synthetic routes have been developed to a variety of analogs. Family members include abbeymycin, chicamycin, DC-81, mazethramycin, neothramycin A and B, porothramycin, prothracarcin, sibanomicin (DC-102), sibiricin, and tomamycin. PBDs and ADCs comprising them are also described in WO 2015/155345 and WO 2015/157592, which are incorporated herein by reference in their entirety. Linkers

In the ADC of the present disclosure, the antibody or antigen-binding fragment can be conjugated to the cytotoxin via a linker.

As used herein, the term "linker" or "spacer" means a bivalent chemical moiety comprising a covalent bond or a 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. Suitable linkers have two reactive ends, one for antibody conjugation and the other for cytotoxin conjugation. Due to the formation of bonds between the linker and/or cytotoxin and between the linker and/or antibody or antigen-binding fragment thereof, one or both reactive ends will not be present or will be incomplete (e.g., because it is only a carbonyl group of a carboxylic acid). Such conjugation reactions are discussed in more detail below.

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.

In some embodiments, the linker is cleavable in an intracellular environment, thereby allowing the drug unit to be released from the antibody in the intracellular environment.

Alternatively, the linker unit may be non-cleavable. In such embodiments, the drug is released, for example, by antibody degradation. However, non-cleavable payloads require complete digestion of the mAb in lysosomes, and the resulting drug-containing product may be too polar, for example, to achieve a bystander effect.

ADCs are preferably stable and intact before being transported or delivered into cells, i.e., the antibody should be attached to the drug moiety. Outside the target cell, linkers are stable, but inside the cell, they can be cleaved at a high rate. Effective linkers will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact (i.e., not cleaved) until the conjugate is delivered or transported to its target site; and (iv) maintain the cytotoxic or cytostatic effect of the cytotoxic moiety. Standard analytical methods (such as mass spectrometry, HPLC, and separation/analysis techniques LC/MS) can be used to assess the stability of ADCs.

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

Linkers that can be hydrolyzed under acidic conditions include, for example, hydrazones, semicarbazides, thiosemicarbazides, uridines, orthoesters, acetals, ketals, and the like (see, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Ohtsuka et al. , 1989, Biol. Chem. 264:14653-14661). Linkers that can be cleaved under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-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 (CW Vogel et al., 1996). ed., Oxford U. Press, 1987).

In certain embodiments, the linker is susceptible to enzymatic hydrolysis. In certain embodiments, such linkers may be superior to pH-sensitive cleavable linkers, which may not be stable enough and prematurely cleave before reaching the target cell, and therefore potential off-target toxicity may be observed. Enzymatically cleavable linkers may be, for example, peptide-containing linkers that are cleaved by intracellular peptidases or proteases (including but not limited to lysosomal or endosomal proteases). One advantage of using intracellular proteolytic release of therapeutic drugs is that the agent is generally weakened when conjugated, and the serum stability of the conjugate is generally 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 comprising the amino acids valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine are examples of suitable peptides. Natural amino acids, minor amino acids, and non-naturally occurring amino acid analogs, such as citrulline, are examples of amino acid residues that constitute the amino acid linker component. 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.

In some embodiments, the linker comprises PEG. A stable protease-cleavable linker containing PEG can limit the hydrophobicity of the effective load and can selectively cleave and release free drugs in target cancer cells. The low hydrophobicity of the linker as described herein can enable high drug loading onto the antibody or antigen-binding fragment (e.g., DAR8) without aggregation, which will be significantly higher than that of Socin-Metuximab (DAR3-4) or its derivatives, such as IMGN151 (DAR3.5). This can enable ADC to deliver significantly higher concentrations of cytotoxic effective loads to target cancer cells by binding to FRα on cancer cells.

In some embodiments, the linker comprises a maleimide. The use of maleimide in the linker can allow the generation of DAR8 and DAR4 ADCs by using the native interchain disulfides in the antibody. This is superior to the fusion of surface amines to lysine residues, which can result in a mixture of DAR species and batch-to-batch variability. If the fusion site interferes with antigen binding, there may also be reproducibility issues that affect ADC efficacy. In addition, other fusion methods, such as azide-alkyne click chemistry involving engineered antibodies may not easily achieve DARs exceeding 4.

In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof in the ADC disclosed herein is linked to a cytotoxin via a linker ^RL selected from the following: (Ia): , where Q is: , wherein Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; and X is: wherein a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, wherein 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); ^GL is a linker for linking to an antibody or an antigen-binding fragment thereof in the ADC of the present disclosure; (Ib): , wherein R ^L1 and R ^L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group; and e is 0 or 1; or (Ib') , where R ^L1 and R ^L2 are as defined above.

By way of example, preferred embodiments of ^GL , X, ^QX (such as in the above-mentioned connector of Ia) and the connector of Ib will be outlined.

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

Various definitions relating to certain terms in this section are provided below under the heading "Chemical Definitions". G ^L G ^L may be selected from: (G ^L1-1 ) (G ^L6 ) (G ^L1-2 ) (G ^L7 ) (G ^L2 ) (G ^L8 ) (G ^L3-1 ) The NO ₂ group is optional (G ^L9 ) (G ^L3-2 ) The NO ₂ group is optional (G ^L10 ) (G ^L3-3 ) The NO ₂ group is optional (G ^L11 ) (G ^L3-4 ) The NO ₂ group is optional (G ^L12 ) (G ^L4 ) Where Hal = I, Br, Cl (G ^L13 ) (G ^L5 ) (G ^L14 ) wherein Ar represents a C5-6 aryl group, such as phenylene, and X represents a C1-4 alkyl group.

In some embodiments, ^GL is selected from ^GL1-1 and ^GL1-2 . In some of these embodiments, ^GL is ^GL1-1 .

X is better: Where a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c = 0 or 1, d = 0 to 5, where at least b1 or b2 = 0 and at least c1 or c2 = 0.

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 other embodiments, a is 0.

b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

b2 may 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.

c1 can be 0 or 1. c2 can be 0 or 1. Preferably, only one of c1 and c2 can be non-zero.

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 other embodiments, d is 2. In other embodiments, d is 5.

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, c2 is 0 and one of a and d is 0. The other of a and d is from 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 from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8. Q ^X

In some embodiments, Q is an amino acid residue. The amino acid can be a natural amino acid or a non-natural amino acid. For example, Q can be selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, wherein Cit is citrulline.

In some embodiments, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. When the linker is a histase-labile linker, the dipeptide is the site of action for histase-mediated cleavage. Then, the dipeptide is the recognition site for histase.

In some embodiments, Q is selected from: ^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 ; wherein Cit is citrulline.

Preferably, Q is selected from: ^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 .

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

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 .

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

In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. When the linker is a tissue protease unstable linker, the tripeptide is the site of action for tissue protease-mediated cleavage. Then, the tripeptide is the recognition site for tissue proteases. 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

In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. When the linker is a tissue protease unstable linker, the tetrapeptide is the site of action for tissue protease-mediated cleavage. Then, the tetrapeptide is the recognition site for tissue proteases. Tetrapeptide linkers of particular interest are: ^NH -Gly-Gly-Phe-Gly ^C=O ; and ^NH -Gly-Phe-Gly-Gly ^C=O .

In some embodiments, the tetrapeptide is: ^NH -Gly-Gly-Phe-Gly ^C=O .

In the above representation of peptide residues, ^NH- represents the N-terminus of the residue, and ^-C=O represents the C-terminus of the residue. The C-terminus is bound to the NH of a "drug unit" (e.g., A* discussed below).

Glu represents the residue of glutamic acid, that is:

αGlu represents the residue of glutamine when bound via the α chain, i.e.:

In some embodiments, the amino acid side chains are chemically protected, where appropriate. The side chain protecting groups can be groups as discussed above. The protected amino acid sequence can be cleaved by an enzyme. For example, a dipeptide sequence containing a Lys residue protected by a Boc side chain can be cleaved by a tissue protease.

Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalog and as described above .

R ^L1 and R ^L2 may be independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group.

In some embodiments, both ^RL1 and ^RL2 are H. In some embodiments, ^RL1 is H and ^RL2 is methyl. In some embodiments, both ^RL1 and ^RL2 are methyl.

In some embodiments, ^RL1 and ^RL2 together with the carbon atoms to which they are bonded form a cyclopropene group. In some embodiments, ^RL1 and ^RL2 together with the carbon atoms to which they are bonded form a cyclobutene group.

In group Ib, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group can be in any available position of the ring. In some of these embodiments, it is located in the ortho position. In other of these embodiments, it is located in the para position. R ^L

In some implementations, R ^L is selected from: (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x)

Preferably, R ^L is .

For example, the antibody-drug conjugate of the present disclosure may have the general formula IV: L-( ^DL ) _p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is the antibody or antigen-binding fragment thereof in the ADC of the present disclosure, ^DL is a "drug unit" (e.g., a cytotoxin, such as TOPOi), which has a linker ^RLL linked to the antibody or antigen-binding fragment thereof in the ADC of the present disclosure, wherein the linker is preferably selected from (Ia'): , wherein Q and X are as defined above, and ^GLL is a linker linked to the antibody or antigen-binding fragment thereof in the ADC of the present disclosure; and (Ib'): , wherein R ^L1 and R ^L2 are as defined above; and p is an integer from 1 to 20.

The drug loading is represented by p, which is the number of "drug units" (e.g., cytotoxins such as TOPOi) per antibody or antigen-binding fragment thereof. The drug loading per antibody or antigen-binding fragment thereof may range from 1 to 20 drug units (D). For a composition, p represents the average drug loading of the conjugates in the composition, and p ranges from 1 to 20. In some embodiments, p ranges from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10; preferably, p is 8. G ^LL

G ^LL can be selected from: (G ^LL1-1 ) (G ^LL8-1 ) (G ^LL1-2 ) (G ^LL8-2 ) (G ^LL2 ) (G ^LL9-1 ) (G ^LL3-1 ) (G ^LL9-2 ) (G ^LL3-2 ) (G ^LL10 ) (G ^LL-4 ) (G ^LL11 ) (G ^LL5 ) (G ^LL12 ) (G ^LL6 ) (G ^LL13 ) (G ^LL7 ) (G ^LL14 ) wherein Ar represents a C _5-6 arylene group, such as phenylene, and X represents a C _1-4 alkyl group.

In some embodiments, ^GLL is selected from ^GLL1-1 and ^GLL1-2 . In some of these embodiments, ^GLL is ^GLL1-1 .

In some embodiments, R ^LL is a group derived from the above-mentioned R ^L group.

Those skilled in the art will recognize that any one or more of the chemical groups, moieties and features disclosed herein can be combined in a variety of ways to form linkers that can be used to conjugate the antibodies and cytotoxins disclosed herein.

In some embodiments where the compounds described herein are provided as a single image isomer or in an image isomer-enriched form, the image isomer-enriched form has a ratio greater than 60:40, 70:30, 80:20, or 90:10. In other embodiments, the image isomer ratio is greater than 95:5, 97:3, or 99:1. Specific ADC embodiments

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor, which is represented by the following compound having Formula “I”: , wherein ^RL is as defined above; wherein the range of DAR is about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In a specific embodiment, the DAR is about 8.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to a topoisomerase I inhibitor, which is represented by the following compound having Formula “I”: , wherein ^RL is as defined above; wherein the range of DAR is about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In a specific embodiment, the DAR is about 4.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG3932. wherein 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 a specific embodiment, the DAR is about 8.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG3932. wherein 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 a specific embodiment, the DAR is about 4.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG4010. wherein 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 a specific embodiment, the DAR is about 8.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG4010. wherein 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 a specific embodiment, the DAR is about 4.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG4057. wherein 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 a specific embodiment, the DAR is about 8.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG4057. wherein 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 a specific embodiment, the DAR is about 4.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG4052. wherein 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 a specific embodiment, the DAR is about 8.

In some embodiments, the anti-FRα ADC disclosed herein comprises an anti-FRα antibody or an antigen-binding fragment thereof described herein conjugated to the topoisomerase I inhibitor SG4052. wherein 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 a specific embodiment, the DAR is about 4.

In some embodiments, (i) an anti-FRα antibody or an antigen-binding fragment thereof comprises a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, a light chain CDR2 (KASGLES) of SEQ ID NO: 5, and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, optionally wherein the anti-FRα antibody or an antigen-binding fragment thereof has a VH and a VL, the VH having 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 the VL having 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. NO:38, wherein the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO:49 and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO:50; (ii) the cytotoxin is a topoisomerase I inhibitor represented by the following compounds having Formula I: , wherein ^RL is as defined above; and (iii) wherein the range of DAR is about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In a specific embodiment, the DAR is about 8.

In some embodiments, (i) an anti-FRα antibody or an antigen-binding fragment thereof comprises a heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, a heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, a heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, a light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, a light chain CDR2 (KASGLES) of SEQ ID NO: 5, and a light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6, optionally wherein the anti-FRα antibody or an antigen-binding fragment thereof has a VH and a VL, the VH having 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 the VL having 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. NO:38, wherein the anti-FRα antibody or antigen-binding fragment thereof has a heavy chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO:49 and a light chain amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical or identical to the amino acid sequence of SEQ ID NO:50; (ii) the cytotoxin is a topoisomerase I inhibitor represented by the following compounds having Formula I: , wherein ^RL is as defined above; and (iii) wherein the range of DAR is about 1 to 20, optionally the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. In a specific embodiment, the DAR is about 4.

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

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

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

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

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

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

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

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

Internalization can be a useful property of an ADC. For example, internalization allows for delivery of a payload to cells. The inventors have shown that the antibodies and ADCs described herein exhibit rapid internalization and lysosomal trafficking.

In some embodiments, the anti-FRα ADCs of the present disclosure bind to FRα on the surface of cells and are internalized into the cells. In some embodiments, internalization of the anti-FRα ADCs of the present disclosure into cells expressing FRα is saturated within about 4 hours or less, about 5 hours or less, about 6 hours or less, about 7 hours or less, about 8 hours or less, about 9 hours or less, about 10 hours or less, about 11 hours or less, or about 12 hours or less. Cytotoxicity

In some embodiments, the anti-FRα ADCs of the present disclosure inhibit or suppress proliferation (e.g., proliferation of a tumor) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% (in specific embodiments, at least 40%) relative to the level of inhibition or suppression in the absence of the anti-FRα ADC. Cell proliferation can be measured using art-recognized techniques that measure the rate of cell division, and/or the fraction of cells in a cell population that undergo cell division, and/or the rate of cell loss in a cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

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

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

In some embodiments, the anti-FRα ADC disclosed herein inhibits or suppresses the proliferation of cell populations with heterogeneous expression of FRα and/or low expression of FRα. In some embodiments, the anti-FRα ADC disclosed herein inhibits or suppresses the proliferation of cell populations with moderate FRα expression (e.g., Jeg-3, OVCAR-3 cell lines, or cells with similar or equivalent levels of FRα expression), the proliferation of cell populations with medium to high FRα expression (e.g., Igrov-1 cell lines, or cells with similar or equivalent levels of FRα expression), and the proliferation of cell populations with high expression of FRα (e.g., KB cell lines, or cells with similar or equivalent levels of FRα expression). Preparation of ADC

The ADCs of the present disclosure can be prepared in a variety of ways using known organic chemical reactions, conditions, and reagents, such as: (1) reacting a reactive substituent of an antibody or antigen-binding fragment with a divalent linker reagent, followed by a cytotoxin (e.g., a topoisomerase I inhibitor); or (2) reacting a reactive substituent of a cytotoxin (e.g., a topoisomerase I inhibitor) with a divalent linker reagent, followed by a reactive substituent of an antibody or antigen-binding fragment thereof.

Reactive substituents that may be present within an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, nucleophilic groups such as (i) an N-terminal amine group, (ii) pendant amine groups such as lysine, (iii) pendant thiol groups such as cysteine, and (iv) sugar hydroxyl groups or amine groups where the antibody is glycosylated. Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amine moieties of lysine residues; carboxyl moieties of aspartic acid and glutamine residues; and thiol moieties of cysteine residues, as well as propargyl, azido, halogenated aryl (e.g., fluoroaryl), halogenated heteroaryl (e.g., fluoroheteroaryl), halogenated alkyl, and halogenated heteroalkyl moieties of non-naturally occurring amino acids. In some embodiments, reactive substituents present in an antibody or antigen-binding fragment thereof as disclosed herein include amine moieties or thiol moieties. Certain antibodies have cysteine bridges, which are reducible interchain disulfides. Antibodies can be made reactive for conjugation with linker agents 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. Reaction of lysine with 2-imidosulfane (Traut's reagent) results in conversion of amines to thiols, which can be used to introduce additional nucleophilic groups into antibodies. One, two, three, four or more cysteine residues can be used to insert reactive thiol groups into antibodies (or fragments thereof) (e.g., to prepare mutant antibodies comprising one or more non-natural cysteine amino acid residues). U.S. Patent No. 7,521,541 describes engineered antibodies having reactive cysteine amino acids and is incorporated herein by reference.

In another aspect, the antibody or antigen-binding fragment thereof may have one or more carbohydrate groups that can be chemically altered to contain one or more hydroxyl groups. The ADC is then formed by fusion of the sulfur atoms of the hydroxyl groups.

In yet another aspect, the antibody may contain one or more carbohydrate groups that can be oxidized to generate an aldehyde group (-CHO) (see, e.g., Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). Conjugation of the corresponding aldehyde results in the formation of the ADC. Additional protocols for modifying proteins, for attaching or conjugating 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) have been discovered, 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.

Conventional conjugation strategies for antibodies or antigen-binding fragments thereof rely on arbitrary or random conjugation of a payload to an antibody or fragment via lysine or cysteine. In some embodiments, an antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxin (e.g., a topoisomerase I inhibitor), for example, by partial reduction of the antibody or fragment followed by reaction with the desired agent (with or without an attached linker moiety). The antibody or fragment may be reduced using DTT or other reducing agents for similar reduction, such as TCEP. The agent, with or without a linker moiety attached, may then be added to the reduced antibody or fragment in molar excess in the presence of DMSO. After ligation, a quencher such as N-acetyl-L-cysteine can be added to quench unreacted reagents. The reaction mixture can then be purified (by, e.g., TFF, SEC-FPLC, CHT, spin filter centrifugation) and the buffer exchanged into PBS or other relevant formulation buffer.

In some embodiments, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof by site-specific conjugation. In some embodiments, site-specific conjugation of the therapeutic moiety to the antibody using reactive amino acid residues at specific positions produces a homogeneous ADC preparation with consistent stoichiometry.

The site-specific conjugation can be carried out via cysteine residues or non-natural amino acids. In a specific embodiment, the cytotoxin is conjugated to the antibody or its antigen-binding fragment via at least one cysteine residue. The cysteine amino acid can be engineered at the reactive site in the antibody (or its antigen-binding fragment) and preferably does not form intra-chain or intermolecular disulfide bonds (Junutula et al. , 2008b Nature Biotech., 26(8):925-932; Dornan et al., (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO 2009/052249). In some embodiments, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof by 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, wherein the numbering corresponds to the EU index in Kabat. In some embodiments, the specific 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 fused to the antibody or antigen-binding fragment thereof via a thiol-maleimide bond. In some aspects, the amino acid side chain is a hydroxy side chain.

When more than one nucleophilic or electrophilic group of an antibody or antigen-binding fragment thereof reacts with a cytotoxic agent, the resulting product may be a mixture of ADCs in which the drug units attached to the antibody are distributed as, for example, 1, 2, 3, etc. Liquid chromatography methods such as hydrophobic interaction (HIC) can separate the compounds in the mixture by drug loading values. ADC preparations with a single drug loading value (p) can be isolated.

The average amount of cytotoxic agent per antibody (or antigen binding fragment) in an ADC preparation resulting from the fusion reaction can be characterized by conventional methods such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of ADCs according to p can also be determined. By ELISA, the average value of p in a particular ADC preparation can be determined (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11:843-852). In some cases, separation, purification, and characterization of homologous ADCs (where p is a value derived from the antibody and the other agent) can be achieved by reverse phase HPLC, electrophoresis, TFF, SEC-FPLC, CHT, spin filter centrifugation, etc. Such techniques are also applicable to other types of conjugates. Chemical Definition

The following definitions apply in particular to the above description of topoisomerase I inhibitors.

C _5-6 arylene group: As used herein, the term “C _5-6 arylene group” relates to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.

As used herein, a prefix (eg, C _5-6 ) indicates the number of ring atoms or a range of numbers of ring atoms, whether carbon atoms or heteroatoms.

The ring atoms may be all carbon atoms, as in a "carbonylene group", in which case the group is phenylene (C ₆ ).

Alternatively, the ring atoms may include one or more heteroatoms as in a "heteroaryl group". Examples of heteroaryl groups include, but are not limited to, those derived from: N ₁ : pyrrole (azole) (C ₅ ), pyridine (azine) (C ₆ ); O ₁ : furan (oxole) (C ₅ ); S ₁ : thiophene (thiole) (C ₅ ); N ₁ O ₁ : oxazole (C ₅ ), isoxazole (C ₅ ), isoxazine (C ₆ ); N ₂ O ₁ : oxadiazole (furazan) (C ₅ ); N ₃ O ₁ : oxatriazole (C ₅ ); N ₁ S ₁ : thiazole (C 5 ), isothiazole (C ₅ ); N ₂ : imidazole (1,3-oxadiazole) (C ₅ ), pyrazole (1,2-oxadiazole) (C ₅ ), indole (1,2-dioxadiazole) (C ₆ ), pyrimidine (1,3-dioxadiazole) (C ₆ ) (e.g., cytosine, thymine, uracil), pyridine (1,4-dioxadiazole) (C ₆ ); and N ₃ : triazole (C ₅ ), trioxadiazole (C ₆ ).

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 alkyl compound having from 1 to 4 carbon atoms, which alkyl compound 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 alkyl compound having from 1 to n carbon atoms, which alkyl compound may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the subclasses discussed below: alkenyl, alkynyl, cycloalkyl, etc.

Examples of saturated alkyl groups include, but are not limited to, methyl (C ₁ ), ethyl (C ₂ ), propyl (C ₃ ) and butyl (C ₄ ).

Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C ₁ ), ethyl (C ₂ ), n-propyl (C ₃ ) and n-butyl (C ₄ ).

Examples of saturated branched alkyl groups include isopropyl (C ₃ ), isobutyl (C ₄ ), dibutyl (C ₄ ) and tertiary butyl (C ₄ ).

C _2-4 alkenyl; As used herein, the term "C _2-4 alkenyl" relates to an alkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl) (-CH=CH ₂ ), 1-propenyl (-CH=CH-CH ₃ ), 2-propenyl (allyl, -CH-CH=CH ₂ ), isopropenyl (1-methylvinyl, -C(CH ₃ )=CH ₂ ) and butenyl (C ₄ ).

C _2-4 alkynyl: As used herein, the term "C _2-4 alkynyl" relates to an alkyl group having one or more carbon-carbon triple bonds.

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

_C3-4 cycloalkyl: As used herein, the term " _C3-4 cycloalkyl" relates to an alkyl group that is also a cycloalkyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic alkyl (carbocyclic) compound, the moiety having from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C ₃ ) and cyclobutane (C ₄ ); and unsaturated monocyclic hydrocarbon compounds: cyclopropene (C ₃ ) and cyclobutene (C ₄ ).

Connection label: In-line In , the superscripts ^C(=0) and ^NH indicate the groups to which the atoms are bonded. For example, an NH group is shown bonded to a carbonyl group (which is not part of the moiety shown), and a carbonyl group is shown bonded to an NH group (which is not part of the moiety shown).

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

For example, if the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be ^-COO- ), a salt can be formed with an appropriate cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca2 ⁺ and Mg2 ⁺ , and other cations such as Al ⁺³ . Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., _NH4 ⁺ ) and substituted ammonium ions (e.g. _{, NH3R} ⁺ , _NH2R2 ⁺ , _NHR3 ⁺ , _NR4 ⁺ ). Some examples of suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

If the compound is cationic, or has a functional group that can be cationic (e.g., _-NH2 can be _-NH3 ⁺ ), then a salt can be formed with an appropriate anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

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

It may be convenient or desirable to prepare, purify, and/or process 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. If the solvent is water, the solvate may be conveniently referred to as a hydrate, such as a monohydrate, a dihydrate, a trihydrate, etc. Isomers

Certain compounds/agents disclosed herein can exist in one or more specific geometric, optical, mirror-image, non-mirror-image, epimeric, atropisomer, stereoisomer, 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-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; cis- and trans-forms; syncline- and anticline-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and semi-chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isoconfigurations").

Unless otherwise indicated, references to a particular compound include all such isomeric forms, including (whole or partial) racemates and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by employing the methods taught herein or known methods in a known manner. PARP1 Inhibitors

The use of PARP inhibitors in BRCA mutant ovarian cancer is the first clinical example of the successful use of synthetic lethality to target tumor suppressor gene deletion (Ledermann, Harter et al . (2014) Lancet Oncol 15(8): 852-861). This finding is based on the finding that PARP and BRCA1 and BRCA2 are components of efficient DNA repair.

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

The term "AZD5305" refers to the following compound: It has the chemical name 5-[4-[(7-ethyl-6-oxo-5H-1,5-oxadiin-3-yl)methyl]piperidin-1-yl]-N-methyl-pyridine-2-carboxamide and the structure is shown below: .

AZD5305 is disclosed in U.S. Publication No. US 2021/0040084 A1, the disclosure of which is incorporated by reference in its entirety. In some embodiments, the PARP1 inhibitor is a 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.

In some embodiments of any aspect of the present disclosure, the PARP1 inhibitor also refers to a pharmaceutically acceptable salt thereof. Pharmaceutical composition

The ADC and/or PARP1 inhibitor of the present disclosure can be administered to a subject as a pharmaceutical composition. Therefore, in one aspect, the present disclosure provides a pharmaceutical composition comprising: (i) the ADC of the present disclosure; (ii) the PARP1 inhibitor of the present disclosure; and (iii) a pharmaceutically acceptable formulation.

The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient and does not contain additional components that are unacceptably toxic to the subject to which the composition is to be administered. Such a composition may be sterile and may contain a pharmaceutically acceptable vehicle, such as saline. Suitable pharmaceutical compositions may contain one or more buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., human albumin), preservatives (e.g., benzyl alcohol), and absorption enhancers for enhancing bioavailability, and/or other conventional solubilizing agents or dispersants.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia, the European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

In some embodiments, the pharmaceutical compositions according to the present disclosure and for use according to the present disclosure may contain, in addition to the active ingredients (ADC and PARP1 inhibitor of the present disclosure), pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredients. The exact nature of the vehicle or other materials will depend on the route of administration, which may be oral, or by injection (e.g., dermal, subcutaneous or intravenous).

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, optionally wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) the cytotoxin is a topoisomerase I inhibitor SG3932 ; (iii) the DAR of the ADC is about 8; and (iv) the PARP1 inhibitor is AZD5305 having the following formula: or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutical compositions disclosed herein may include a pharmaceutically acceptable nontoxic sterile carrier, such as physiological saline, nontoxic buffer, preservatives, etc. Suitable formulations for use in the treatment methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd edition, edited by Lloyd V. Allen, Jr. (2012).

Examples of suitable excipients include one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and any combination thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol, or sodium chloride, in the composition.

Those skilled in the art will appreciate that the appropriate choice of one or more excipients for use with the ADCs and/or PARP1 inhibitors of the present disclosure will depend on the desired properties of the drug composition.

In some embodiments, the pharmaceutical compositions of the present disclosure may be contained in one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, lyophilized powders (which may be reconstituted into suspensions or solutions before use), or combinations thereof.

In some embodiments, the pharmaceutical composition comprises more than one type of ADC and/or PARP1 inhibitor of the present disclosure.

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

The pharmaceutical compositions disclosed herein are used, but not limited to, for use in diagnosing, detecting or monitoring a disease, for use in preventing, treating, controlling or ameliorating a disease or one or more symptoms thereof, and/or for use in research. The pharmaceutical compositions disclosed herein may be suitable for veterinary use or human pharmaceutical use.

The pharmaceutical compositions disclosed herein can be administered to a patient by any suitable systemic or local administration route. For example, administration can be oral, buccal, sublingual, ocular, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular, or intratumoral.

The pharmaceutical compositions disclosed herein may be formulated for administration by any appropriate means, such as by topical or transdermal patches, ointments, lotions, creams or gels; by nebulizers, vaporizers or inhalers; 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 of administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. Tablets may contain a solid vehicle or adjuvant. Liquid pharmaceutical compositions generally contain a liquid vehicle, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. They may include physiological saline solutions, dextrose or other sugar solutions or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol. Capsules may contain a solid vehicle, such as gelatin.

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

In some embodiments, the ADC disclosed herein and the PARP1 inhibitor disclosed herein are formulated as two separate drug compositions. Therefore, in some embodiments, the ADC disclosed herein and the PARP1 inhibitor disclosed herein are administered separately or sequentially. Kit, therapeutic combination, article of manufacture, method for manufacturing a drug

In one aspect, a kit is provided, the kit comprising an ADC of the present disclosure and a PARP1 inhibitor of the present disclosure. Further contemplated is the use of the kit in a method of the present disclosure.

In one aspect, provided herein is a therapeutic combination for treating cancer in a human subject in need thereof, wherein the therapeutic combination comprises an ADC of the present disclosure and a PARP1 inhibitor of the present disclosure.

In one aspect, provided herein is an article of manufacture comprising: (i) an ADC of the present disclosure; (ii) a PARP1 inhibitor of the present disclosure; (iii) a pharmaceutically acceptable formulation; and (iv) instructions for administering the ADC and the PARP1 inhibitor in combination to a human subject in need of treatment for cancer.

In one aspect, provided herein are articles of manufacture comprising: (i) a pharmaceutical composition of the present disclosure; and (ii) instructions for administering a combination of an ADC of the present disclosure and a PARP1 inhibitor to a human subject in need of treatment for cancer.

In one aspect, the present invention provides a method for manufacturing a medicament, wherein the method comprises: (a) using the ADC disclosed herein and the PARP1 inhibitor disclosed herein; and (b) combining the ADC and the PARP1 inhibitor in a pharmaceutically acceptable carrier.

In some embodiments of any aspect of the disclosure, the ADC may have any antibody or antigen-binding fragment thereof as described herein. In some embodiments of any aspect of the disclosure, the ADC may have any linker as described herein. In some embodiments of any aspect of the disclosure, the ADC may have any cytotoxic agent as described herein.

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, optionally wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) the cytotoxin is the topoisomerase I inhibitor SG3932 ; (iii) the DAR of the ADC is about 8; and (iv) the PARP1 inhibitor is AZD5305 having the following formula: or a pharmaceutically acceptable salt thereof.

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

In some embodiments, the kit comprises one or more containers filled with one or more of the ADC and/or PARP1 inhibitors disclosed herein. Optionally, associated with such one or more containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products, which notice reflects the agency's approval of the manufacture, use, or sale for human administration. For example, instructions on how to use the provided pharmaceutical composition to treat cancers such as 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 may also be included or made available to patients or healthcare providers.

In some embodiments, a kit may provide an antigen or antigen-binding fragment and a cytotoxin (which is not fused to the antibody or antigen-binding fragment, but is in a form suitable for fusion therewith); optionally, wherein the kit further provides instructions and/or reagents for fusion of the cytotoxin to the antibody or antigen-binding fragment. In some embodiments, the kit contains all elements necessary and/or sufficient to perform a detection assay, including all controls, instructions for performing the assay, and any necessary software for analyzing and presenting the results. Therapy

The present disclosure encompasses methods for preventing, treating, or ameliorating symptoms associated with a disease, disorder, or infection, which methods involve administering to a subject a combination of an anti-FRα antibody-drug conjugate (ADC) of the present disclosure and a PARP1 inhibitor, a pharmaceutical composition comprising the anti-FRα ADC of the present disclosure and a PARP1 inhibitor, or a therapeutic combination comprising the anti-FRα ADC of the present disclosure and a PARP1 inhibitor.

"Treatment" refers to therapeutic measures that cure, slow down, reduce the symptoms of, and/or stop the progression of a diagnosed pathological condition or disorder. Therefore, those in need of treatment include those who already have a disorder. In some embodiments, if the patient shows, for example, complete, partial or temporary reduction or elimination of symptoms associated with the disease or disorder (preferably cancer), the subject's disease or disorder (preferably cancer) is successfully "treated" according to the methods provided herein.

"Prevention" refers to a preventive or prophylactic measure that prevents and/or slows the development of a targeted pathological condition or disorder. Thus, persons in need of prevention include those who are predisposed to or susceptible to the disorder. In some embodiments, a disease or disorder (preferably cancer) is successfully prevented according to the methods provided herein if the patient temporarily or permanently exhibits, for example, fewer or less severe symptoms associated with the disease or disorder, or the onset of symptoms associated with the disease or disorder is delayed compared to a patient not subjected to the methods of the present disclosure.

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, competition animal, and 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 a specific embodiment, the subject is a human. In the methods disclosed herein, the subject may not have been previously diagnosed as having cancer. Alternatively, the subject may have been previously diagnosed as having cancer. The subject may also be a person who exhibits risk factors for the disease, or a person who has no symptoms of cancer. The subject may also be a person who has cancer or is at risk of cancer. In some embodiments, the subject has been previously administered cancer therapy.

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

In one aspect, provided is a combination of an anti-FRα ADC of the present disclosure and 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, such as for treating a disease or disorder (e.g., cancer).

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

In one aspect, a PARP1 inhibitor of the present disclosure is provided for use in therapy, such as for treating a disease or disorder (such as cancer), wherein the treatment comprises administering an anti-FRα ADC of the present disclosure and a PARP1 inhibitor to a subject.

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

In one aspect, provided is a combination of an anti-FRα ADC of the present disclosure and 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 preventing the onset of a disease or disorder (e.g., cancer).

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

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

The term "therapeutically effective amount" is an amount sufficient to show benefit to the patient. This benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and schedule of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment (e.g., determination of dosage) is the responsibility of general practitioners and other physicians.

In one aspect, a method for treating cancer is provided, the method comprising administering to a subject a combination of an anti-FRα ADC of the present disclosure and a PARP1 inhibitor of the present disclosure; a pharmaceutical composition of the present disclosure; or a therapeutic combination of the present disclosure.

In one aspect, provided is a combination of an anti-FRα ADC of the present disclosure and 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 treating cancer.

In one aspect, an anti-FRα ADC of the present disclosure for use in treating cancer is also provided, wherein the treatment comprises administering the anti-FRα ADC of the present disclosure and a PARP1 inhibitor to a subject.

In one aspect, the PARP1 inhibitor of the present disclosure is also provided for use in treating cancer, wherein the treatment comprises administering the anti-FRα ADC of the present disclosure and the PARP1 inhibitor to a subject.

In some embodiments, the subject is a human subject in need.

In some embodiments, cancer is associated with FRα expression. In other words, the cancer mentioned herein may include cancer cells expressing FRα. The cancer cells may be contained within a tumor. In some embodiments, the cancer is a tumor or other malignant cell cluster comprising cancer cells expressing FRα. In some embodiments, the cancer comprises cancer cells with heterogeneous expression of FRα and/or low expression of FRα. On the other hand, the FRα molecule is expressed in cancer cells at a level similar to that in non-cancerous cells. On the other hand, the FRα molecule is expressed in cancer cells at a level lower than that in non-cancerous cells. In some embodiments, the cancer further comprises cancer cells that do not express FRα.

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 (NSCLCs), which in certain embodiments can be selected from squamous NSCLCs, adenocarcinoma NSCLCs, or combinations thereof.

Additional examples of cancer include, but are not limited to, benign, pre-malignant and malignant cell proliferations, including, but not limited to, tumors and neoplasms (e.g., histiocytoma, neuroglioma, 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 kidney cancer). Any type of cell may be treated, including, but not limited to, lung, gastrointestinal, breast (breast), ovarian, renal, and pancreatic.

In some embodiments, the cancer is a homologous recombination defective (HRD) cancer. In some embodiments, the cancer comprises one or more cells having mutations in HRD genes selected from the following: BRCA1 , BRCA2 , ATM , BRIP1 , BARD1 , CDK12 , CHEK1 , CHEK2 , FANCL , PALB2 , PPP2R2A , RAD51B , RAD51C , RAD51D and RAD54L . Mutations in HRD genes can also be referred to as mutations in genes 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 .

In one aspect, a method for eliminating a FRα-positive cell population in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a combination of an anti-FRα ADC of the present disclosure and a PARP1 inhibitor of the present disclosure; a pharmaceutical composition of the present disclosure; or a therapeutic combination of the present disclosure.

In one aspect, provided is a combination of an anti-FRα ADC of the present disclosure and 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 eliminating a FRα-positive cell population in a subject.

In one aspect, an anti-FRα ADC of the present disclosure is also provided for use in a method for depleting a FRα-positive cell population in a subject, wherein the method comprises administering to the subject an anti-FRα ADC of the present disclosure and a PARP1 inhibitor.

In one aspect, the PARP1 inhibitor of the present disclosure is also provided for use in a method for eliminating a FRα-positive cell population in a subject, wherein the method comprises administering the anti-FRα ADC of the present disclosure and the PARP1 inhibitor to the subject.

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

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.

In an embodiment 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, optionally wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) the cytotoxin is the topoisomerase I inhibitor SG3932 ; (iii) the DAR of the ADC is about 8; and (iv) the PARP1 inhibitor is AZD5305 having the following formula: or a pharmaceutically acceptable salt thereof.

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

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.

The above embodiments are to be understood as illustrative examples. Additional embodiments are contemplated. It should be understood that any feature described with respect to any of the embodiments may be used alone or in combination with other features described, and may also be used in combination with one or more features of any other embodiment or aspect, or any combination of any other embodiment or aspect. In addition, equivalents and modifications not described above may also be employed without departing from the scope of the present disclosure as defined by the appended claims.

In the context of the present disclosure, other embodiments and variations of the antibodies and methods described herein will be apparent to those skilled in the art. Other embodiments and variations are within the scope of the present disclosure as shown in the appended claims.

All references cited herein are incorporated herein by reference in their entirety, including all data, tables, figures and text presented in the references. Examples Example 1 - Generation of anti-FRα antibodies Objectives

Humanized transgenic mice were used for hybridoma campaigns to generate high-affinity, fully human antibodies that bind to folate receptor α (FRα). Materials and Methods Production of human FRα for immunization

The human FRα gene was inserted into the pDEST12.2 OriP vector. A sequence corresponding to the soluble region of the folate receptor was further cloned, with an N-terminal CD33 leader sequence and C-terminal Avi and His6 tags. The sequence responsible for GPI anchoring was removed to generate a soluble construct.

Human FRα protein was expressed and purified using standard methods. Briefly, plasmid DNA was prepared and transfected into a self-suspension adapted CHO cell line using PEI-mediated delivery at a cell density of 4 x 10 ⁶ cells/ml at the point of transfection. Cells were cultured for 7 days at 34°C, 5% CO ₂ , 140 rpm, 70% humidity. Conditioned media was harvested and purified using a 5 ml HisTrap excel column (Cytiva) for affinity capture and then polished on a HiLoad Superdex 75 16/600 pg column (Cytiva) equilibrated in DPBS. Fractions were analyzed for purity by SDS-PAGE, pooled and their concentrations determined by UV absorbance, and flash frozen in liquid nitrogen before storage at -80°C. For site-specific biotinylation of the protein on its Avi tag, the protein was incubated with recombinant BirA enzyme, ATP and biotin and then purified on size exclusion chromatography as described above. Antibodies were expressed as described for the target antigen and purified using protein A chromatography. Immunization

A repeated immunization multisite (RIMMS) strategy was employed, in which Del-1 humanized mice and CD1 wild-type mice were immunized as follows: -   4 days: before bleed -   0 days: initial immunization -   7 days: second boost -   13 days: first bleed -   15 days: third boost -   20 days: second bleed -   22 days: fourth boost -   24 days: fifth boost -   28 days: terminal bleed and spleen (SP) and lymph node (LN) fusion For immunization, six humanized Del-1 mice and four CD1 wild-type mice were divided into two groups (Group 1: Del-1 mice 1-6, Group 2: CD1 mice 7-10). Animals were immunized with the recombinant human FRα extracellular domain as discussed 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 next three injections, the immunogen was emulsified in incomplete Freund's adjuvant and injected as above. The final boost was performed on day 24 by intraperitoneal injection of the recombinant protein in PBS.

Tail vein blood was obtained from mice 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. Evaluation of the immune response of mice 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 HRP-labeled multiplexed goat anti-mouse IgG-specific secondary antibodies (Jackson Immunolabs), and assays were developed using TMB substrate (Sigma), followed by the addition of 0.5 M sulfuric acid to stop the reaction. Plates were then read using a PerkinElmer EnVision 2103 multilabel plate reader.

Serum titration curves against human FRα and negative protein control were plotted and the corresponding area under the curve (AUC) was calculated. Single-selection mouse IgG isolation (hybridoma generation)

Four days after the last boost, lymph nodes were harvested aseptically and cells were separated by mechanical disruption and then counted. The cells were mixed with SP2/0 myeloma cells and fused using an electrofusion device. The resulting fusion was mixed with a methylcellulose-based semi-solid medium and plated into an OmniTray plate. The cells in the semi-solid medium were cultured at 37°C in a 5% _CO2 incubator for 13 days. During this incubation period, selected colonies were formed by single pioneer cell fusion tumor cells. The colonies secreted IgG, which was captured near the colonies by FITC-conjugated anti-IgG present in the semi-solid medium. When visualized by the ClonePix FL colony picker (Molecular Devices), the resulting immune complex formation can be observed around the cells as a fluorescent 'halogen'. The halogenated colonies are then picked into 96-well microtiter plates. After 3-5 days of culture, the supernatants of picked colonies are harvested and screened for human FRα binding. DNA Sequencing of Mouse IgG

Messenger RNA (mRNA) was extracted from fusion tumor cells using magnetic oligo(dT) particles and reverse transcribed into cDNA. PCR amplification was performed using poly-C and constant region VH or VL primers specific for all mouse IgG subclasses. PCR amplicon was sequenced by Sanger sequencing. Mouse IgG Phynexus Purification

Cells were propagated in 24-well plates and after 10 days, supernatants were transferred to 96-well master blocking buffer and all subclasses of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) were purified from the overgrown cell culture supernatant using a PerkinElmer Minitrack on ProPlus resin (Phynexus). The 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 reading at 280 nm in a UV-Star 384-well plate. Reconstitution of mouse IgG

For each hybridoma, mouse hybridoma IgG clones were molecularly reshaped to generate constructs expressing mouse VH and VL domains and relevant mouse IgG constant domains, essentially as described by Persi et al., Gene 187:9-18, 1997. The VH domains were cloned into relevant vectors containing mouse heavy chain constant domains and regulatory elements to express complete IgG1 heavy chains in mammalian cells. Similarly, the VL domains were cloned into vectors for expressing appropriate mouse light chain (λ or κ) constant domains and regulatory elements to express complete IgG light chains in mammalian cells. To obtain IgG, mammalian suspension CHO cells were transfected with heavy chain and light chain IgG vectors. IgG was expressed and secreted into the culture medium. IgG was purified from the clarified supernatant using MabSelect SuRe columns (GE Healthcare Lifesciences Catalog No. 11003493 (for 1 ml columns); 11003495 (for 5 ml columns)) and the AktaXpress™ Purification System from GE Healthcare Lifesciences. The eluted material was buffer exchanged into PBS using a PD-10 desalting column (GE Healthcare Lifesciences; Catalog No. 17085101). The concentration of IgG was determined spectrophotometrically using the extinction coefficient based on the IgG amino acid sequence (Pace et al., Protein Sci. 4:2411-2423, 1995), and the purity of the purified IgG was analyzed using SDS-PAGE and HP SEC analysis. Results Anti- FRα IgG titer in serum after immunization

Many approaches can be used for mAb discovery, including phage display, immunization, and the use of binding repertoires (which will involve the identification of mAbs that compete with folate for binding to FRα). Here, a dual approach was used to generate anti-FRα antibodies by immunization. The first approach involved immunization of human transgenic mice (i.e., Del-1) that contain complete human VH and Vk domains in the Ig locus. This ensured that a high diversity of mAbs would be generated and that they would moreover be fully humanized and could be used for development without the need for humanization. The second approach involved immunization of non-transgenic mice (i.e., CD-1), where humanization of the mAbs would be required.

On days 4 (pre-bleed), 13 (bleed 1), and 20 (bleed 2) of the immunization schedule, serum samples were collected from all mice and tested for the presence of anti-FRα specific antibodies. All mice responded to the immunization by producing anti-FRα specific antibodies.

A total of 10,510 fusion tumor colonies were generated, of which 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 picked colonies were harvested and screened for human FRα binding. Lead antibodies generated (see Example 2) were all obtained from Del-1 transgenic mice. Example 2 - Species Cross-Reactivity of Anti-FRα Antibodies OBJECTIVE

The antibodies generated are characterized by their binding strength, heterologous and homologous specificity to the target. Materials and Methods Antigen Generation for Assays

Table 9 indicates the species from which the protein was derived, the vector into which the construct was cloned, the signal peptide and the epitope tag fused to the protein. Table 10 further shows the sequence of each insert. In each case, a sequence corresponding to the soluble region of each folate receptor was cloned, with an N-terminal CD33 leader sequence and C-terminal Avi and His6 tags. The sequence responsible for GPI anchoring was removed to generate a soluble construct. [ Table 9 ]: Summary of cloned inserts Selected inserts Carrier Signaling peptide Epitope tag Human FRα pDEST12.2 OriP CD33 C-terminal Avi-His6 Cynomolgus monkey FRα pDEST12.2 OriP CD33 C-terminal Avi-His6 Mouse FRα pDEST12.2 OriP CD33 C-terminal Avi-His6 Human FRβ pDEST12.2 OriP CD33 C-terminal Avi-His6 Human FRγ pDEST12.2 OriP CD33 C-terminal Avi-His6 [ Table 10 ]: Sequence of insert SEQ ID NO Sequence Content Description sequence SEQ ID NO: 138 Human FRα ECD-Avi-His RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWED CRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGGGGSGLNDIFEAQKIEWHEAAHHHHHHHHHH SEQ ID NO: 139 Human FRβ ECD-Avi-His QDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVPLCKEDCQRWWEDC HTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVARFYAAAMHGGGGSGLNDIFEAQKIEWHEAAHHHHHHHHHH SEQ ID NO: 140 Human FRγ ECD-Avi-His SARARTDLLNVCMNAKHHKTQPSPEDELYGQCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKERILNVPLCKEDCERWWEDCRTSYT CKSNWHKGWNWTSGINECPAGALCSTFESYFPTPAALCEGLWSHSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGAPSRGIIDSGGGGSGLNDIFEAQKIEWHEAAHHHHHHHHHHH SEQ ID NO: 141 Cynomolgus FRα ECD-Avi-His RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQW WEDCRTSYTCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGLNDIFEAQKIEWHEAAHHHHHHHHHH SEQ ID NO: 142 Mouse FRα ECD-Avi-His TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTNSCCSTNTSQEAHKDISYLYRFNWNHCGTMTSECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERILDVPLCKEDCQQWWEDC QSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTSAALCEEIWSHSYKLSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAEAMSGGGGSGLNDIFEAQKIEWHEAAHHHHHHHHHHH

All proteins in Table 10 were expressed and purified using the methods discussed for human FRα in Example 1. HTRF assay

HTRF assays were performed in 384-well white shallow-well non-binding plates (Corning, 4513) in a 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). After the indicated incubation periods, time-resolved fluorescence at 590 and 665 nm was measured after excitation at 320 nm on an Envision (PerkinElmer) plate reader. The ratio value of (665 nm emission/590 nm emission) × 10,000 was used to calculate % ΔF according to the following equation:

Negative controls were obtained from nonspecific binding (NSB) control wells. The curves were analyzed using GraphPad Prism software using a four-parameter logistic curve fit equation. HTRF Antigen Binding Assay

Supernatants containing mouse IgG from fused tumor cells were incubated at a final assay dilution of 25% with 0.5 nM streptavidin cryptate (CisBio, 610SAKLB), anti-mouse IgG AF647 (Jackson ImmunoResearch, 115-605-164), and 1 nM biotinylated human FRα, cynomolgus FRα, mouse FRα, human FRβ, or human FRγ to give a final assay volume of 10 μl and incubated for 4 h at room temperature before fluorescence was measured. Antigen Binding Assay for Rat FRα

Species binding assays were performed on Octet RED384 (FoteBio) using tilt bottom black 384-well plates (FoteBio, 18-5076) at 25°C in assay buffer containing PBS, 0.1% v/v BSA (Sigma, A9576), 0.01% v/v Tween-20 (Sigma, P9416), pH 7.4. Assays were set up using either protein A or anti-human capture biosensors (AHC) (FoteBio, 18-5089) according to the manufacturer's instructions. 10 µg/ml of anti-rat FRα IgG (Yixuan Shenzhou Technology Co., Ltd., 81073-RP01) was coated onto a protein A biosensor (Fotebio, NC9490476), and 10 µg/ml of test human IgG was loaded onto an anti-human capture biosensor (AHC) (Fotebio, 18-5089) for 180 seconds. Association was measured by incubating the loaded biosensors with 500 nM human FRα (in-house) or 500 nM rat FRα (Yixuan Shenzhou Technology Co., Ltd., 81073-R08H). Dissociation was measured after transfer into assay buffer. Data were analyzed using Octet Data Analysis Software Version 7.0. Results

2586 fusion tumor supernatants were tested for binding to human FRα, cynomolgus FRα, mouse FRα, human FRβ or human FRγ in an HTRF assay format. This experiment was performed to isolate antibodies that cross-reacted with cynomolgus FRα while ensuring that no binding to the homologs: FRβ and FRγ was observed. To determine the sequence similarity of the human folate receptor with homologs and heterologs, a multiple sequence alignment was determined using the Clustal Omega v1.2.2 algorithm. The sequence homology of human FRα with homologs ( Table 11 ) and heterologs ( Table 12 ) is shown below. [ Table 11 ] : Percent sequence identity of mature human FRβ and FRγ with FRα polypeptide FRγ_people FRβ_human FRα_People 77.8% 78.9% FRγ_people N/A 87.7% N/A – Not applicable [ Table 12 ]: Percent sequence identity of mature human, cynomolgus monkey, mouse and rat FRα FRα_People FRα_mouse FRα_rat FRα_Cynomolgus monkey 97.6% 84.3% 85.7% FRα_People N/A 83.3% 84.8% FRα_mouse N/A N/A 94.8% N/A – Not Applicable

Binders were defined as IgGs with > 30% ΔF assay signal. A total of 129 IgGs showed binding only to human and cynomolgus monkey FRα, 9 IgGs showed binding only to human, cynomolgus monkey and mouse FRα, and another 30 IgGs showed binding only to human, cynomolgus monkey or mouse FRα. The data for the 6 lead IgGs are shown in Table 13. [ Table 13 ] : HTRF IgG binding assay of 6 lead IgGs Reference IgG HTRF IgG binding assay (% ΔF) Human FRα Cynomolgus monkey FRα Mouse FRα Human FRβ Human FRγ AB1370026 270.58 213.68 1.63 -0.38 1.31 AB1370035 225.19 141.47 -5.22 -3.26 9.2 AB1370049 187.81 94.06 -9.23 -8.11 -0.75 AB1370083 139.98 85.16 -3.65 3.81 -2.92 AB1370095 126.25 85.33 -5.66 -5.87 -1.17 AB1370117 79.81 67.23 -3.41 -1.07 -1.77

168 fusion tumor IgGs identified in the fusion tumor supernatant screening were made into Phynexus purified IgGs. These were tested in the antigen binding assay at 4 dilutions of IgG. 115 IgGs were identified as IgGs that cross-reactive to human and cynomolgus monkey FRα and did not bind to human FRβ and FRγ. The data for the 6 lead IgGs are shown in Figures 1A-1F .

In addition, for the rat FRα antigen binding assay, the six lead IgGs also showed binding to human FRα but not to rat FRα.

Overall, it has been demonstrated that a diverse range of anti-FRα IgG antibodies can be generated through extensive and high-throughput antibody generation and screening activities. Example 3 - High-throughput internalization assay Objective

The antibodies generated were characterized by their internalization rate. Materials and Methods

PhyNexus purified IgG samples from fusion tumor output were evaluated in the internalization assay. Frozen stocks of KB cells were inoculated overnight prior to the addition of IgG samples. The assay was run using the fixation acquisition method with a 2.5 hour incubation prior to fixation. The assay was run using pHrodo Green labeled detection reagent and Cell Mask Red (Thermo Fisher Scientific). Day 1

Frozen vials of KB cells were thawed, diluted in medium, centrifuged, and resuspended in fresh medium before counting. Cells were seeded at 10,000 cells/well in MEM + NEAA + 10% FCS (Thermo Fisher Scientific) and the prepared plates were incubated overnight at 37°C in a humidified incubator with 5 % CO _2. Day 2

Prepare antibodies and secondary detection reagents and pre-incubate at room temperature for 30 min. Remove medium from cells and add pre-mixed antibodies and detection reagents. Incubate plates at 37°C for 2.5 hours. After incubation, add 30 µl/well of 7.2% formaldehyde (Thermo Fisher Scientific) + Hoechst (Thermo Fisher Scientific) diluted 1:5000 to obtain 3.7% formaldehyde fixation and 1:10000 dilution of nuclear stain. Incubate plates at room temperature for 20 minutes, then wash with 1x HBSS (Thermo Fisher Scientific) and add 30 µl/well of cell mask red (1:5000 dilution into 0.1% Triton X-100 (Merck)). The plates were incubated at room temperature in the dark for 20 minutes, washed twice with HBSS and imaged on the Opera High Content Imaging system ( see Table 14 for Opera acquisition parameters). [ Table 14 ]: Opera acquisition parameters Laser efficacy Camera Filter Categories Time ( ms ) Lens Exposure 1 ( Hoechst ) 405 3100 1 450/50 2 80 20xA Exposure 1 (Cell Mask Red) 640 3440 3 600/40 2 80 20xA Exposure 2 (pHrodo Green) 488 10400 2 540/75 2 2000 20xA result

A high throughput internalization assay was run to determine the rate of antibody internalization into KB cells. Phynexus purified antibodies were labeled, added to KB cells, and cells were fixed after a 2.5 hour time course. Image analysis was performed using Columbus and output analyzed within the High Content Profiler in Spotfire, allowing for multi-parametric analysis of acquired image data and development of a “hit list” containing mAbs that exhibited enhanced uptake ( Figure 2 ). All 6 lead antibodies showed internalization above the cutoff determined by the positive control sample. Example 4 – Epitope Classification Target Generation of Anti-FRα Antibodies

The purpose of this experiment was to perform epitope classification on the generated anti-FRα antibodies, specifically the 6 exemplary antibodies. Materials and Methods

Three reference anti-FRα IgGs were labeled for use in homogeneous time-resolved fluorescence (HTRF) assays using the Micro DyLight 650 Antibody Labeling Kit (Thermo Fisher Scientific, 84536) according to the manufacturer's instructions (for general setup of HTRF assays, see Example 2).

HTRF epitope competition assays were set up using test IgG, 1 nM streptavidin cryptate, biotinylated human FRα, and DyLight 650-labeled IgG titrated to an assay volume of 10 μl. For the Comparator Antibody 1 and Comparator Antibody 2 assays, 0.5 nM biotinylated human FRα and 0.5 nM DyLight 650-labeled Comparator Antibody 1 or Comparator Antibody 2 were used. For the Comparator Antibody 3 assay, 1.25 nM biotinylated human FRα and 1.25 nM DyLight 650-labeled Comparator Antibody 3 were used. The assay plates were incubated at room temperature for 3-4 hours prior to fluorescence measurement. Results

115 human/cynomolgus folate receptor-specific fusion tumor IgGs were analyzed in three HTRF epitope competition assays to determine epitope diversity. Eight IgGs inhibited in all assays, 41 in both the comparator 1 and comparator 2 assays, 39 in both the comparator 2 and comparator 3 assays, 12 in both the comparator 1 and comparator 3 assays, 4 in the comparator 1 assay only, 6 in the comparator 3 assay only, and 5 IgGs did not inhibit in any assay.

The six exemplary antibodies were reshaped, expressed and purified as human IgG1 and analyzed in HTRF epitope competition assays to confirm potency. In particular, all lead exemplary antibodies inhibited the comparative antibody 2 competition assay. AB1370117 and AB1370035 were 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 ). [ Table 15 ] : Summary of IC50 analysis in HTRF epitope competition analysis antibody HTRF epitope competition assay, IC50 (M) geometric mean, n > 2 Comparator 1 IgG Comparator 2 IgG Comparator 3 IgG AB1370026 hIgG1 3.20E-11 1.77E-10 No inhibition AB1370035 hIgG1 Inhibitory but not S-shaped 1.46E-09 2.85E-10 AB1370049 hIgG1 No inhibition 3.37E-08 6.35E-09 AB1370083 hIgG1 2.96E-11 8.06E-11 No inhibition AB1370095 hIgG1 3.63E-10 4.15E-09 No inhibition AB1370117 hIgG1 Inhibitory but not S-shaped 9.33E-09 1.83E-09 Conclusion

Six antibodies were analyzed by epitope competition assay. A range of behavior and epitope diversity was observed, with some molecules competing with all three comparator antibodies and others competing with only a subset. AB1370049 showed no competition with comparator antibody 1 and demonstrated the weakest competition with comparator antibody 2 while competing with comparator antibody 3. Example 5 – Antigen Binding Affinity of Generated Anti-FRα Antibodies OBJECTIVE

Using surface plasmon resonance analysis, the binding kinetics and equilibrium dissociation constants of a panel of generated antibodies (specifically, 6 exemplary antibodies) to human and cynomolgus monkey FRα were determined. Materials and Methods

Antibody affinity to human and cynomolgus FRα proteins was measured using a Biacore T200 surface plasmon resonance system (Cytiva) at 25°C. Protein A was covalently immobilized to the 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 to the protein A surface in HBS-EP+ buffer, pH 7.4, at 10 µl/min to ensure FRα ECD binding. FRα ECD was serially diluted (0.4 nM-100 nM human FRα ECD, 0.8 nM-200 nM cynomolgus FRα ECD, 30 nM-4000 nM mouse FRα ECD, and rat FRα ECD) in HBS-EP+ buffer (pH 7.4) and flowed over the chip at 50 µl/min, associated for 2 min, and dissociated for 8 min. The chip surface was fully regenerated with _a pulse of 3 M MgCl2 to remove captured antibody, as well as any bound FRα ECD. Multiple buffer-only injections were performed under the same conditions to allow double reference subtraction of the final sensorgrams that were analyzed using the Biacore T200 Evaluation software to derive equilibrium dissociation constants. Results

The antibody affinity of a panel of antibodies to human and cynomolgus monkey FRα proteins was measured by SPR. All antibodies bound to both human and cynomolgus monkey proteins. The affinity to human FRα was in the range of 1-16 nM, and the affinity to cynomolgus monkey FRα was in the range of 1-37 nM. Table 16 below summarizes the kinetic binding parameters of the six exemplary antibodies. [ Table 16 ] : Summary of Antibody Affinity Data antibody Protein ka ( 1/Ms ) kd ( 1/s ) KD ( nM ) AB1370026 Human FRα 2.79E+05 3.54E-04 1.3 Cynomolgus monkey FRα 1.95E+05 3.42E-04 1.8 AB1370035 Human FRα 5.68E+05 5.27E-04 0.93 Cynomolgus monkey FRα 4.75E+05 5.08E-04 1.1 AB1370049 Human FRα 2.05E+05 3.22E-03 15.7 Cynomolgus monkey FRα 2.15E+05 7.94E-03 36.9 AB1370083 Human FRα 1.45E+05 1.35E-04 0.93 Cynomolgus monkey FRα 1.01E+05 1.60E-04 1.56 AB1370095 Human FRα 9.57E+04 1.29E-03 13.5 Cynomolgus monkey FRα 9.58E+04 1.83E-03 19.1 AB1370117 Human FRα 2.93E+05 7.62E-04 2.6 Cynomolgus monkey FRα 3.64E+05 1.87E-03 5.1 Conclusion

All six antibodies had dissociation constants in the low nM range and in all cases bound to cynomolgus monkey FRα with an affinity within 2.5-fold of that to human FRα. Therefore, from an affinity perspective, all six exemplary antibodies appear suitable for further development as therapeutic agents. Example 6 - Physicochemical properties of generated anti-FRα antibodies Objective

The generation of therapeutic antibodies that are developable and aligned to manufacturing requirements requires a comprehensive evaluation of the antibody's physicochemical properties. In this experiment, a panel of generated antibodies was evaluated for their performance titer, stability, and reversible self-association tendency. 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 x 35 mm column with approximately 1 mg/ml of each lead mAb sample diluted 1:1 in 1.5 M ammonium sulfate in 25 mM sodium phosphate (pH 7.40) buffer and eluted with a gradient of 1.5 M to 0 M ammonium sulfate and 0 to 20% acetonitrile in 25 mM sodium phosphate (pH 7.40). The more hydrophobic the species, the later they elute and therefore have a longer retention time. Affinity capture self-interaction 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 incubation with the antibody at 0.4 mg/ml for 1 h at room temperature. Nanoparticles were blocked by incubation with 100 nM PEG 2000 (Merck) for 1 h at room temperature and subsequently concentrated 10-fold. Antibodies for analysis were prepared in a total volume of 120 µl at a final concentration of 45 µg/ml in a solution containing 12 µl of captured nanoparticles and 20 mM histidine, 120 mM sucrose, 80 mM arginine, pH 6 (HSA) buffer in a 96-well plate. Samples were incubated for 20 min at room temperature and duplicate 50 µl aliquots were transferred to 384-well polystyrene plates (Nunc 384-well clear polystyrene plates, Thermo Fisher Scientific). Sample absorbance was measured in a plate reader and the wavelength red shift was determined for each sample compared to a buffer-only control well. A shift of > 5 nm was flagged as a risk of self-association. Bacitravirus ELISA

Nonspecific binding of antibodies to bacillary virus particles was determined by ELISA as described by Hotzel et al. (Hotzel et al. 2012 mAbs 4:6, 753-760). Preparations of each antibody were prepared at 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 1% bacillary virus extract in 50 mM NaCO (BV plates) or with 50 mM NaCO (blank plates). After washing with PBS, the plates were blocked with 300 µl/well of PBS + 0.5% BSA for 1 hour at room temperature and washed three times with PBS. 50 µl/well of PBS + 0.5% BSA (background) or test antibody dilution was added and incubated for 1 h at room temperature. After washing three times in PBS, 50 µl/well of the detection antibody (anti-human Fc-specific-HRP Sigma A0170) diluted 1:5000 in PBS + 0.5% BSA was added. The samples were incubated for 1 hour at room temperature and the plates were washed three times in PBS. HRP substrate – TMB (SureBlue Reserve, KPL 53-00-03) was then added at 50 µl/well and after the color change, the reaction was stopped by adding 50 µl/well of 0.5M sulfuric acid. The absorbance was measured at 450 nm. The BV score was calculated by averaging the absorbance at 450 nm for each antibody sample at 10 nM and 100 nM concentrations and then dividing by the secondary control sample alone. A BV score > 5 may indicate a risk of increased clearance due to nonspecific binding. Accelerated Thermal Stability 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 centrifuge column (Millipore, UFC30HVNB). High performance size exclusion chromatography (HP-SEC) was performed by loading 70 µl of IgG onto a TSKgel G3000SWXL5 µm, 7.8 mm x 300 mm column using a flow rate of 1 ml/min and 0.1 M anhydrous sodium hydrogen phosphate dibasic and 0.1 M sodium sulfate, pH 6.8 as an isocratic running buffer. Larger molecules are excluded from the pores of the size exclusion column to a greater extent than smaller molecules and therefore elute earlier. Peaks eluting earlier than the monomer peak were recorded as aggregates. Peaks eluting after the monomer peak (excluding buffer-related peaks) were recorded as fragments. At the same time, the potency changes of the antibodies were analyzed in the HTRF epitope competition assay. Results Hydrophobic interaction chromatography ( HIC ) HPLC

Higher HIC retention times correspond to increased hydrophobicity, which may indicate a risk of increased aggregation and clearance due to nonspecific uptake. In addition, more hydrophobic mAbs may produce more hydrophobic ADCs, which may lead to aggregation during conjugation, instability as an ADC, and potentially more nonspecific uptake into normal tissues, leading to toxicity.

By HPLC-HIC, the generated panel of antibodies showed a retention time in the range of about 2.0-2.8 minutes. In particular, the six exemplary antibodies showed an acceptably low retention time compared to the tested panel of antibodies ( see Table 17 ). [ Table 17 ] : Summary of HIC-HPLC data Sample Retention time by HIC-HPLC (min) AB1370026 hIgG1 1.96 AB1370035 hIgG1 2.25 AB1370049 hIgG1 2.27 AB1370083 hIgG1 2.27 AB1370095 hIgG1 2.12 AB1370117 hIgG1 2.27 Affinity capture self-interacting nanoparticle spectroscopy ( AC-SINS )

The self-interaction tendency of the antibodies was also tested by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS). This behavior underlies or is associated with undesirable properties such as reversible self-association, aggregation, stickiness, opalescence, and phase separation. By monitoring the peak absorption wavelength (plasma wavelength) of gold particles coated with antibodies, AC-SINS indirectly measures the tendency of proteins to self-interact in an environment simulating high protein concentration. The red shift of the exemplary antibodies indicated that the level of self-interaction of all tested antibodies in HSA buffer was negligible, 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 6 exemplary antibodies tested. Bacivirus ELISA

To test for non-specific binding, which could be indicative of increased in vivo clearance, the level of binding of the antibodies to bacilli particles was determined in an ELISA format. In all cases, the antibodies showed negligible levels of non-specific binding, all below the cut-off assay threshold of 5. Advantageously, this indicates a low risk of poor in vivo clearance. Accelerated Thermal Stability Assay

Thermostability of a panel of 6 exemplary antibodies was assessed by HP-SEC and in parallel the potency changes were assessed in the HTRF epitope competition assay ( Table 18 ). In all cases, any potency changes were < 2-fold and the monomer reduction was < 3%, indicating that all antibodies were thermostable under the conditions tested. [ Table 18 ] : Summary of accelerated stability study data Sample HP SEC monomer % MOv19 HTRF epitope competition assay, IC50(M) geometric mean 45°C 4°C 45°C 4°C Fold Difference AB1370026 hIgG1 97.3 100 7.08E-10 7.11E - 10 1.00 AB1370035 hIgG1 97.3 100 1.16E-09 9.45E - 10 1.23 AB1370049 hIgG1 97.0 100 9.62E-09 1.63E-08 0.59 AB1370083 hIgG1 98.3 99.6 6.65E - 11 5.11E - 11 1.30 AB1370095 hIgG1 96.6 99.2 4.31E-09 5.07E-09 0.85 AB1370117 hIgG1 97.0 99.4 5.48E-09 3.75E-09 1.46 Conclusion

All 6 lead mAbs showed good thermal stability, low risk of self-association, negligible non-specific binding and low hydrophobicity, thus all lead mAbs showed acceptable developability parameters. Example 7-Pharmacokinetics of anti-FRα antibodies generated in SCID mice Objective

To investigate the pharmacokinetics of anti-FRα antibodies generated in wild-type SCID mice after a single intravenous dose. Materials and Methods

Wild-type SCID mice (n = 3 per group) were dosed intravenously (bolus) with exemplary anti-FRα antibodies (5 mg/kg). Blood samples were taken at 15 minutes, 4 hours, 1 day, 2 days, 3 days, 6 days, 10 days, 14 days, and 21 days after dosing, centrifuged to obtain plasma, and analyzed for total antibody. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara).

Plasma antibody concentrations were determined by universal ELISA IgG assay. Results

Although the screening process based on the above examples appears to be exemplary, a range of PK profiles were observed within a broader panel in mice. Through PK screening, lead compounds with undesirably high clearance rates and short half-lives were eliminated.

The tested antibodies gave a clearance rate in the range of about 5-25 ml/kg/day and a half-life in the range of about 4-20 days. Among the tested antibodies, 6 exemplary anti-FRα antibodies showed relatively low clearance rates and long half-lives. Table 19 shows the pharmacokinetic parameters of 6 exemplary anti-FRα antibodies in mice. [ Table 19 ] : Pharmacokinetic parameters of exemplary anti-FRα antibodies (total antibodies) in wild-type SCID mice Lead Antibody Cmax _{[ µg/ml]} AUC _{last time [ day *µg/ml]} CL _{[ml/kg/ day ]} V _{ss [ml/kg]} t _{1/2 [ day ]} AB1370026 91.7 553 5.3 124 16.6 AB1370035 73.2 455 5.6 160 19.5 AB1370049 80.3 528 5.2 135 18.0 AB1370086 86.0 430 8.6 132 11.0 AB1370095 123.2 646 6.6 71 7.8 AB1370117 110.9 762 5.2 71 10.0 _Cmax = maximum observed plasma concentration _AUClast = area under the plasma concentration vs. time curve (up to the last measurable time point); CL = plasma clearance _Vss = steady-state distribution volume t _1/2 = elimination phase half-life Conclusion

The exemplary anti-FRα antibodies exhibited a range of pharmacokinetic properties in wild-type SCID mice, with AB1370035 exhibiting the longest half-life in this model, followed closely by AB1370049, which had the lowest clearance rate as AB1370117. Example 8 - Pharmacokinetics of AB1370049 in hFcRn Tg32 mice Objective

The pharmacokinetics of AB1370049 in hFcRn Tg32 mice after a single intravenous administration were investigated. Materials and Methods

Human FcRn Tg32 mice (n = 3 per group) were intravenously (bolus) administered AB1370049 (5 mg/kg). Blood samples were collected at 15 minutes, 4 hours, 1 day, 2 days, 3 days, 6 days, 10 days, 14 days, and 21 days after administration, and plasma was centrifuged and analyzed for total antibodies. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara).

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 beads. After capture, the beads were washed multiple times and then digested with trypsin in the presence of an internal standard. Acid was added to quench the digestion. Aliquots of the trypsin-digested liquid contents were then transferred to injection plates for antibody analysis by LC-MS/MS.

A signature tryptic peptide (VVSVLTVLHQDWLNGK) on the Fc region of a human antibody is used to calculate the concentration of total antibody in the selected matrix. Tryptic digests from immunoaffinity enriched samples are separated using reverse phase chromatography (RPLC) and the signature peptide is detected using multiple reaction monitoring (MRM). The internal standard used in this experiment is an isotopically labeled peptide. The peak area ratio of the analyte to the internal standard is used for calculations based on a standard curve created by spiking ADC reference material in the desired matrix.

Standard curves and QCs were prepared by spiking different concentrations of target compound (ADC reference substance) into the same matrix as the sample matrix. The quantitation range covered 100 ng/ml-12,000 ng/ml, and the dilution QCs covered up to 50-fold dilution. The standard curves were fitted with linear regression with a weight of 1/x2. Results

Table 20 shows the pharmacokinetic parameters of AB1370049 in hFcRn Tg32 mice. [ Table 20 ] : Pharmacokinetic parameters of AB1370049 in hFcRn Tg32 mice antibody Cmax _{[ µg/ml]} AUC _{last time [ day *µg/ml]} CL _{[ml/kg/ day ]} V _{ss [ml/kg]} t _{1/2 [ day ]} AB1370049 68 584 5.5 107 13.3 _Cmax = maximum observed plasma concentration _AUClast = area under the plasma concentration vs. time curve (up to the last measurable time point); CL = plasma clearance _Vss = steady-state distribution volume t _1/2 = elimination phase half-life Conclusion

AB1370049 exhibits similar pharmacokinetic properties in hFcRn Tg32 and wild-type SCID mice. The hFcRn Tg32 mouse is a transgenic mouse model in which mouse FcRn has been knocked out and human FcRn has been knocked in. Therefore, it represents a model particularly suitable for predicting the human pharmacokinetics of AB1370049. Based on the pharmacokinetics of hFcRn Tg32 mice, AB1370049 may have a lower clearance rate in humans and a half-life similar to other clinically used antibodies, thus promising convenient dosing. Example 9 - Internalization/lysosomal trafficking of exemplary anti-FRα antibodies in SCID mice Target

Internalization of lead FRα mAb into FRα-positive KB and JEG-3 cells was assessed. Materials and methods

KB and Jeg-3 cells were stained with cell trace violet (Thermo Fisher Scientific) and plated in 96-well plates and incubated overnight. Monoclonal antibodies were incubated with 50 nM Fab-pHast human (ATS bio, Carlsbad) at room temperature before addition to plated cells. Cell images (20x) were taken every 30 min and fluorescent areas were automatically analyzed for up to 48 h in the CellInsight CX7 high-content screening platform (Thermo Fisher Scientific). Results

All lead antibodies tested were rapidly internalized into cell lines expressing FRα, reaching saturation in approximately 6–7 h in JEG-3 cells ( Fig. 5A ) and approximately 5 h in KB cells ( Fig. 5B ).

Despite lower 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, indicating their potential utility in ADC format. Example 10 – ADC Generation in DAR8 Format Objectives

To generate DAR8 ADCs with lead mAbs bearing SG3932 payload for in vitro and in vivo activity evaluation for lead selection. Materials and methods

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

UHPLC-RP analysis was performed on a Shimadzu Prominence system using a Thermo Fisher Scientific MAbPac 50 mm x 2.1 mm column with gradient elution using water and acetonitrile, showing a mixture of unconjugated light chains (L0), light chains attached to a single molecule of SG3932 (L1), unconjugated heavy chains (H0), and heavy chains attached to up to three molecules of SG3932 (H1, H2, H3) at 214 nm and 330 nm on a reduced sample of the ADC (SG3932 specific), with the major species being L1 and H3, which is consistent with a drug/antibody ratio (DAR) of 7.58-7.94 SG3932 molecules per antibody as calculated in the formula below based on the 214 nm peak integration. The effective load of free NAC quenching was < LOD/LOQ. Representative UHPLC-RP chromatograms of AB1370049-SG3932 DAR8 are shown in Figures 6A-6B , showing a DAR of 7.82.

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

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

UV (Nanodrop, Thermo) showed that the final ADC concentration in 5.0-6.0 ml was 0.92-3.10 mg/ml, and the obtained ADC quality was 11.0-37.0 mg (23%-74% yield). Conclusion

The lead antibodies are all suitable for DAR8 conjugation, showing high conjugation efficiency DAR>7.5, no DAR loss during purification, no aggregation/fracture during manufacturing, and monomer purity>98%. Therefore, the ADC disclosed herein is able to deliver significantly higher concentrations of cytotoxic payloads to target cancer cells by binding to FRα on cancer cells. DAR8 ADC is also homogeneous and provides the advantages of reproducibility and limited batch-to-batch variation in the manufacturing process. This will allow higher amounts of less potent drugs (such as TOPOi) to be delivered to target cancer cells while maintaining tolerance.

Additionally, the relatively low hydrophobicity of the resulting ADCs may result in less nonspecific uptake into normal tissues and thus may lead to improved tolerability compared with comparator ADCs that deliver more hydrophobic drugs (e.g., sutrin-mituximab or IMGN151).

The exemplary lead ADC was then further evaluated for thermal and serum stability, in vivo PK in mice, and in vitro and in vivo efficacy (see Examples below). Example 11 – DAR4 format ADC generation Objectives

To generate DAR4 ADCs with lead mAbs bearing SG3932 payload for in vitro and in vivo activity evaluation of lead selection and comparison with DAR8 ADCs. Materials and Methods

A 5 mM solution of 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 nmol, 83.2-117.7 µl) to 9.2-12 ml of antibody (AB1370049 lead, AB1370026, AB1370035, AB1370083, AB1370095, or AB1370117; 23-30 mg each, 153-200 nmol) in reducing buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA, Molekula) (final antibody concentration 2.5 mg/ml). The reduced mixture was heated in an incubated orbital shaker at +37°C for 3 hours with gentle (60 rpm) shaking. After cooling to room temperature, SG3932 was added as a DMSO solution (7 molar equivalents/antibody, 2.91-4.12 micromolar in 1.02-1.33 ml DMSO) to the reduced antibody solution (23-30 mg, 153-200 nanomolar) to a final DMSO concentration of 10% (v/v). The solution was mixed for 17 hours at room temperature, then quenched by the addition of N-acetylcysteine (5.4-7.1 μM, 54-71 μl at 100 mM) for >30 minutes at room temperature and purified on an AKTA™ Start FPLC using a GE Healthcare HiLoadTM 26/600 column (packed with Superdex 200 PG) and eluted with 2.6 ml/min PBS. 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. Results

UHPLC-RP analysis using a Thermo Fisher Scientific MAbPac 50 mm x 2.1 mm column on a Shimadzu Prominence system with a gradient elution of water and acetonitrile showed a mixture of unconjugated light chains (L0), light chains attached to a single molecule of SG3932 (L1), unconjugated heavy chains (H0), and heavy chains attached to up to three molecules of SG3932 (H1, H2, H3) at 214 nm and 330 nm on a reduced sample of the ADC (SG3932 specific), consistent with a drug/antibody ratio (DAR) of 4.13-4.27 molecules of SG3932 per antibody as calculated in Example 10. Payload for free NAC quenching was < LOD. Representative UHPLC-RP chromatograms of AB1370049-SG3932 DAR4 are shown in Figures 7A-7B , showing a DAR of 4.24.

UHPLC-SEC analysis was performed on a Shimadzu Prominence system using a Tosoh Life Sciences TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column with a 4 µm 3.0 x 20 mm guard column, eluting with 0.3 ml/min of sterile filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v/v), showing a monomer purity of >99.5% at 280 nm for the ADC sample. A representative UHPLC-SEC chromatogram of AB1370049-SG3932 DAR4 is shown in Figure 7C .

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

UV (Stunner, Unchained Labs) analysis showed that the final ADC concentration in 5.0-6.0 ml was 3.59-4.06 mg/ml, and the obtained ADC quality was 18.0-24.0 mg (68%-79% yield). Conclusion

Lead antibodies are suitable for adjusting TCEP equivalents for random DAR4 conjugation, with no DAR loss during purification, no aggregation/fragmentation during manufacturing, >99% monomer purity and high yield.

The exemplary lead ADC was then further evaluated for in vivo PK in mice and in vivo efficacy (see Examples below). Example 12 - In vitro serum stability/dehybridization of an exemplary ADC Objective

Determine the stability of AB1370049-SG3932 DAR8 and other lead DAR8 ADCs against a range of sera. Materials and Methods IgG depletion of human sera

Human serum (100 ml) was collected twice by passing it through a column packed with MabSelect SuRe™ LX Protein A resin (42 ml, 1 CV) and equilibrated in PBS. The column was regenerated each time by washing with citrate buffer (100 mM sodium citrate, pH 3.0, 210 ml, 5 CV). Incubation of ADC in serum

ADC (1.00 mg/ml, diluted in PBS as needed) was spiked into serum (mouse (ab7486, abcam), rat (C13SDZ, BioRad), IgG-depleted human (DHP-2001, Access Biologicals), and cynomolgus monkey (custom order, 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 different time points (0, 1, 3, and 7 days) and stored at -80°C prior to analysis. Preparation of capture resin

PureProteome™ streptavidin beads (4.90 ml) were washed in PBS (5 times) and CaptureSelect™ human IgG-Fc PK biotin conjugate (750 µl of a 1.0 mg/ml solution) was added. The mixture was further diluted with PBS (60 µl, pH 7.4) before mixing up and down (3 hours, 40 rpm). Residual CaptureSelect was removed, the beads were washed in PBS (5 times), made up to the original volume with PBS, and stored at 2-8°C before further use. Capture of ADCs from serum

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

Reduction of samples (17 µl per sample) was continued for 15 min at 37°C using a reaction mixture of the reducing mixture (1.0 M sodium phosphate pH 6.0, 150 mM sodium borate pH 8.4, 1.0 M DTT, and LC-MS grade water in a ratio of 15:20:6:10). Quenching by the 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. LC-MS Analysis

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

Mass spectra were deconvoluted using Thermo BioPharma Finder. Peaks in the deconvoluted spectra that were considered chemically significant had their percentage height values extracted so that the DAR and percentage of significant chemical modification could be determined for each sample. Comparison of the DAR values for a given ADC in a given serum over time allowed the percentage of deconjugation to be determined for each sample. Results

All ADCs showed low levels of payload deconjugation in all sera ( Figure 8A ). This was due in part to the fact that all ADCs showed significant levels of maleimide hydrolysis, at least in mouse, rat, and cynomolgus monkey sera ( Figure 8B ). Maleimide hydrolysis chemically stabilizes the fused payloads, preventing them from deconjugating from the ADC's antibody. However, in IgG-depleted human sera, the incidence of maleimide hydrolysis was lower. Therefore, the reduced protection conferred by maleimide hydrolysis here demonstrates the intrinsic stability of these ADCs against deconjugation. Conclusion

All ADCs showed good overall stability against all sera. Example 13 – In vivo stability/dehydation of AB1370049-SG3932 DAR8 Objective

AB1370049-SG3932 DAR8 antibody integrity and warhead dehysteresis were assessed under in vivo conditions throughout the duration of the DRF study in cynomolgus monkeys. Materials and Methods

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

Blood samples were taken by venous puncture and centrifuged to obtain plasma. Total antibody and ADC determinations were performed by LBA-LCMS method. AB1370049-SG3932 DAR8 was immunocaptured by biotinylated anti-human antibodies conjugated to magnetic beads and further subjected to multiple washing steps. It was then digested with trypsin and a portion of the supernatant was injected into LCMS for total antibody determination. Another aliquot was further enzymatically cleaved with papain and the solution was used for ADC determination. The injected samples were separated using reverse phase chromatography (RPLC) and then detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard was used for calculation based on a standard curve created by spiking the ADC reference material in the desired matrix.

For the total antibody assay, two characteristic tryptic peptides on the AB1370049-SG3932 DAR8 antibody were used to calculate the concentration of total antibody in the selected matrix: heavy chain (VVSVLTVLHQDWLNGK) and light chain (DSTYSLSSTLTLSK). For the ADC assay, the warhead released by papain was used to calculate the concentration. Isotope-labeled peptides or warheads were used as internal standards. Results

The ratio of ADC to total antibody was approximately 1, indicating that there was no significant warhead decoupling in vivo. Conclusion

Under in vivo conditions, the AB1370049-SG3932 DAR8 ADC remained stable with no significant degradation observed. Example 14 - Pharmacokinetics of an exemplary DAR8 ADC in SCID mice Objective

To investigate the pharmacokinetics of six exemplary anti-FRα DAR8 ADCs in wild-type SCID mice following a single intravenous dose. Materials and Methods

Wild-type SCID mice (n = 3 per group) were dosed intravenously (bolus) with an exemplary anti-FRα ADC (5 mg/kg). Blood samples were taken at 15 minutes, 4 hours, 1 day, 2 days, 3 days, 6 days, 10 days, 14 days, and 21 days after dosing, centrifuged to obtain plasma, and analyzed for total ADC. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara).

Total ADC concentrations were measured using an immunocapture LC-MS/MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 µl of plasma samples were then diluted in TBS and incubated with the beads. After capture, the beads were washed multiple times and then digested with trypsin. Aliquots of the trypsin-digested supernatant were enzymatically digested overnight with papain in the presence of an internal standard. The digested samples were quenched by the addition of acid prior to analysis by LC-MS/MS.

The bullet released by papain is used to calculate the concentration of the ADC. The internal standard used in this experiment is an isotopically labeled bullet. Tryptic digests from immunoaffinity enriched samples are separated using reverse phase chromatography (RPLC) and the released bullets are detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard is used for calculations based on a standard curve created by spiking the ADC reference material in the desired matrix.

Standard curves and QCs were prepared by spiking different concentrations of target compound (ADC reference substance) into the same matrix as the sample matrix. For rodent studies (FcRn and Tg32), the quantitation range covered 100 ng/ml-12,000 ng/ml, and dilution QCs covered up to 50-fold dilution. Standard curves were fit with linear regression with weighting of 1/x2. Results

Table 21 shows the pharmacokinetic parameters of an exemplary anti-FRα DAR8 ADC in small SCID mice. [ Table 21 ] : Pharmacokinetic parameters of DAR8 ADC in SCID mice C _max [µg/ml] AUC _{last time} [day*µg/ml] CL [ml/kg/day] V _ss [ml/kg] t _1/2 [day] ADC 26 96.13 576.51 5.15 125.69 18.46 ADC 35 114.24 578.45 4.17 135.57 23.21 ADC 49 127.48 696.25 4.56 97.63 15.94 ADC 95 100.77 609.16 2.95 139.85 34.13 ADC 117 117.14 654.19 5.98 87.21 11.35 ADC 83 106.97 798.87 3.63 93.63 20.05 _Cmax = maximum observed plasma concentration _AUClast = area under the plasma concentration vs. time curve (up to the last measurable time point); CL = plasma clearance _Vss = steady-state distribution volume t _1/2 = elimination phase half-life Conclusion

Exemplary anti-FRα ADCs exhibit a range of pharmacokinetic properties in wild-type SCID mice. SCID mouse pharmacokinetics of all exemplary antibodies exhibited low plasma clearance and long plasma half-life in SCID mice. Example 15 - Pharmacokinetics of DAR4 and DAR8 ADCs formed from AB1370049 and SG3932 in hFcRn Tg32 mice Objective

The pharmacokinetics of DAR4 and DAR8 ADCs formed by AB1370049 and SG3932 in hFcRn Tg32 mice were investigated after a single intravenous administration. Materials and Methods

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

AB1370049-SG3932 DAR4 or AB1370049-SG3932 DAR8 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 magnetic beads. After capture, the beads were washed multiple times and then digested with trypsin. Aliquots of the trypsin-digested supernatant were enzymatically digested overnight with papain in the presence of an internal standard. The digested samples were quenched by the addition of acid prior to analysis by LC-MS/MS.

The warhead released by papain is used to calculate the concentration of DAR4 or DAR8 ADC. The internal standard used in this experiment is an isotopically labeled warhead. Tryptic digests from immunoaffinity enriched samples are separated using reverse phase chromatography (RPLC) and the released warheads are detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard is used for calculations based on a standard curve created by spiking DAR4 or DAR8 ADC reference material in the desired matrix.

Standard curves and QCs were prepared by spiking different concentrations of DAR4 or DAR8 ADC (ADC reference material) into the same matrix as the sample matrix. The quantitation range covered 100 ng/ml-12,000 ng/ml, and the dilution QCs covered up to 50-fold dilution. Standard curves were fit by linear regression with weighting of 1/x2. Results

Table 22 shows the pharmacokinetic parameters of DAR4 or DAR8 ADC in hFcRn Tg32 mice. [ Table 22 ] : Pharmacokinetic parameters (mean ADC) of DAR4 or DAR8 forms of AB1370049-SG3932 in hFcRn Tg32 mice ADC Cmax _​ AUC _{last time} CL V _ss t _1/2 [µg/ml] [day*µg/ml] [ml/kg/day] [ml/kg] [sky] DAR-4 ADC 121 504 7.2 126 12.2 DAR-8 ADC 110 405 10.4 120 8.0 _Cmax = maximum observed plasma concentration _AUClast = area under the plasma concentration vs. time curve (up to the last measurable time point); CL = plasma clearance _Vss = steady-state distribution volume t _1/2 = elimination phase half-life Conclusion

AB1370049-SG3932 DAR8 exhibits slightly higher clearance and lower half-life in hFcRn Tg32 than in wild-type SCID mice. The hFcRn Tg32 mouse is a transgenic mouse model in which the mouse FcRn has been knocked out and the human FcRn has been knocked in. Therefore, it represents a model suitable for predicting the human pharmacokinetics of AB1370049-SG3932 DAR8. Based on the pharmacokinetics of hFcRn Tg32 mice, the human clearance of AB1370049-SG3932 DAR8 may be lower, the half-life will be the same as other clinically used ADCs, and it should be easy to administer. This data is consistent with the pharmacokinetic data obtained with the exemplary mAbs of the present disclosure. DAR4 ADCs exhibited slightly longer half-life in hFcRn Tg32 mice, driven by slightly lower plasma clearance. Example 16 - In vitro cytocidal activity of exemplary DAR8 ADCs Objectives

The cytotoxic activity of exemplary anti-FRα DAR8 ADCs was evaluated against cancer cell lines expressing different levels of FRα. Materials and Methods

Cancer cell lines with different levels of FRα expression were obtained from ATCC (American Tissue Culture collection). KB cells are cervical cancer cell lines with HeLa contaminants, while IGROV-1 is an ovarian cancer cell line and JEG-3 is a choriocarcinoma cell line. Cells were plated in duplicate onto 96-well plates. After 24 hours of incubation, cells were treated with lead ADC or non-targeted ADC control NIP228 in the range of 0-66.66 nM (0-10 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 culture medium without cells and subtracted from experimental values. IC50 values were calculated on Graph Pad Prism. Results

The exemplary ADCs were tested in multiple cancer cell lines with different FRα expression levels: KB (high), IGROV-1 (high-intermediate), and JEG-3 (intermediate). As shown in Figures 9A-9C , all exemplary ADCs had cytotoxic activity in the 3 tested FRα-positive cell lines. The ADC lead candidate was more potent in the high FRα expressing cell line KB (IC50 = 0.13 to 0.23 µg/ml) and the intermediate JEG-3 (IC50 = 0.01-0.14 µg/ml). Conclusion

All ADCs showed high potency in these in vitro cytotoxicity assays, indicating their potential efficacy in treating cancers that overexpress FRα. In particular, AB1370049-SG3932 DAR8 had the best cytotoxic activity in all 3 FRα-expressing cell lines. Example 17 – Bystander Killing of AB1370049-SG3932 DAR8 Target

Evaluation of the bystander activity of AB1370049-SG3932 DAR8 in co-culture experiments with FRα-positive and -negative KB cells. Materials and methods

To assess the bystander activity of AB1370049-SG3932 DAR8, a mixture of antigen-positive KB cells (KB WT) and antigen-negative KB FRα knockout GFP-tagged cells (KB FRα k/o GFP) were incubated in the presence of AB1370049-SG3932 DAR8. After 6 days of incubation, 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 to untreated samples. Results

AB1370049-SG3932 DAR8 showed potent bystander killing activity when mixed in a 1:1 co-culture experiment with FRα-expressing and FRα-negative KB cells ( Figure 10 ).

AB1370049-SG3932 DAR8 can induce apoptosis and ultimately cell death not only in tumors that homologously express FRα, but also in tumors that heterologously express FRα, as shown in co-culture experiments where 50% of the cells express FRα and 50% do not. The ADC is internalized in FRα-positive cells, and the free warheads are released and kill the target cancer cells. The free warheads can also diffuse into neighboring cancer cells that do not express FRα and kill them as well. Example 18 - In vivo anticancer activity of exemplary ADCs in cell-derived xenograft models Objectives

The anti-tumor activity of exemplary ADCs, specifically AB1370049-SG3932 DAR8, was evaluated in a panel of CDX models of human cancer, representing a range of target expression levels. Materials and Methods

For the KB xenograft model, 6 x 10 ⁶ cells/mouse were inoculated subcutaneously into female CB17-SCID mice (Charles River Laboratories). When tumors reached approximately 150-200 mm ³ , mice were randomized. Each of the 6 lead DAR8 ADCs was administered intravenously at a single dose (day 7) of 0.3125 mg/kg, 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, and 10 mg/kg and DAR4 ADCs were administered at 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, and 20 mg/kg together with the isotype control NIP228 with corresponding dose levels.

For the Caco-2 xenograft model, 5 x 10 ⁶ cells/mouse in 50% Matrigel were inoculated subcutaneously into female athymic nude mice (Harlon Laboratories). When tumors reached approximately 150 mm ³ , mice were randomized into groups. AB1370049-SG3932 DAR8 was administered intravenously at a single dose (day 17) of 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, the corresponding isotype control NIP228 was administered at 1.25 mg/kg, 5 mg/kg, and FRα-DM4 ADC (a "biosimilar of sutrin-mituximab" with a DAR of approximately 3) was administered at 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg, and the corresponding isotype control NIP228 was administered at 1.25 mg/kg and 5 mg/kg.

For the IGROV-1 xenograft model, 1 x ¹⁰⁷ cells/mouse in 50% Matrigel were subcutaneously inoculated into female severe combined immunodeficient (SCID) mice (Envigo). When tumors reached approximately 285 ^mm3 (day 32), mice were randomized into groups based on tumor volume. Control animals received 100 ul of vehicle intravenously, while treated animals received a single intravenous injection of AB1370049-SG3932 DAR8 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 4 ml/kg, the corresponding isotype control NIP228 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, and FRα-DM4 ADC (a "biosimilar of sutrin-mituximab" with a DAR of approximately 3) at 5 mg/kg, and the corresponding isotype control NIP228 at 5 mg/kg.

For the OVCAR3 xenograft model, AB1370049-SG3932 DAR8 was administered at a single dose of 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg, and FRα-DM4 ADC (a “soxin-mituximab biosimilar” with a DAR of approximately 3) was administered at 1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg.

Tumor volume was measured twice weekly with calipers. Results

For all DAR 8 ADCs tested, complete tumor regression was observed at 5 mg/kg (KB and OVCAR-3) and 1.25 mg/kg (OVCAR-3 only) ( Figures 11-12 ). In IGROV-1 xenografts with moderate to high FRα expression, AB1370049-SG3932 DAR8 was active at 5 mg/kg, with partial tumor regression within 70 days before tumor regrowth began ( Figure 13 ). The lead ADC AB1370049-SG3932 DAR8 was selected because it had a more durable response compared to other lead candidates, particularly in the KB and OVCAR-3 xenograft models.

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

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

The exemplary lead ADC showed potent anti-tumor activity in a variety of different xenograft models and at different doses. The anti-tumor activity was similar to or better than the comparator molecule FRα-DM4. Example 19-AB1370049-SG3932 In vivo anti-cancer activity of DAR8 in Champions and in-house patient-derived xenograft models Target

To evaluate the anti-tumor activity of AB1370049-SG3932 DAR8 in low-passage Champions PDX models and in-house PDX models of human non-small cell lung cancer, ovarian cancer, colorectal cancer, and endometrial cancer representing a range of target expression levels .

Thirty PDX models (19 NSCLC models, 9 ovarian cancer models, and 2 endometrial cancer models) were selected for study. Tumor tissue fragments were implanted subcutaneously into 6-8 week old female athymic nude Foxn1nu stock mice. When enough stock ^animals reached 1000 – 1500 mm3, tumors were harvested and fragments were implanted into pre-study animals. When tumors reached an average volume of 150-300 ^mm3 , animals were randomized into 5 groups based on 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 5 mg/kg or 2.5 mg/kg of the isotype control antibody NIP228-SG3932, respectively. All animals received a single intravenous dose on day 0.

Animals were observed daily and tumor size and body weight were measured and recorded twice weekly. Tumor volume was measured by digital calipers and calculated using the following formula: Tumor volume = [Length (mm) x Width (mm) ² x 0.52, where length and width are the longest and shortest diameters of the tumor, respectively.

Study endpoints were performed in all models when the mean tumor volume in the control group reached 1200 ^mm3 , or up to 60 days if maximum tumor volume had not been reached. If 1200 ^mm3 volume occurred before day 28, the treated group was measured until day 28. After randomization of animals to the study, tumor samples were collected using 3 animals per model. Tumors of approximately 400-600 ^mm3 were collected, with half of the tumors processed for immunohistochemistry analysis and the other half processed for genomic analysis. Internal Models

In vivo patient-derived xenograft (PDX) efficacy studies were performed using different tumor models (e.g., ovarian cancer, colorectal cancer, endometrial cancer, non-small cell lung cancer). Tumor fragments from these PDX models were implanted subcutaneously into female NOD-SCID mice (Envigo) using a trocar. Mice were randomized into groups when tumors reached approximately 150-200 ^mm3 . AB1370049-SG3932 DAR8 was administered intravenously at single doses of 2.5 mg/kg, 5 mg/kg along with corresponding dose levels of isotype control NIP228. Tumor volume was measured twice a week with a caliper. Tumor growth was calculated using the formula ½ x L x W2 (L = length; W = width). Body weight was measured twice weekly to assess tolerability of treatment. Determination of Antitumor Response

The response from initial tumor volume (ITV) to final tumor volume (FTV) was calculated at the time that resulted in the greatest reduction from the initial tumor volume. The anti-tumor response for each tumor-bearing mouse was calculated using the following formula: Tumor growth (%) = [(FTV-ITV)/ITV] x 100

Tumor growth (%) was calculated for each animal in the study group, and group responses were calculated as the median response of all treated mice.

For each treatment group, the response to the test agent was determined. Response criteria were based on RECIST 1.1, where response was based on a 30% reduction in tumor size from baseline tumor measurements. Results

Table 23 and Figure 16A summarize the median percentage of tumor growth resulting from a single administration of 5.0 mg/kg AB1370049-SG3932 DAR8 or NIP228-SG3932 in 51 PDX models. Tumor growth inhibition was observed after a single dose of 5.0 mg/kg AB1370049-SG3932 DAR8, with 54% of models (29 of 54) demonstrating a 30% or greater reduction in tumor size from baseline. A single dose of 5.0 mg/kg NIP228-SG3932 resulted in a 30% or greater reduction in tumor size from baseline in 22% (11 of 49) of the models tested. In the ovarian cancer PDX models tested, 5.0 mg/kg AB1370049-SG3932 DAR8 resulted in a 30% or greater reduction in tumor size from baseline in 78.3% (18 of 23, 95% CI 58% - 91%) of the models tested. 5.0 mg/kg NIP228-SG3932 resulted in a 30% or greater reduction in tumor size from baseline in 43.5% (10 of 23, 95% CI 26% - 63%) of the models tested. [ Table 23 ] : Evaluation of in vivo anti-tumor response to AB1370049-SG3932 DAR8 (5.0 mg/kg) in 54 models (30 Champions models and 24 in-house models) Tumor Name Cancer Indications AB1370049-SG3932 DAR8 5.0 mg/kg NIP228-SG3932 5.0 mg/kg Median tumor growth % Response Median tumor growth % Response MEDI-COL-06 Colorectal cancer 59.0% NR -6.4% NR MEDI-COL-41 Colorectal cancer 52.3% NR Not tested Not tested MEDI-COL-37 Colorectal cancer 46.3% NR 54.6% NR MEDI-COL-08 Colorectal cancer 43.4% NR Not tested Not tested MEDI-COL-29 Colorectal cancer 5.6% NR Not tested Not tested MEDI-COL-28 Colorectal cancer -0.3% NR 60.9% NR MEDI-COL-43 Colorectal cancer -1.6% NR Not tested Not tested MEDI-COL-14 Colorectal cancer -5.9% NR Not tested Not tested MEDI-COL-05 Colorectal cancer -67.3% R -59.0% R MEDI-COL-01 Colorectal cancer -81.7% R -1.9% NR CTG-1577 Endometrial cancer 120.8% NR 27.3% NR CTG-2268 Endometrial cancer -49.0% R 76.0% NR CTG-0502 NSCLC 438.3% NR 273.7% NR CTG-1014 NSCLC 33.8% NR 29.6% NR CTG-0192 NSCLC 29.6% NR 31.3% NR CTG-2532 NSCLC 19.0% NR -1.6% NR CTG-2393 NSCLC 6.6% NR 13.1% NR CTG-2579 NSCLC 3.5% NR 77.1% NR CTG-2542 NSCLC 2.8% NR 43.4% NR CTG-2610 NSCLC -2.4% NR 15.2% NR CTG-1194 NSCLC -5.3% NR 26.3% NR CTG-2535 NSCLC -20.1% NR 25.3% NR CTG-2548 NSCLC -30.5% R -1.3% NR CTG-0848 NSCLC -49.4% R 5.5% NR CTG-0184 NSCLC -50.5% R -9.6% NR CTG-2530 NSCLC -57.0% R -8.6% NR CTG-2534 NSCLC -79.7% R -18.8% NR CTG-2367 NSCLC -85.4% R -13.8% NR CTG-1878 NSCLC 16% NR -twenty four% NR CTG-2143 NSCLC -87% R 6% NR CTG-2180 NSCLC -36% R -13% NR CTG-0252 Ovarian cancer 182.5% NR 211.9% NR OV419 Ovarian cancer 100.4% NR 14.5% NR CTG-1423 Ovarian cancer 48.4% NR 71.0% NR CTG-0992 Ovarian cancer 3.1% NR 14.1% NR CTG-3331 Ovarian cancer -7.5% NR -18.1% NR OV1110F Ovarian cancer -59.5% R -41.8% R OV0452F Ovarian cancer -61.8% R -30.5% R CTG-0486 Ovarian cancer -63.7% R -32.2% R CTG-1678 Ovarian cancer -66.8% R 40.4% NR HOXF-031 Ovarian cancer -70.6% R 6.6% NR MEDI-OVA-05 Ovarian cancer -72.4% R 52.3% NR OV1828F Ovarian cancer -76.4% R -68.6% R CTG-3383 Ovarian cancer -77.8% R -100.0% R CTG-3226 Ovarian cancer -87.3% R -68.0% R OV2022F Ovarian cancer -90.4% R -57.4% R CTG-0956 Ovarian cancer -92.2% R -12.9% NR CTG-1166 Ovarian cancer -93.6% R -39.7% R CTG-0711 Ovarian cancer -96.9% R -0.5% NR MEDI-OVA-13 Ovarian cancer -100.0% R -74.0% R MEDI-OVA-14 Ovarian cancer -100.0% R 15.3% NR HOXF-050 Ovarian cancer -100.0% R 24.0% NR HOXF-053 Ovarian cancer -100.0% R -17.8% NR OV0857 CIS Ovarian cancer -100.0% R -76.0% R

Table 24 and Figure 16B summarize the median tumor growth percentages resulting from a single administration of 2.5 mg/kg AB1370049-SG3932 DAR8 or NIP228-SG3932 in 39 PDX models. Specific tumor growth inhibition was observed following a single intravenous injection of 2.5 mg/kg AB1370049-SG3932 DAR8 in 49% of the PDX models tested (19 of 39), while 51% of the models did not respond (20 of 39). Only two of the 37 models tested responded to the same dose of the non-targeted ADC NIP228-SG3932 DAR8. The activity of the non-targeted ADC was dose-dependent, as fewer models responded to NIP228-SG3932 DAR8 (5%) at lower dose concentrations. [ Table 24 ] : Evaluation of in vivo anti-tumor response to AB1370049-SG3932 DAR8 (2.5 mg/kg) in 39 models (10 in-house models) Tumor Name Cancer Indications AB1370049-SG3932 DAR8 2.5 mg/kg NIP228-SG3932 2.5 mg/kg Median tumor growth % Response Median tumor growth % Response MEDI-COL-28 Colorectal cancer 31.0% NR 82.1% NR MEDI-COL-43 Colorectal cancer 15.3% NR 20.2% NR MEDI-COL-05 Colorectal cancer -61.6% R 28.2% NR MEDI-COL-01 Colorectal cancer -64.0% R 42.0% NR CTG-1577 Endometrial cancer 40.4% NR 79.5% NR CTG-2268 Endometrial cancer -0.4% NR 86.0% NR CTG-0502 NSCLC 310.9% NR 279.0% NR CTG-2579 NSCLC 72.7% NR 94.4% NR CTG-0192 NSCLC 54.5% NR 55.3% NR CTG-1194 NSCLC 49.3% NR 73.1% NR CTG-1014 NSCLC 33.1% NR 53.4% NR CTG-2532 NSCLC 21.2% NR 41.4% NR CTG-2393 NSCLC 16.7% NR -1.8% NR CTG-2548 NSCLC 10.6% NR -14.8% NR CTG-0184 NSCLC 2.1% NR 26.8% NR CTG-2535 NSCLC -1.3% NR 19.0% NR CTG-2610 NSCLC -23.0% NR 8.2% NR CTG-2542 NSCLC -24.4% NR 41.8% NR CTG-2530 NSCLC -28.2% NR 19.7% NR CTG-0848 NSCLC -58.4% R 8.9% NR CTG-2367 NSCLC -80.9% R -23.1% NR CTG-2534 NSCLC -83.0% R 11.9% NR CTG-0252 Ovarian cancer 151.3% NR 288.6% NR CTG-1423 Ovarian cancer 64.7% NR 64.6% NR CTG-0992 Ovarian cancer 3.6% NR 37.9% NR OV1110F Ovarian cancer -29.5% R -8.5% NR CTG-3331 Ovarian cancer -34.2% R -5.8% NR CTG-0956 Ovarian cancer -40.1% R -5.6% NR OV2022F Ovarian cancer -54.0% R Not tested Not tested CTG-3383 Ovarian cancer -57.3% R -68.7% R CTG-0486 Ovarian cancer -60.4% R 45.4% NR CTG-1678 Ovarian cancer -62.6% R 45.4% NR OV1828F Ovarian cancer -63.0% R -42.2% R CTG-3226 Ovarian cancer -70.0% R -11.1% NR MEDI-OVA-14 Ovarian cancer -76.4% R Not tested Not tested CTG-1166 Ovarian cancer -77.5% R -25.8% NR CTG-0711 Ovarian cancer -77.8% R -21.1% NR OV0857 CIS Ovarian cancer -83.6% R Not tested Not tested MEDI-OVA-13 Ovarian cancer -100.0% R 83.1% NR

AB1370049-SG3932 DAR8 has demonstrated anti-tumor responses in multiple models of 4 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).

The objective remission rate was 80% for ovarian cancer, 30% for NSCLC, 20% for CRC, and 50% for endometrial cancer. AB1370049-SG3932 DAR8 FRα expression was positively correlated with activity. Conclusion

AB1370049-SG3932 DAR8 was highly effective in tumors expressing high, medium, and medium-low levels of FRα. In general, the anti-tumor activity of AB1370049-SG3932 DAR8 was positively correlated with FRα expression levels. Example 20 - In vitro safety studies using exemplary ADCs Objectives

The in vitro safety of AB1370049-SG3932 DAR8 was evaluated using primary hematopoietic stem and precursor cells (HSPCs) differentiated into erythroid, myeloid, and megakaryocytes. Materials and Methods

Cryopreserved human bone marrow CD34 ⁺ precursor cells (Lonza) were thawed and cultured overnight in a humidified incubator at 37°C, 5% CO 2 in a 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). The next day, cells were resuspended in Cell Expand medium that supports erythroid differentiation (Preferred Cell Systems, SEC-BFU1-40H), myeloid differentiation (Preferred Cell Systems, SEC-GM1-40H), or megakaryocyte differentiation (Stem Cell Technologies, 09707) in the presence of drug at a concentration of 5000 cells/ml for erythroid and myeloid cells or 15,000 cells/ml for megakaryocytes.

Cells (100 µl) were plated in triplicate into white-walled, clear-bottom 96-well tissue culture plates (Corning Incorporated) with exemplary ADCs (e.g., AB1370049-SG3932 DAR8) or NIP228 isotype control ADCs (200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03, and 0 µg/ml) and cultured in a humidified incubator at 37°C, 5% _CO2 for 5 days.

In addition, the effects of exemplary ADCs on expanding and differentiating cells were evaluated. To this end, cells were seeded at a concentration of 5000 cells/ml in erythroid differentiation for 5 days and then seeded in triplicate into white-walled, clear-bottomed 96-well tissue culture plates for 5 days to which exemplary ADCs (e.g., AB1370049-SG3932 DAR8), NIP228 control ADCs that do not target FRα, or unconjugated mAbs (NIP228 and AB1370049, respectively) were added and cultured at the following concentrations: 200, 66.66, 22.22, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03, and 0 µg/ml in a humidified incubator at 37°C, 5% _CO2 for 5 days. For myeloid differentiation or megakaryocyte differentiation, cells were seeded at 10,000 cells/ml and 15,000 cells/ml, respectively, for 5 days, then centrifuged and seeded into fresh medium at 10,000 cells/ml and 30,000 cells/ml, respectively, for another 5 days, and then seeded in myeloid differentiation medium or megakaryocytes at 20,000 cells/ml and 30,000 cells/ml 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) for 4 days.

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

A 2D in vitro culture system was used to measure toxicity in primary human CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs). In the presence of one or more compounds, HSPCs proliferate and differentiate along the erythroid, megakaryocyte, and myeloid (granulocyte/monocyte) lineages. Inhibition was determined using ATP quantification as a surrogate for cell viability. Using this approach, toxicity induced by AB1370049-SG3932 DAR8 and its associated non-FRα-targeting ADC control was evaluated to assess any exacerbated toxicity of AB1370049-SG3932 DAR8.

AB1370049-SG3932 DAR8 exhibited similar levels of toxicity as non-FRα-targeted ADC molecules carrying the same payload ( FIGS. 18A-18F ). This observation was seen in primary CD34 ⁺ bone marrow cells differentiated into any lineage, regardless of their differentiation level. 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. The unconjugated mAb (AB1370049) did not induce any significant reduction in ATP levels in HSPCs when administered under similar culture conditions as described for AB1370049-SG3932 DAR8. Furthermore, HSPCs treated with AB1370049 conjugated to the same TOPOi payload (at DAR4) showed comparable levels of toxicity to HSPCs treated with the non-FRα-targeting ADC DAR4.

The results showed no target-mediated toxicity and no exacerbation of lead antibody-driven toxicity compared to non-FRα-targeted ADC molecules. Example 21 - In vivo pharmacokinetic study of DAR8 using AB1370049 and AB1370049-SG3932 Objective

Plasma pharmacokinetic (PK) analysis of AB1370049-SG3932 DAR8, including peak and total exposure, clearance, and half-life, was performed in cynomolgus monkeys. PK samples were collected from cynomolgus monkeys at different dose levels for the lead ADC candidate and the unconjugated antibody. Noncompartmental analyses were performed to estimate PK parameters. Materials and Methods Administration of AB1370049 and AB1370049-SG3932 DAR8 to cynomolgus monkeys

Male cynomolgus monkeys (N = 3) were intravenously administered 2 single doses (3 weeks apart, i.e., day 1 and day 22) of AB1370049 at 15 mg/kg or AB1370049-SG3932 DAR8 at 15 and 25 mg/kg.

Blood samples were obtained by venous puncture before dosing, on day 1, and on day 22, and at 0.04, 0.25, 1, 3, 7, 14, and 21 after dosing. Plasma was obtained by centrifugation and analyzed for total antibody, total ADC, and unarticulated bullet. Pharmacokinetic parameters were determined using Phoenix 64 software (Certara). Total antibody and ADC determination

Total ADC and total Ab 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 beads. After capture, the beads were washed multiple times and then digested with trypsin in the presence of an internal standard. Acid was added to quench the digestion. Aliquots of the trypsin-digested liquid contents were then transferred to injection plates for total Ab analysis.

For ADC assays, an aliquot of the tryptic supernatant was enzymatically digested overnight with papain in the presence of an internal standard. The digested samples were quenched by the addition of acid prior to analysis by LC-MS/MS. The bullets released by papain were used to calculate the concentration. The internal standard used in this experiment was an isotopically labeled bullet. The tryptic digests from the immunoaffinity enriched samples were separated using reverse phase chromatography (RPLC) and the released bullets were then detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard was used for calculations based on a standard curve created by spiking the ADC reference material in the desired matrix.

For total Ab determination, a signature tryptic peptide (VVSVLTVLHQDWLNGK) on the Fc region of a human antibody is used to calculate the concentration of total antibody in the chosen matrix. Tryptic digests from immunoaffinity enriched samples are separated using reverse phase chromatography (RPLC) and the signature peptide is detected using multiple reaction monitoring (MRM). The internal standard used in this experiment is an isotopically labeled peptide. The peak area ratio of the analyte to the internal standard is used for calculations based on a standard curve created by spiking ADC reference material in the desired matrix.

Standard curves and QCs were prepared by spiking different concentrations of the target compound (ADC reference substance) into the same matrix as the sample matrix. For the NHP study, the quantitation range was 100-15000 ng/ml with dilutions up to 100-fold. The standard curves were fit to a linear regression with a weighting of 1/x2 and the assay accuracy and precision were within 20% for all levels except the LLOQ (25%). Unharnessed Bullet Assay

The unconjugated bullet assay is performed by a precipitation procedure. The internal standard used in this experiment is an isotopically labeled bullet. The sample is precipitated using a buffer containing a high percentage of organic solvent and spiked with the internal standard. The supernatant is then dried under nitrogen and then reconstituted into an appropriate buffer for injection into the LCMS. The samples are then separated using reverse phase chromatography (RPLC) and then detected using multiple reaction monitoring (MRM). The peak area ratio of the analyte to the internal standard is used for calculations based on a standard curve created by spiking the ADC reference material in the desired matrix.

Standard curves and QCs were prepared by spiking unharnessed bullets at different concentrations into a matrix identical to the sample matrix. For the NHP study, the quantitation range of the assay was 0.059 - 29.5 ng/ml. The standard curves were fitted by linear regression. The accuracy and precision of the assay were within 15% at all levels except at LLOQ (20%). Results

The mean PK parameters based on non-compartmental analysis (NCA) are summarized in Table 25. The unconjugated mAb (AB1370049) exhibited linear PK at 15 mg/kg in cynomolgus monkeys, with half-life and clearance consistent with the literature for human IgG in monkeys. The ADC (AB1370049-SG3932 DAR8) exhibited linear PK at 15 and 25 mg/kg in cynomolgus monkeys, with dose-proportional exposure ( _Cmax and AUC), and comparable CL and t1 _/2 were observed. The unconjugated antibody had higher exposure compared to the dose-matched ADC; with slower clearance and longer half-life than the ADC, which may be due to the addition of eight cytotoxic warheads. However, the PK of the ADC was well within the acceptance criteria for ADCs in cynomolgus monkeys. [ Table 25 ] : Average NCA PK parameters of unconjugated and conjugated mAbs in monkeys after single administration Dosage ( mg/kg ) Unconjugated mAb ( AB1370049 ) 15 mg/kg AB1370049-SG3932 DAR8 15 mg/kg AB1370049-SG3932 DAR8 25 mg/kg Analytes Total mAb Total ADC Total mAb Total ADC Total mAb Cmax ₍ ug/ml) 377 396 340 773 609 AUC (ug* day /ml) 2470 1620 1390 3990 2910 t½ ( day ) 11.5 6.6 6.4 6.2 6.9 CL (ml/kg/ day ) 4.49* n = 1 8.4 9.8 5.8 7.8

No significant ADC accumulation was seen between the first and second doses, and there was no decrease in exposure after the second dose, indicating that no significant neutralizing anti-drug antibodies were formed ( Figure 19 ). From Day 0 to Day 42, total antibody and total ADC levels measured for AB1370049-SG3932 DAR8 (25 mg/kg) samples were similar to those of the unconjugated mAb (15 mg/kg) samples. Total antibody and total ADC levels measured for AB1370049-SG3932 DAR8 (15 mg/kg) samples were relatively low. Overall, total antibody and total ADC were similar within groups, resulting in minimal free warheads seen over time—all of which indicates limited deconjugation and high in vivo stability. Conclusions

The DAR8 ADC (AB1370049-SG3932 DAR8) was advanced into a dose-ranging finding study in monkeys, where dose-dependent exposures were seen with a persistently long half-life and slow clearance, as previously shown in mouse PK studies.

In particular, AB1370049-SG3932 DAR8 exhibits a longer half-life and slower clearance than the state-of-the-art FRα ADC (Olga et al. Cancer Res 2020 Aug 15 (80) (Suppl 16) 2890; DOI: 10.1158/1538-7445.AM2020-2890; WO 2020223221A1). The toxicity profile of the ADCs described in this disclosure may lead to more tolerable therapies and potential clinical advantages. Example 22 - In vitro cytotoxic activity of anti-FRα ADCs when combined with PARP1 inhibitors

PARP1 limits the cytotoxicity of TOPOi by enhancing the excision and repair of the topoisomerase I cleavage complex. Therefore, this example aimed to evaluate the cytotoxic activity of AZD5335 (AB1370049-SG3932 DAR8) in combination with a PARP1 inhibitor (AZD5305) in cancer cell lines expressing different levels of FRα. Materials and Methods Cell lines

Cancer cell lines with different FRα expression were used. As described in Example 16, KB cells (about 37 million FRα/cell) are cervical cancer cell lines with HeLa contamination, while IGROV-1 (about 1.3 million FRα/cell), OVCAR-3 (about 180,000 FRα/cell), and SKOV-3 (402,000 FRα/cell) cells are ovarian cancer cell lines. All cell lines were obtained from ATCC.

KB cells were grown in MEM medium (ThermoFisher) with 10% FBS (ThermoFisher) at 37°C, 5% CO ₂ in a humidified incubator. IGROV-1 cells were grown in RPMI-1640 medium (ThermoFisher) with 10% FBS at 37°C, 5% CO ₂ in a humidified incubator. OVCAR-3 cells were grown in RPMI-1640 medium with 20% FBS at 37°C, 5% CO ₂ in a humidified incubator. SKOV-3 cells were grown in McCoy's 5a medium with 10% FBS at 37°C, 5% CO ₂ in a humidified incubator. Cytotoxicity assay

KB, IGROV-1, OVCAR-3, and SKOV-3 cells were plated at 350 cells/well in 20 µl of complete growth medium in white polystyrene tissue culture treated 384-well plates (Corning, Corning, NY, USA). After 24 h, 4x stocks of AZD5335 and AZD5305 were prepared in complete growth medium (KB cells: MEM medium; IGROV-1 and OVCAR-3 cells: RPMI-1640 medium; SKOV-3 cells: McCoy's 5a medium) containing 10% heat-activated FBS (Thermo Fisher Scientific). Cells were treated with 10 µl of suboptimal doses of AZD5335 (for KB: 3 nM to 0.5 pM, for IGROV-1: 10 nM to 1.5 pM, for OVCAR-3: 50 nM to 32 pM, and for SKOV-3: 700 nM to 0.1 nM) or AZD5305 (for KB and IGROV-1: 10 µM to 123 nM, for OVCAR-3: 2 nM to 0.02 nM, and for SKOV-3: 30 µM to 30 nM) or a combination of AZD5335 and AZD5305. The final volume was adjusted to 40 µl/well. Cells were then incubated at 37°C in a humidified atmosphere of 5% _CO2 for 6 days.

At the end of the experiment, 40 µl of Cell Titer-Glo® (Promega, Madison, WI, USA) was added to each well. The plate was then incubated at room temperature for 20 min to ensure complete cell lysis. Luminescence was measured using an EnVision 2104 multilabel plate reader (Perkin-Elmer, Waltham, MA, USA). The potency of AZD5335 was determined by generating half maximal inhibitory concentration (IC50) values using a nonlinear regression model [log inhibitor relative to response] in GraphPad Prism, version 8.1 (GraphPad, San Diego, CA, USA) and expressed as the percentage of cell viability relative to mock-treated control cells (medium only) using the formula [(treated cells)/(mock-treated control cells)] x 100.

The Bliss synergy model was used to assess the dual-drug combination effect and pharmacological synergy (Bliss score > 0) or antagonism (Bliss score < 0) responses of the combination of AZD5335 and AZD5305, determined using Combenefit software (Cambridge Cancer Research Institute, UK) (Veroli et al. Bioinformatics. 2016 Sep 15; 32(18): 2866-2868).

The highest single agent (HSA) synergy model (Berenbaum 1989) was also used to assess dual-drug combination effects and pharmacological synergy. This model quantifies the higher effect of two single compounds at their respective concentrations. The combination effect is compared to the effect of each single agent at the concentration used in the combination. Exceeding the highest single agent effect (i.e., a positive HSA score) indicates synergy. HSA does not require that the compounds affect the same target. Synergy scores were calculated using the software Genedata Screener®. Results

Figures 20A-20D show the results of cytotoxicity assays in which KB, IGROV-1, OVCAR-3 and SKOV-3 cells were treated with suboptimal doses of AZD5335 or AZ5305 (as single agents) or a combination of AZ5335 and AZD5305.

KB, IGROV-1, OVCAR-3, and SKOV-3 cells were all sensitive to AZD5335 as a single agent, i.e., the percentage of cell viability decreased as the concentration of AZD5335 increased ( Figures 20A-20D ). Even at the highest concentration tested (10 µM), KB and IGROV-1 cell lines were insensitive to AZD5305 as a single agent ( Figures 20A and 20B ), and SKOV-3 cells were slightly sensitive to AZD5305 as a single agent at high concentrations (30 µM) ( Figure 20D ). OVCAR-3 cells were sensitive to AZD5305, and 2 nM AZD5305 produced approximately 50% cytotoxicity to OVCAR-3 cells ( FIG. 20C ). Therefore, 2 nM AZD5305 was used as the highest concentration in the OVCAR-3 cytotoxicity assay.

Surprisingly, as shown in Figures 20A-20D , the combination of suboptimal doses of AZD5335 and AZD5305 appeared more cytotoxic to all KB, IGROV-1, OVCAR-3 and SKOV-3 cell lines than treatment with either AZD5305 or AZD5335 alone.

By applying the Bliss synergy model, a synergistic interaction was found between AZD5335 and AZD5305 in all four cell lines ( Figures 21A-21D ). The strongest Bliss synergy score was observed in KB cells (56), followed by OVCAR-3 cells (51), SKOV-3 cells (34) and IGROV-1 cells (26). This was further supported by applying an additional synergy model (HSA model). In this model, the combination of AZD5335 and AZD5305 again showed synergistic activity in all three cell lines ( Table 26 ). [ Table 26 ]: HSA synergy scores of AZD5335 + AZD5305 in multiple cell lines Cell lines HSA Synergy Score KB 7.31 IGROV-1 6.01 OVCAR-3 3.79 Conclusion

The combination of AZD5335, an ADC comprising an anti-FRα antibody and a TOPOi payload, and the PARP1 inhibitor AZD5305 was found to exhibit synergistic effects in killing FRα-expressing cancer cell lines, including KB, IGROV-1, OVCAR-3, and SKOV-3. These results were particularly pronounced for cells that were insensitive to AZD5305 alone. Therefore, there is great potential for the application of this combination in cancer treatment, especially for cells that are resistant to PARP1 inhibitors. Example 23 – In vivo anticancer activity of a combination of an anti-FRα ADC and a PARP1 inhibitor in the cell-derived xenograft (CDX) model NIH-OVCAR3

To evaluate the antitumor activity of the combination of AZD5335 and AZD5305 in the ovarian cancer CDX model NIH-OVCAR3.

All animal studies were performed in an Association for Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility. Mice were housed in individually ventilated cages (Techniplast Greenline GM500) with corncob bedding. Each cage was cleaned once a week, and the animals were examined twice daily. Mice were fed Envigo Teklad Global 18% protein chow with food and water ad libitum. The animal housing environment was maintained at a constant temperature of 22.2°C ± 4°C and a humidity of 50% ± 20% with a 12-h light-dark cycle.

On the day of transplantation, cells were isolated using TrypLE™ Express (Thermo Fisher Scientific, Waltham, MA, USA), centrifuged at 1200 rpm for 5 min, counted and resuspended in a 1:1 mixture of PBS: Matrigel® (Corning Incorporated) to a final concentration of 50 x 10 ⁶ cells/ml. Female NSG mice were each injected subcutaneously in the right flank with 10 × 10 ⁶ cells in a volume of 200 μl. Tumors were allowed to reach approximately 175 mm ³ before randomization. Mice were randomized to treatment groups using the built-in deterministic randomization method in Study Director software (Studylog Systems, Inc., South San Francisco, CA, USA) without animal replacement. Administration of ADC ( AZD5335 or ADC isotype control) and AZD5305

Test and control ADC preparations (i.e., AZD5335 and NIP228-SG3932, respectively) were diluted from stock solutions with vehicle buffer (25 mM histidine, 7% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH 6.0) and administered as a single intravenous (IV) injection at a volume of 10 ml/kg based on body weight. AZD5305 was prepared by sonication of the dry compound in an appropriate volume of water/HCl to obtain a 1 mg/kg (0.1 mg/ml) dosing solution. AZD5305 was administered daily by oral gavage (PO) for 28 days and was administered at a volume of 10 ml/kg based on body weight. Group Name

Female NSG mice were randomly assigned to groups on day 33. Animals in Group 1 were untreated. Animals in Groups 2 and 4 were administered 1.25 mg/kg NIP228-SG3932 as a single IV administration. Animals in Groups 3-4 and 7-8 were administered 1 mg/kg AZD5305 and Groups 9-10 were administered 0.1 mg/kg AZD5305 daily for 28 days as an oral foetus. Animals in Groups 5, 7, 9 and 6, 8, 10 were administered 1.25 mg/kg and 0.625 mg/kg AZD5335 as a single IV dose, respectively. Group names and dose levels are shown in Table 27 . [Table 27]: Group names and dose levels in the NIH-OVACR3 study Group treatment Dosage Level (mg/kg) ^# Investment channels Number of animals 1 Unprocessed NA NA 6 2 NIP228-SG3932 1.25 IV, single dose 6 3 AZD5305 1 PO, QDx28 6 4 NIP228-SG3932 1.25 IV, single dose 6 AZD5305 1 PO, QDx28 5 AZD5335 1.25 IV, single dose 6 6 AZD5335 0.625 IV, single dose 6 7 AZD5335 1.25 IV, single dose 6 AZD5305 1 PO, QDx28 8 AZD5335 0.625 IV, single dose 6 AZD5305 1 PO, QDx28 9 AZD5335 1.25 IV, single dose 6 AZD5305 0.1 PO, QDx28 10 AZD5335 0.625 IV, single dose 6 AZD5305 0.1 PO, QDx28 IV: intravenous; PO : oral; QDx28: once daily for 28 days; NA: not applicable. # Dose volume: 10 ml/kg Tumor size and body weight measurement

Tumor measurements and body weight were collected twice weekly from day 33 to day 89. Tumors were measured by caliper, and tumor volume was calculated using the following formula: tumor volume = [ length ( mm ) x width ( mm ) ² ]/2 where length and width are the longest and shortest diameters of the tumor, respectively.

Monitor animal welfare and determine humane endpoints per IACUC. Animals meeting any of the following criteria will be removed from the study prior to the planned endpoint date: •   Weight loss ≥ 20% •   General changes in behavior or other health issues (e.g., erect hair, hunched back, or persistent loss of appetite) •   Tumors that have reached critical severity, e.g., due to development of an open eschar •   Mean tumor diameter ≥ 15 mm

Additional tumor volume measurements were performed in individual animals if tumor status or tumor size approached the welfare limits of the study.

Tumor growth in each experimental group is expressed as mean tumor volume (mm ³ ) ± SEM for the number of animals used. To compare the effects of different treatments on tumor growth, tumor volumes were compared between groups on the last day that more than 75% of the animals remained in the vehicle-treated control group. This resulted in a tumor growth inhibition (TGI) analysis at day 58 in the NIH-OVCAR3 study.

The effects of different treatments on tumor growth were analyzed by one-way analysis of variance using GraphPad Prism 9.0.0 software (GraphPad Software, La Jolla, CA). If there were significant differences in the sample means of different experimental groups (p < 0.05), multiple comparisons of the means of the selected experimental groups were performed using Dunnett's test. A p value of < 0.05 indicated a significant difference in the mean tumor volume between the groups.

TGI was calculated by comparing each treatment group to the vehicle-treated control group. Percent inhibition was calculated from the group mean using the following formula: TGI (%) = (CT)/C x 100 where C is the mean tumor volume of the vehicle control group and T is the mean tumor volume of the treatment group. Results

As summarized in Table 28 and shown in Figure 22 , AZD5335 treatment produced significant %TGI at both dose levels compared to the untreated group, producing 86.14% (p < 0.0001) and 50.64% (p < 0.0001) TGI at 1.25 mg/kg and 0.625 mg/kg, respectively.

The individual isotypes NIP-228-SG3932 (1.25 mg/kg) and AZD5305 PARP1 inhibitor (1 mg/kg) also showed modest TGI of 44.99% (p < 0.0001) and 37.45% (p < 0.0001) at the 1.25 mg/kg and 1 mg/kg dose levels, respectively, compared to the untreated group. The modest TGI of the isotype ADCs could be explained by non-specific uptake of the antibodies into cells.

Compared with the untreated group, the combination of AZD5335 and AZD5305 showed TGI of 92.83% (p < 0.0001), 85.61% (p < 0.0001), 89.97% (p < 0.0001) and 80.16% (p < 0.0001) at 1.25 mg/kg + 1 mg/kg, 0.625 mg/kg + 1 mg/kg, 1.25 mg/kg + 0.1 mg/kg, and 0.625 mg/kg + 0.1 mg/kg, respectively.

In particular, the combination of AZD5335 (1.25 mg/kg) and AZD5305 (0.1 or 1 mg/kg) showed better efficacy as compared to AZD5335 (1.25 mg/kg) monotherapy. The combination of AZD5335 (0.625 mg/kg) and AZD5305 (0.1 or 1 mg/kg) also showed improved efficacy as compared to AZD5335 (0.625 mg/kg) monotherapy.

For example, the higher dose combination group, AZD5335 (1.25 mg/kg) + AZD5305 (1 mg/kg) showed slightly better efficacy (92.83% TGI) compared with the lower dose combination group, AZD5335 (0.625 mg/kg) + AZD5305 (0.1 mg/kg) (80.16% TGI).

The isotype control NIP228-SG3932 + AZD5305 combination produced a TGI of 87.53% at 1.25 + 1 mg/kg. However, this combination effect was reduced during the regrowth phase of the tumor (i.e., the phase when the tumor "escapes" treatment and begins to regrow) when compared to the higher dose AZD5335 + AZD5305 combination group (Group 7). [ Table 28 ] : Differences in NIH-OVCAR3 xenograft tumor growth between experimental groups at day 58 Group treatment Dosage Level (mg/kg) ^# Mean tumor volume (mm ³ ) ± SEM % TGI, compared with untreated p-value (ANOVA) ^* Treatment vs. vehicle ^{^} Mean tumor volume (mm ³ ) ± SEM ⁺ 1 Unprocessed NA 1822.02 ± 173.16 NA NA 0.0001 2 NIP228-SG3932 1.25 1002.35 ± 135.18 44.99 0.0001 NA 3 AZD5305 1 1139.6 ± 138.94 37.45 0.0001 0.8742 (NS) 4 NIP228-SG3932 1.25 227.24 ± 21.59 87.53 0.0001 0.0001 AZD5305 1 5 AZD5335 1.25 252.55 ± 26.98 86.14 0.0001 0.0001 6 AZD5335 0.625 899.42 ± 112.99 50.64 0.0001 0.9719 (NS) 7 AZD5335 1.25 130.57 ± 15.59 92.83 0.0001 0.0001 AZD5305 1 8 AZD5335 0.625 262.12 ± 27.16 85.61 0.0001 0.0001 AZD5305 1 9 AZD5335 1.25 182.77 ± 23.98 89.97 0.0001 0.0001 AZD5305 0.1 10 AZD5335 0.625 361.41 ± 20.93 80.16 0.0001 0.0001 AZD5305 0.1 ANOVA: analysis of variance; NA: not applicable; NS: not significant; SEM: standard error of the mean; TGI: tumor growth inhibition, NIP288-SG3932: isotype control ADC # Dose volume: 10 ml/kg; * p value < 0.05 indicates a significant difference in mean tumor volume; ^ Comparison between untreated and treated groups; + Comparison between NIP228-SG3932 control (Group 2) and other groups. Conclusion

In the in vivo CDX model NIH-OVCAR-3, the activity of the AZD5335/AZD5305 combination was significantly higher than either single agent. Example 24 – In vivo anticancer activity of the combination of an anti-FRα ADC and a PARP1 inhibitor in the patient-derived xenograft (PDX) model CTG-3718

To evaluate the antitumor activity of the combination of AZD5335 and AZD5305 in the ovarian cancer PDX model CTG-3718.

All animal studies were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility. Female mice were housed in individual ventilated cages (Techniplast Greenline GM500) with corn cob bedding. Each cage was cleaned once a week, and the animals were examined twice daily. Mice were fed Envigo Teklad Global 18% protein chow with food and water ad libitum. The animal housing environment was maintained at a constant temperature of 22.2°C ± 4°C and a humidity of 50% ± 20% with a 12-h light-dark cycle.

Seed expansion PDX tumors were grown for 76 days to a volume of approximately 530 mm ³ before expansion for use in studies. Donor mice were first euthanized by CO ₂ asphyxiation followed by cervical dislocation. Mice were immersed in 70% isopropyl alcohol containing 2% chlorhexidine gluconate (Sigma) for 5 minutes. Mice were then placed on a sterile field and wiped with povidone iodine followed by 70% isopropyl alcohol. The entire tumor was then excised and placed in RPMI medium containing 50 µg/ml tadalafil (Thermo Fisher Scientific) and subsequently cut into 3 x 3 mm ³ fragments using sterile forceps and a scalpel. Mice receiving donor 3 x 3 ^mm3 tumors were swabbed with povidone-iodine and 70% isopropyl alcohol and subsequently implanted into the right flank using a 10-gauge or 11-gauge trocar (Innovative Research of America). Tumors were allowed to reach approximately 180 ^mm3 before randomization. Mice were randomized to treatment groups using the deterministic randomization method built into Study Director software (Research Diary Systems, South San Francisco, CA, USA) and there was no animal replacement. Administration of ADC ( AZD5335 or ADC isotype control) and AZD5305

Same as Example 23. Group Name

On day 26, female NSG mice were randomly assigned to groups. Animals in Group 1 were untreated. Animals in Groups 2, 5, 6, 8 and 9 were administered 1 mg/kg AZD5305 orally for 28 days. Animals in Groups 3 and 5 were administered 1.25 mg/kg AZD5335 as a single IV dose, and Groups 4 and 6 were administered 3.5 mg/kg AZD5335. Animals in Groups 7 and 9 received 3.5 mg/kg NIP228-SG3932 as a single IV dose. Animals in Group 8 received 1.25 mg/kg NIP228-SG3932 as a single IV dose. Group names and dose levels are shown in Table 29. [Table 29]: Group names and dose levels in CTG-3718 study Group treatment Dosage Level (mg/kg) ^# Investment channels Number of animals 1 Unprocessed NA NA 4 2 AZD5305 1 PO, QDx28 4 3 AZD5335 1.25 IV, single dose 4 4 AZD5335 3.5 IV, single dose 4 5 AZD5335 1.25 IV, single dose 4 AZD5305 1 PO, QDx28 6 AZD5335 3.5 IV, single dose 4 AZD5305 1 PO, QDx28 7 NIP228-SG3932 3.5 IV, single dose 4 8 NIP228-SG3932 1.25 IV, single dose 4 AZD5305 1 PO, QDx28 9 NIP228-SG3932 3.5 IV, single dose 4 AZD5305 1 PO, QDx28 IV: intravenous; PO : oral; QDx28: once daily for 28 days; NA: not applicable. # Dose volume: 10 ml/kg Tumor size and body weight measurement

Same as Example 23, except that tumor measurements and body weight were collected twice weekly from Day 26 to Day 89. Results

As summarized in Table 30 and shown in Figure 23 , AZD5335 monotherapy resulted in 6.65% and 8.76% TGI at 1.25 mg/kg and 3.5 mg/kg, respectively, compared to the untreated group. However, both values were not significant. The AZD5305 and NIP-228-SG3932 isoforms alone also did not provide a TGI benefit compared to the untreated group.

The combination of AZD5335 and AZD5305 produced better efficacy than either agent alone, with a maximum response of 49.5% TGI observed in the higher dose combination group (3.5 mg/kg AZD5335 + 1 mg/kg AZD5305) and 20.47% TGI observed in the lower dose combination group (1.25 mg/kg AZD5335 + 1 mg/kg AZD5305). [ Table 30 ] : Differences in CTG-3718 xenograft tumor growth between experimental groups at day 55 Group treatment Dosage Level (mg/kg) ^# Mean tumor volume (mm ³ ) ± SEM % TGI, compared with untreated p-value (ANOVA) ^* 1 Unprocessed NA 633.59 ± 184.4 NA NA 2 AZD5305 1 749.53 ± 206.95 NA 0.9950 3 AZD5335 1.25 591.43 ± 121.33 6.65 0.9998 4 AZD5335 3.5 578.09 ± 96.89 8.76 0.9997 5 AZD5335 1.25 503.88 ± 40.91 20.47 0.8914 AZD5305 1 6 AZD5335 3.5 319.96 ± 107.18 49.50 0.5519 AZD5305 1 7 NIP228-SG3932 3.5 715.92 ± 110.45 NA 0.9963 8 NIP228-SG3932 1.25 575.41 ± 81.15 9.18 0.9985 AZD5305 1 9 NIP228-SG3932 3.5 458.35 ± 57.61 27.66 0.8730 AZD5305 1 ANOVA: analysis of variance; NA: not applicable; SEM: standard error of the mean; TGI: tumor growth inhibition, NIP288-SG3932: isotype control ADC # Dose volume: 10 ml/kg; * Comparison between untreated and treated groups; p-value < 0.05 indicates a significant difference in mean tumor volume. Conclusion

In the CTG-3718 PDX model, although significance was not reached, the activity of the AZD5335/AZD5305 combination at high doses (3.5 mg kg AZD5335 + 1 mg/kg AZD5305) was significantly more active than either AZD5335 or AZD5305 monotherapy.

Pharmacological inhibition of the DNA damage response pathway is an attractive approach to maximize the response to TOPOi ADCs and provide greater anti-tumor activity. This study evaluated the combination of AZD5335, a potent PARP1-selective inhibitor that exhibited reduced hematologic toxicity in preclinical models compared to non-selective PARP1 inhibitors, and AZD5305. The combination of AZD5335 and AZD5305 provided greater anti-tumor activity than either monotherapy, resulting in greater tumor regression or improved duration of response. Example 25 - In vivo anti-cancer activity of the combination of an anti-FRα ADC and a PARP1 inhibitor in additional patient-derived xenograft (PDX) models

Folate receptor α (FRα) is a cell surface GPI-anchored protein that is overexpressed in several solid tumors, with the highest incidence in ovarian cancer and lung adenocarcinoma, but with restricted expression in normal tissues. AZD5335, an antibody targeting FRα conjugated to AZ's proprietary topoisomerase 1 inhibitor (TOP1i) payload AZ14170132, is currently being studied in the FONTANA Phase 1/2 clinical trial (NCT05797168). Because homologous recombinant repair deficiency (HRD) and mutations in homologous recombinant repair-related genes (HRRm+) are associated with sensitivity to DNA damaging agents, we evaluated the efficacy of AZD5335 in ovarian cancer patient-derived xenograft (PDX) models evaluating HRRm as described in Clarke et al. 2022, NEJM Evid 2022;1(9) DOI: 10.1056/EVIDoa2200043. Sixteen ovarian cancer PDX models were treated with a single dose of AZD5335 (5 mg/kg IV). In the HRRm- group, 8/11 (73%) responded to treatment with >30% tumor regression from baseline. In the HRRm+ group, 4/5 (80%) responded to AZD5335. PARP1 can act as a positive regulator of genomic stability by counteracting TOP1-induced DNA damage (Malanga and Althaus, JBC 2004; 279(7) DOI: 10.1074/jbc.C300437200). Therefore, the inventors hypothesized that the efficacy of TOP1i drugs could be enhanced by combining with PARP inhibitors.

To evaluate the potential synergistic effects of AZD5335 and the PARP1 inhibitor saruparib (AZD5305), three FRα-expressing cancer cell lines (KB, IGROV-1, and OVCAR-3) were treated with AZD5335 +/- saruparib, revealing evidence of synergistic cytotoxicity (see also Example 22 ). These in vitro results encouraged us to study this combination in preclinical HRRm+ in vivo xenograft models (OVCAR-3 and OV2022F). The OVCAR-3 xenograft model was essentially as described in Example 23 : single agent activity with saruparib (1 mg/kg PO once daily for 28 days; 1 mg/kg PO QD x28) was 37% tumor growth inhibition (TGI), and single agent activity with AZD5335 (0.625 mg/kg intravenously as a single dose; IV x1) was 50% TGI. However, combination administration of the same doses increased the TGI to 86%. Similar combination activity was observed in OV2022F, with TGIs of 61%, 54%, and 97% for saruparib, AZD5335, and saruparib + AZD5335, respectively ( see Table 31 and Figure 24 ). [ Table 31 ]: Differences in OV2022F xenograft tumor growth between experimental groups treatment Mean tumor volume (mm ³ ) ± SEM % TGI (compared to untreated) p -value (ANOVA ) Treatment versus no treatment Treatment vs. Nip228 control Unprocessed 1771.5 ± 290.45 NA NA 0.0169 Nip228 1.25 mg/kg 980.47 ± 148.81 44.65 0.0169 NA AZD5305 1 mg/kg 680.58 ± 200.77 61.58 0.0005 0.7708 Nip228+AZD5305 1.25 + 1 mg/kg 100.89 ± 23.87 94.30 < 0.0001 0.0063 AZD5335 1.25 mg/kg 816.48 ± 290.31 53.91 0.0026 0.9902 AZD5335+AZD5305 1.25 + 1 mg/kg 50.44 ± 21.34 97.15 < 0.0001 0.0035 AZD5335+AZD5305 1.25 + 0.1 mg/kg 71.57 ± 35.9 95.96 < 0.0001 0.0045

In conclusion, AZD5335 exhibited similar levels of single-agent antitumor activity in both HRRm+ and HRRm- preclinical models, and its efficacy could be enhanced by combination with the PARP inhibitor sarupari (AZD5305). Materials and Methods

OVCAR-3 xenograft materials and methods were as described in Example 23.

For OV2022F, the materials and methods are as follows: All animal studies were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility. Female mice were housed in individual ventilated cages (Techniplast Greenline GM500) with corn cob bedding. Each cage was cleaned once a week, and the animals were examined twice daily. Mice were fed Envigo Teklad Global 18% protein chow diet with food and water ad libitum. The animal housing environment was maintained at a constant temperature of 22.2°C ± 4°C and a humidity of 50% ± 20% with a 12-h light-dark cycle.

Seed-expanded PDX tumors were grown for 76 days to a volume of approximately 530 mm3 before expansion for use in studies. Donor mice were first euthanized by CO2 asphyxiation followed by cervical dislocation. Mice were immersed in 70% isopropyl alcohol containing 2% chlorhexidine gluconate (Sigma) for 5 minutes. Mice were then placed on a sterile field and wiped with povidone-iodine followed by 70% isopropyl alcohol. The entire tumor was then excised and placed in RPMI medium containing 50 µg/ml tadalafil (Thermo Fisher Scientific) and subsequently cut into 3 x 3 mm3 fragments using sterile forceps and a scalpel. Mice receiving donor 3 x 3 mm3 tumors were swabbed with povidone-iodine and 70% isopropyl alcohol and subsequently implanted into the right flank using a 10-gauge or 11-gauge trocar (Innovative Research, Inc., USA). Tumors were allowed to reach approximately 180 mm3 before randomization. Mice were randomized to treatment groups using the deterministic randomization method built into Study Director software (Research Diary Systems, Inc., South San Francisco, CA, USA) without animal replacement.

Test ADC products (AZD5335 or Nip228-SG3932 DAR8 ("Nip228")) were diluted from stock solutions in vehicle buffer (25 mM histidine, 7% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH 6.0) and administered as a single intravenous (IV) injection at a volume of 10 ml/kg based on body weight. AZD5305 was prepared by sonicating the dry compound in an appropriate volume of water/HCl to obtain a 1 mg/kg (0.1 mg/ml) dosing solution. AZD5305 was administered daily by oral gavage (PO) for 28 days and was administered at a volume of 10 ml/kg based on body weight.

Female NSG mice were randomly assigned to groups on day 34. Animals in Group 1 were untreated. Animals in Groups 2 and 4 were administered 1.25 mg/kg Nip228 as a single IV administration. Animals in Groups 3, 4, and 6 were administered 1 mg/kg AZD5305 orally for 28 days, and Group 7 was administered 0.1 mg/kg AZD5305. Animals in Groups 5, 6, and 7 were administered 1.25 mg/kg AZD5335 as a single IV administration. Group names and dose levels are shown in Table 32. [Table 32]: Group names and dose levels in the OV2022F study Group treatment Dosage Level (mg/kg) ^# Investment channels Number of animals 1 Unprocessed NA NA 5 2 Nip228 1.25 IV, single dose 5 3 AZD5305 1 PO, QDx28 5 4 Nip228 1.25 IV, single dose 5 AZD5305 1 PO, QDx28 5 AZD5335 1.25 IV, single dose 5 6 AZD5335 1.25 IV, single dose 5 AZD5305 1 PO, QDx28 7 AZD5335 1.25 IV, single dose 5 AZD5305 0.1 PO, QDx28 IV: intravenous; PO : oral; QDx28: once daily for 28 days; NA: not applicable. #Dose volume: 10 ml/kg

Tumor measurements and body weight were collected twice weekly from day 33 to day 89, as described in Example 23 .

without

The present disclosure will now be described in more detail with reference to the accompanying drawings, in which:

[ Figure 1 ] shows the binding of (A) AB1370026; (B) AB1370035; (C) AB1370049; (D) AB1370083; (E) AB1370096; and (F) AB1370117 to human FRα, cynomolgus monkey FRα, mouse FRα, human FRβ, or human FRγ as determined by HTRF.

[ Figure 2 ] shows a High Content Profiler multiparameter analysis of antibody internalization into KB cells, plotted with intensity on the vertical axis and sample concentration on the horizontal axis. Antibodies with absorbance above the cutoff value determined by the positive control sample (circle) are shortlisted and shown as gray diamonds, with antibodies below the cutoff value shown as white diamonds. Data points for the six exemplary antibodies were run in duplicate in the assay, represented by black diamonds and labeled with the sample name. At least one sample for each exemplary antibody was above the cutoff value.

[ FIG. 3 ] shows HTRF epitope competition assays of six exemplary IgGs with: (A) Comparator 1 IgG; (B) Comparator 2 IgG; and (C) Comparator 3 IgG.

[ Figure 4 ] shows the results of the AC-SINS self-interaction assay. The propensity of antibodies to self-associate in HSA (black bars) buffer was measured. As expected, the negative control antibody exhibited low levels of self-interaction, while the positive control antibody showed high levels of self-interaction in HSA. The threshold (> 5 nm) used to mark antibodies at risk is indicated by the horizontal dashed line.

[ Figure 5 ] shows the internalization of anti-FRα antibody into (A) Jeg-3 cells (with medium FRα expression) and (B) KB cells (with high FRα expression), measured by the increase in fluorescence on the CX7 instrument over time.

[ Figure 6 ] shows the chromatograms of AB1370049-SG3932 DAR8 obtained from: (A) UHPLC-RP at 214 nm (in reduced form); (B) UHPLC-RP at 330 nm (in reduced form); (C) UHPLC-SEC at 280 nm; and (D) UHPLC-HIC at 214 nm.

[ Figure 7 ] shows the chromatograms of AB1370049-SG3932 DAR4 obtained from: (A) UHPLC-RP at 214 nm (in reduced form); (B) UHPLC-RP at 330 nm (in reduced form); (C) UHPLC-SEC at 280 nm; and (D) UHPLC-HIC at 214 nm.

[ Figure 8 ] shows the chemical transformation to a series of DAR8 ADCs upon exposure to various sera. The chemical processes observed are (A) deconjugation, and (B) maleimide hydrolysis.

[ FIG. 9 ] shows a 6-day cytotoxicity assay using the lead DAR8 ADC on the following cells: (A) KB cells (with high FRα expression); (B) Jeg-3 cells (with medium to high FRα expression); and (C) Igrov-1 cells (with medium FRα expression).

[ Figure 10 ] shows the bystander killing activity of AB1370049-SG3932 DAR8. FRα-positive and FRα-negative KB cells were treated with 1 nM AB1370049-SG3932 DAR8 ADC alone or at a 1:1 ratio for 6 days. Cytotoxic activity was measured by flow cytometry after 6 days.

[ Figure 11 ] shows a KB xenograft study using the lead DAR8 ADC. On day 6, a single intravenous injection of (A) 1.25 mg/kg or (B) 5 mg/kg was administered.

[ Figure 12 ] shows an OVCAR-3 xenograft study using the lead DAR8 ADC. On day 33, a single intravenous injection of (A) 1.25 mg/kg or (B) 5 mg/kg was administered.

[ Figure 13 ] shows an IGROV-1 xenograft study using AB1370049-SG3932 DAR8. On day 33, a single intravenous injection of 5 mg/kg was administered.

[ Figure 14 ] shows a KB xenograft study using the lead DAR4 ADC. On day 7, a single intravenous injection of 5 mg/kg was administered.

[ FIG. 15 ] shows (A) OVCAR-3 and (B) CaCo-2 xenograft studies in which AB1370049-SG3932 DAR8 or FRα-DM4 ADCs were administered at single doses of 1.25, 2.5, and 5 mg/kg.

[ FIG. 16 ] shows the median percentage of tumor growth resulting from: (A) a single administration of 5 mg/kg AB1370049-SG3932 DAR8 in 51 PDX models, and (B) a single administration of 2.5 mg/kg AB1370049-SG3932 DAR8 in 39 PDX models.

[ FIG. 17 ] shows PDX model studies in which a single dose of 2.5 mg/kg or 5 mg/kg of AB1370049-SG3932 DAR8 was administered to (A) ovarian PDX model CTG-0711; (B) NSCLC PDX model CTG-2367; and (C) endometrial PDX model CTG-2268.

[ Figure 18 ] shows cell viability information measured on the following cells: (A) Pioneer cells in the megakaryocyte lineage; (B) Pioneer cells in the myeloid lineage; (C) Pioneer cells in the erythroid lineage; (D) Expanding and differentiating cells in the megakaryocyte lineage; (E) Expanding and differentiating cells in the myeloid lineage; and (F) Expanding and differentiating cells in the erythroid lineage. The X-axis represents drug concentration and the Y-axis represents percent survival (values are mean values of triplicates -/+SD). AB1370049-SG3932 DAR8 did not show enhanced toxicity compared to a non-targeted ADC control in primary CD34 ⁺ bone marrow-derived hematopoietic stem cell precursor cells induced to differentiate into erythroid, myeloid, or megakaryocyte lineages.

[ Figure 19 ] Shows the mean (±SD) of the concentration-time curves of unconjugated mAb compared to AB1370049-SG3932 DAR8 in cynomolgus monkeys. PK profiles of 15 and 25 mg/kg plasma samples collected and processed using immunocapture LC-MS/MS assays and non-compartmental PK. Total mAb is a measure of intact antibody (including ADC or deconjugated mAb in the case of ADC). Total ADC is a measure of intact ADC only.

[ Figure 20 ] shows the results of a 6-day cytotoxicity assay performed on the following cells: (A) KB cells treated with 0.000457 to 3 nM AZD5335, 123.45 to 10000 nM AZD5305, or a combination of the two; (B) IGROV-1 cells treated with 0.00152 to 10 nM AZD5335, 123.45 to 10000 nM AZD5305, or a combination of the two; (C) OVCAR-3 cells treated with 0.00152 to 50 nM AZD5335, 0.0247 to 2 nM AZD5305, or a combination of the two; and (D) 0.1 nM to 700 nM AZD5335, 30 nM to 30 SKOV-3 cells treated with AZD5305 at 4 µM. Only data points for AZD5305 are included in the figure for comparison purposes and they do not correspond to the concentrations provided on the x-axis.

[ FIG. 21 ] Shows a heat map of the synergy matrix of AZD5335 and AZD5305 in the following cells: (A) KB cells; (B) IGROV-1 cells; (C) OVCAR-3 cells; and (D) SKOV-3 cells. Synergy was calculated using Bliss model analysis. A higher positive score indicates a stronger synergy. A score of 0 indicates additivity, and a negative value indicates antagonism. The darker the black coloring, the higher the synergy score.

[ Figure 22 ] shows studies of the OVCAR-3 CDX model 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 ^mm3 . The mice were then randomized and dosed on day 33. Tumor volume was measured twice a week. NIP228-SG3932 (abbreviated as "Nip228") and AZD5305 (abbreviated as "PARPSel") alone showed very weak to no anti-tumor activity. Modest tumor growth inhibition (TGI) was observed with AZD5335 (1.25 mg/kg) as a single agent, but the combination of AZD5335 and AZD5305 (1.25 mg/kg + 1 mg/kg) produced greater additive efficacy. The combination of the NIP228-SG3932 isoform with AZD5305 showed antitumor activity early in the study but failed to show a sustained response. The low-dose group AZD5335 1.25 mg/kg + AZD5305 0.1 mg/kg showed modest tumor growth inhibition with additive effects. Values are mean ± SEM tumor volume for n: 6 animals per group. Dashed lines indicate dosing days.

[ Figure 23 ] shows a CTG3718 PDX model study using single therapy with AZD5335 or AZD5305 or a combination therapy with AZD5335 and AZD5305. CTG3718 PDX tumors were grown in female NSG mice until tumor volume reached approximately 180 ^mm3 . Mice were then randomized and dosed on day 26. Tumor volume was 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 significant tumor growth inhibition was produced with the combination of 3.5 mg/kg AZD5335 + AZD5305. NIP228-SG3932 isotype ADC (also abbreviated "NIP228") did not exhibit any TGI, although a small level of tumor growth inhibition was observed with the group combined with AZD5305. Values are mean ± SEM tumor volume, for n: 4 animals per group.

[ Figure 24 ] shows OV2022F PDX model studies using single therapy with AZD5335 or AZD5305 or combination therapy with AZD5335 and AZD5305. OV2022F xenograft tumors were grown in female NSG mice until they reached approximately 180 ^mm3 in size. Mice were then randomized and dosed on day 34. Tumor volume was measured twice a week. NIP228-SG3932 (abbreviated as "Nip228") and AZD5305 ("PARPSel") alone showed some anti-tumor activity. Moderate tumor growth inhibition (TGI) was observed with AZD5335 (1.25 mg/kg) as a single agent, but better efficacy was produced with the combination of AZD5335 and AZD5305 (1.25 mg/kg + 1 mg/kg). The low-dose group AZD5335 (1.25 mg/kg) + AZD5305 (0.1 mg/kg) as a combination also showed strong tumor growth inhibition. Values are mean ± SEM tumor volume, for n: 5 animals per group.

without

TW202448517A_113105350_SEQL.xml

Claims (34)

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 linked to a cytotoxin; and (b) a PARP1 inhibitor. The method of claim 1, wherein the cancer comprises cancer cells having heterogeneous expression of FRα and/or low expression of FRα; optionally, wherein the cancer cells have FRα expression similar to that of the Igrov-1 cell line. 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, and optionally wherein the cancer is selected from ovarian cancer and lung cancer. The method of claim 3, wherein the lung cancer is non-small cell lung cancer (NSCLC), optionally wherein the NSCLC is selected from squamous NSCLC, adenocarcinoma NSCLC, or a combination thereof. A kit comprising: (a) an ADC comprising an anti-FRα antibody or an antigen-binding fragment thereof linked to a cytotoxin; and (b) a PARP1 inhibitor. The method of any one of claims 1 to 4, or the kit of claim 5, wherein the PARP1 inhibitor is AZD5305 having the following formula: or a pharmaceutically acceptable salt thereof. A method as described in any one of claims 1-4 or 6, or a kit as described in claim 5 or claim 6, wherein the anti-FRα antibody or antigen-binding fragment thereof comprises: (a) heavy chain CDR1 (SDSATWN) of SEQ ID NO: 1, heavy chain CDR2 (RTYYRSKWYNDYAVSVKS) of SEQ ID NO: 2, heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, light chain CDR2 (KASGLES) of SEQ ID NO: 5, and light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6; (b) heavy chain CDR1 (SYAMS) of SEQ ID NO: 7, heavy chain CDR2 (SISSGRSYIYYADSVKG) of SEQ ID NO: 8, heavy chain CDR3 (GVGSFDY) of SEQ ID NO: 3, light chain CDR1 (RASQSISSWLA) of SEQ ID NO: 4, light chain CDR2 (KASGLES) of SEQ ID NO: 5, and light chain CDR3 (QQYNSYSQLT) of SEQ ID NO: 6; 9 heavy chain CDR3 (EMQQLALDY), SEQ ID NO: 10 light chain CDR1 (RASQGISNFLA), SEQ ID NO: 11 light chain CDR2 (AASSLQS), and SEQ ID NO: 12 light chain CDR3 (QQYNSYPFT); (c) SEQ ID NO: 13 heavy chain CDR1 (SNSAAWN), SEQ ID NO: 14 heavy chain CDR2 (RTYYRSNWYNDYTLSVKS), SEQ ID NO: 15 heavy chain CDR3 (GVGRFDS), SEQ ID NO: 16 light chain CDR1 (RASQSISSWLA), SEQ ID NO: 17 light chain CDR2 (KASSLES), and SEQ ID NO: 18 light chain CDR3 (QEYKTYSIFT); (d) SEQ ID NO: 19 heavy chain CDR1 (SYNMN), SEQ ID NO: 20 heavy chain CDR2 (SISSGSSYIYYADSMKG), SEQ ID NO: 21 heavy chain CDR3 (GMTTLTFDY), SEQ ID NO: 22 light chain CDR1 (RASQGISTFLA), SEQ ID NO: 23 light chain CDR2 (AASSLQS), and SEQ ID NO: 24 light chain CDR3 (QQYISYPLT); (e) SEQ ID NO: 25 heavy chain CDR1 (SYSMN), SEQ ID NO: 26 heavy chain CDR2 (SISSRSSYVYYADSVKG), SEQ ID NO: 27 heavy chain CDR3 (GMTTLTFDY), SEQ ID NO: 28 light chain CDR1 (RASQGISSFLA), SEQ ID NO: 29 light chain CDR2 (AASSLQS), and SEQ ID NO: 30 light chain CDR3 (QQYNSYPLT); or (f) SEQ ID NO: 31 heavy chain CDR1 (SDSATWN), SEQ ID NO: 32 heavy chain CDR2 (RTYYRSKWYSDYAVSVKS), SEQ ID NO: 33 heavy chain CDR3 (GGAPFDY), SEQ ID NO: 34 light chain CDR1 (RASQSISSWLA), SEQ ID NO: 35 light chain CDR2 (KASSLES), and SEQ ID NO: 36 light chain CDR3 (QQYNSYSMYT). A method as described in any one of claims 1-4 or 6-7, or a kit as described in any one of claims 5-7, wherein the anti-FRα antibody or antigen-binding fragment thereof comprises: (a) a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 37 and a VL comprising an amino acid sequence 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 at least 90% identical to the amino acid sequence of SEQ ID NO: 45 and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 46; or (f) a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 47 and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 48. The method as described in claim 8, or the kit as described in claim 8, wherein the anti-FRα antibody or antigen-binding fragment thereof comprises: (a) L at the N-terminus (e.g., position 1) of the VH; (b) E at the N-terminus (e.g., position 1) of the VH; or (c) Q at the N-terminus (e.g., position 1) of the VH. A method as described in any one of claims 1-4 or 6-9, or a kit as described in any one of claims 5-9, wherein the anti-FRα antibody or antigen-binding fragment thereof comprises: (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) VH of SEQ ID NO: 45 and VL of SEQ ID NO: 46; or (f) VH of SEQ ID NO: 47 and VL of SEQ ID NO: 48. A method as described in any one of claims 1-4 or 6-10, or a kit as described in any one of claims 5-10, wherein the anti-FRα antibody comprises a constant heavy chain and a constant light chain, the constant heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 109 or 111, and the constant light chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 110. The method as described in any one of claims 1-4 or 6-11, or the kit as described in any one of claims 5-11, wherein the anti-FRα antibody comprises a constant heavy chain amino acid sequence of SEQ ID NO: 109 or 111 and a constant light chain amino acid sequence of SEQ ID NO: 110. A method as described in any one of claims 1-4 or 6-12, or a kit as described in any one of claims 5-12, wherein the anti-FRα antibody comprises: (a) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 49 and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 50; (b) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 51 and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 52; (c) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 53 and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 54; (d) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 55 and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 56; (e) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 57 and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 58; or (f) a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 59 and a light chain comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 60. A method as described in any one of claims 1-4 or 6-13, or a kit as described in any one of claims 5-13, wherein the anti-FRα antibody comprises: (a) a heavy chain amino acid sequence of SEQ ID NO: 49 and a light chain amino acid sequence of SEQ ID NO: 50; (b) a heavy chain amino acid sequence of SEQ ID NO: 51 and a light chain amino acid sequence of SEQ ID NO: 52; (c) a heavy chain amino acid sequence of SEQ ID NO: 53 and a light chain amino acid sequence of SEQ ID NO: 54; (d) a heavy chain amino acid sequence of SEQ ID NO: 55 and a light chain amino acid sequence of SEQ ID NO: 56; (e) a heavy chain amino acid sequence of SEQ ID NO: 57 and a light chain amino acid sequence of SEQ ID NO: 58; or (f) The heavy chain amino acid sequence of SEQ ID NO: 59 and the light chain amino acid sequence of SEQ ID NO: 60. A method as described in any one of claims 1-4 or 6-12, or a kit as described in any one of claims 5-12, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment or a F(ab')2 fragment. The method of any one of claims 1-4 or 6-15, or the kit of any one of claims 5-15, wherein the anti-FRα antibody or antigen-binding fragment thereof is fully human. A method as described in any one of claims 1-4 or 6-16, or a kit as described in any one of claims 5-16, wherein the anti-FRα antibody or antigen-binding fragment thereof is monoclonal, polyclonal, recombinant or multispecific. The method as described in any one of claims 1-4 or 6-17, or the kit as described in any one of claims 5-17, wherein the anti-FRα antibody or its antigen-binding fragment is of IgG1 type, IgG2 type, IgG3 type or IgG4 type. The method as described in claim 18 or the kit as described in claim 18, wherein the anti-FRα antibody or antigen-binding fragment thereof is of IgG1 type. The method as described in any one of claims 1-4 or 6-19, or the kit as described in any one of claims 5-19, wherein the cytotoxin is linked to the anti-FRα antibody or antigen-binding fragment thereof via a linker ^RL selected from the following: (Ia): , where Q is: , wherein Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; and X is: , wherein a = 0 to 5, b1 = 0 to 16, b2 = 0 to 16, c1 = 0 or 1, c2 = 0 or 1, d = 0 to 5, wherein 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); ^GL is a linker for linking the anti-FRα antibody or antigen-binding fragment thereof; (Ib): , wherein R ^L1 and R ^L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group; and e is 0 or 1; or (Ib'): wherein R ^L1 and R ^L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group. The method of claim 20, or the kit of claim 20, wherein ^GL is . The method of claim 20 or claim 21, or the kit of claim 20 or claim 21, wherein R ^L is . The method of any one of claims 1-4 or 6-22, or the kit of any one of claims 5-22, wherein the cytotoxin is selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof. The method of claim 23 or the kit of claim 23, wherein the cytotoxin is a topoisomerase I inhibitor. The method of claim 23 or 24, or the kit of claim 23 or 24, wherein the topoisomerase I inhibitor is represented by formula (I): and its salts and solvates; wherein ^RL is as defined in any one of claims 20-22. The method of any one of claims 23-25, or the kit of any one of claims 23-25, wherein the topoisomerase I inhibitor is: ; ; ; and/or . The method of claim 26 or the kit of claim 26, wherein the topoisomerase I inhibitor is: . The method of any one of claims 1-4 or 6-27, or the kit of any one of claims 5-27, wherein the drug-to-antibody ratio (DAR) of the ADC ranges from about 1 to 20, optionally wherein the range of DAR is selected from about 1 to 10, about 2 to 10, about 2 to 8, about 2 to 6, and about 4 to 10. The method of claim 28, or the kit of claim 28, wherein the DAR is about 8 or about 4. The method of claim 29, or the kit of claim 29, wherein the DAR is approximately 8. The method of any one of claims 1-4 or 6-30, or the kit of any one of claims 5-30, wherein (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 wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) The cytotoxin is the topoisomerase I inhibitor SG3932 ; and (iii) the DAR of the ADC is approximately 8. The method of any one of claims 1-4 or 6-31, or the kit of any one of claims 5-31, wherein (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 wherein the anti-FRα antibody or antigen-binding fragment thereof has a VH of SEQ ID NO: 37 and a VL of SEQ ID NO: 38; (ii) The cytotoxin is the topoisomerase I inhibitor SG3932 (iii) the DAR of the ADC is about 8; and (iv) the PARP1 inhibitor is AZD5305 having the formula: or a pharmaceutically acceptable salt thereof. The method of any one of claims 1-4 or 6-32, wherein the ADC and the PARP1 inhibitor are administered separately or sequentially. The method of any one of claims 1-4 or 6-32, wherein the ADC and the PARP1 inhibitor are administered together.