Anti-folate receptor alpha antibodies and methods of use

Abstract

Antibody-drug conjugates (ADCs) comprising antibody constructs that bind to human folate receptor alpha (FRα or FOLR1) and anti-FRα antibody constructs conjugated to drugs such as cytotoxins or immunomodulators, and their use as therapeutic or diagnostic agents, for example, in the treatment or diagnosis of cancer.

Description

[Technical field]

[0001] The present disclosure relates to the field of antibody therapeutics, and in particular to antibodies that target the human folate receptor alpha (hFRα). [Background technology]

[0002] Folate receptor alpha (FRα) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein encoded by FOLR1 and is a member of a family of high-affinity FRs that also includes FRβ (FOLR2), FRγ (FOLR3), and FRδ (FOLR4). FRα has been identified as a relevant cancer therapy target due to its overexpression in various cancers, including ovarian cancer, triple-negative breast cancer (TNBC), endometrial cancer, mesothelioma, and lung cancer, and minimal expression in non-malignant tissues.

[0003] Several clinical studies are currently underway involving FRα-targeted agents in the treatment of cancer, including the anti-FRα antibody, farletuzumab, and the FRα-targeted antibody-drug conjugates (ADCs), mirvetuximab soravtansine (ImmunoGen, Inc.), MORAb-202 (Eisai Inc.), and STRO-002 (Sutro Biopharma, Inc.).

[0004] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. Summary of the Invention

[0005] Described herein are anti-FRα antibodies and methods of use. One aspect of the disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to human folate receptor alpha (hFRα), where the antibody construct competes for binding to hFRα with a reference antibody that specifically binds to an epitope within hFRα that comprises amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO:15.

[0006] Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to an epitope within human folate receptor alpha (hFRα) comprising amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO:15.

[0007] Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to human folate receptor alpha (hFRα), wherein the antigen-binding domain comprises heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8.

[0008] Another aspect of the present disclosure relates to an antibody construct comprising two antigen binding domains operably linked to an IgG Fc region, each of the antigen binding domains specifically binding to human folate receptor alpha (hFRα); (a) the VL amino acid sequence set forth in SEQ ID NO: 39 and the VH amino acid sequence set forth in SEQ ID NO: 19, or (b) a VL amino acid sequence set forth in SEQ ID NO: 124 and a VH amino acid sequence set forth in SEQ ID NO: 91; or (c) the VL amino acid sequence set forth in SEQ ID NO: 64, and (i) the VH amino acid sequence set forth in SEQ ID NO: 50, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 54, or (iii) the VH amino acid sequence set forth in SEQ ID NO: 57, or (iv) the VH amino acid sequence set forth in SEQ ID NO: 61, or (v) the VH amino acid sequence set forth in SEQ ID NO: 76, or (vi) the VH amino acid sequence set forth in SEQ ID NO: 79, or (vii) the VH amino acid sequence set forth in SEQ ID NO: 82; or (viii) the VH amino acid sequence set forth in SEQ ID NO: 85, or (ix) the VH amino acid sequence set forth in SEQ ID NO: 88, or (x) the VH amino acid sequence set forth in SEQ ID NO: 106, or (d) the VL amino acid sequence set forth in SEQ ID NO: 130, and (i) the VH amino acid sequence set forth in SEQ ID NO: 99, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 106, or (iii) the VH amino acid sequence set forth in SEQ ID NO: 113, or (iv) the VH amino acid sequence set forth in SEQ ID NO: 116, or (v) the VH amino acid sequence set forth in SEQ ID NO: 133, or (vi) the VH amino acid sequence set forth in SEQ ID NO: 136, or (e) the VL amino acid sequence set forth in SEQ ID NO: 119, and (i) the VH amino acid sequence set forth in SEQ ID NO: 106, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 116 Includes.

[0009] Another aspect of the disclosure pertains to a polynucleotide or set of polynucleotides that encodes the anti-FRα antibody constructs described herein.

[0010] Another aspect of the disclosure relates to an expression vector or a set of expression vectors comprising a polynucleotide or a set of polynucleotides encoding an anti-FRα antibody construct as described herein. Another aspect of the disclosure relates to a host cell comprising the expression vector or a set of expression vectors.

[0011] Another aspect of the disclosure relates to antibody-drug conjugates comprising an anti-FRα antibody construct described herein conjugated to one or more drug moieties.

[0012] Another aspect of the present disclosure is a compound represented by general formula I: A-(L-(D) m ) n (I) In an embodiment of the invention, an antibody-drug conjugate having the formula: A is an anti-FRα antibody construct as described herein; L is a linker, D is a drug moiety; m is from 1 to about 8; n is between 1 and about 12.

[0013] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an anti-FRα antibody construct described herein, or an antibody-drug conjugate described herein, and a pharma- ceutically acceptable carrier or diluent.

[0014] Another aspect of the disclosure relates to an anti-FRα antibody construct as described herein or an antibody-drug conjugate as described herein for use in therapy, eg, the treatment of cancer.

[0015] Another aspect of the disclosure relates to the use of an anti-FRα antibody construct described herein or an antibody-drug conjugate described herein in the manufacture of a medicament for the treatment of cancer.

[0016] Another aspect of the disclosure relates to a method of inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an anti-FRα antibody construct described herein or an antibody-drug conjugate described herein.

[0017] Another aspect of the disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of an anti-FRα antibody construct described herein or an antibody-drug conjugate described herein. [Brief description of the drawings]

[0018] [Figure 1A] Figure 1A shows the sequence of the rabbit heavy chain variable domain CDRs (SEQ ID NO: 155) of the chimeric antibody v23924 grafted onto a human VH framework (IGHV3-23*01), and Figure 1B shows the sequence of the rabbit light chain variable domain CDRs (SEQ ID NO: 156) of the chimeric antibody v23924 grafted onto a human VL framework (IGKVI-39*01). The CDRs are assigned by AbM definition and marked in bold italics. [Figure 1B] See legend to Figure 1A. [Figure 2A] Figures 2A-D show the profiles of the purified parent chimeric variant v23924 and the purified representative humanized variant v30384 analyzed by electrophoresis and UPLC-SEC. Figures 2A and C show the profiles from electrophoresis under non-reducing (NR) and reducing (R) conditions after preparative SEC purification (post-preparative SEC) or after Protein A purification (post-pA) of the parent chimeric variant v23924 (2A) and the purified representative humanized variant v30384 (2C), and Figures 2B and D show the UPLC-SEC profiles of the parent chimeric variant v23924 after preparative SEC purification (2B) and the purified representative humanized variant v30384 after Protein A purification (2D). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Diagram 3] 3A and B show biolayer interferometry (BLI) sensorgrams of the parental chimeric variant v23924 (3A) and a purified representative humanized variant v30384 (3B). [Figure 4A] 4A-D show intact LC / MS profiles of representative humanized variants v30384 (4A, with accompanying zoom of the main peak in 4B) and v31422 (4C, with accompanying zoom of the main peak in 4D). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Diagram 5] 5A and B show the receptor-mediated internalization capacity of chimeric antibody v23924, a representative humanized variant v30384, and the FRα-targeting antibodies mirvetuximab and farletuzumab at various concentrations in the FRα-expressing cell line IGROV-1 as determined by flow cytometry after 6 hours (5A) and 24 hours (5B). The anti-RSV antibody palivizumab was included as a negative control. [Figure 6] 6A and B show the receptor-mediated internalization capacity of chimeric antibody v23924, a representative humanized variant v30384, and FRα-targeting antibodies mirvetuximab and farletuzumab at various concentrations in the FRα-expressing cell line OVCAR-3 as determined by flow cytometry after 6 hours (6A) and 24 hours (6B). The anti-RSV antibody palivizumab was included as a negative control. [Figure 7] 1 shows coverage of the hFRα sequence (SEQ ID NO: 15) by peptides generated by pepsin digestion of hFRα. Each bar below the sequence represents a peptide. [Figure 8] 8A and B show summary plots (8A) and difference plots (8B) of hydrogen / deuterium exchange mass spectrometry (HDX-MS) kinetics of peptides generated by pepsin digestion of hFRα:hFOLR1 (hFRα) versus hFOLR1-v23924 complexes. [Figure 9] 9A-C show the amide deuteration levels of peptide 119-126 (WEDCRTSY) (SEQ ID NO: 152) after 1 hour of hydrogen / deuterium exchange mass spectrometry (HDX-MS): hFOLR1 (9A) vs. hFOLR1-v23924 complex (9B), and the difference plot (9C). [Figure 10]Figures 10A and B show the receptor-mediated internalization capacity of the parent humanized variant, v30384, and a representative affinity matured variant, v35356, in the FRα-expressing cell lines IGROV-1 (10A) and JEG-3 (10B) after 5 and 24 hour incubation periods as determined by flow cytometry. Palivizumab was included as a non-targeting control. [Figure 11-1] Figures 11A-D show the intracellular payload delivery capabilities of representative ADCs in cell lines JEG-3 (11A), Caov-3 (11B), H2110 (11C), and HEC-1-A (11D) as assessed by mass spectrometry. The ADCs were humanized antibody variant v30384 and affinity matured variant v35356, respectively, conjugated to drug linker DL1. [Figure 11-2] See description of Figure 11-1. [Figure 12A] Figures 12A-H show the in vivo anti-tumor activity of chimeric anti-FRα antibody v23924 conjugated to drug linker DL1 or DL7 evaluated in xenograft models: CTG-0848 PDX (12A), OV90 CDX (12B), OVCAR-3 CDX (12C), LXFA737 PDX (12D), JEG3 CDX (12E), HCC1954 CDX (12F), SKOV3 CDX (12G), and KB CDX (12H). The control ADC was v17717 (mirvetuximab Fab with HetFc) conjugated to drug linker DL1 or DL6. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. [Figure 12F] See legend to Figure 12A. [Figure 12G] See legend to Figure 12A. [Figure 12H] See legend to Figure 12A. [Figure 13]1 shows the in vivo antitumor activity of ADCs comprising chimeric antibody v23924 or humanized variants v30384 or v30399, respectively, conjugated to drug linker DL1, administered at 4 mg / kg or 9 mg / kg in a medium / high level FRα-expressing OVCAR3 ovarian cancer model. [Figure 14A] Figures 14A-E show the in vivo antitumor activity of ADCs containing humanized variant v30384 conjugated to drug linker DL1 or DL8 administered at the indicated doses as assessed in xenograft models: H2110 CDX (14A), SKOV3 CDX (14B and 14C), IGROV-1 CDX (14D), and LXFA737 PDX (14E). Control ADCs were v17717 (mirvetuximab Fab with HetFc) conjugated to drug linker DL6, v17716 (mirvetuximab Fab with HomoFc) conjugated to drug linker DL6, and v31629 (farletuzumab) conjugated to drug linker DL4 for H2110 CDX. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 15A] Figures 15A-D show the in vivo antitumor activity of ADCs containing humanized variant v30384 conjugated to drug linker DL5 administered in xenograft models at the indicated doses: OV90 (15A), H2110 (15B), and OVCAR-3 (15C and 15D). [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 16A]16A-H show the results of pharmacokinetic analyses showing the concentration of IgG or ADC in serum over time in serum collected from animals treated with various anti-FRα antibodies; chimeric anti-FRα antibody v23924 conjugated to drug linker DL1 in the OV90 model (16A), OVCAR-3 model (16B), LXFA737 model (16C), JEG3 model (16D), or SKOV-3 model (16E); humanized variant v30384 conjugated to drug linker DL1 in the H2110 model (16F), and humanized variant v30384 conjugated to drug linker DL5 in the OV90 model (16G) or H2100 model (16H). [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 16E] See legend to Figure 16A. [Figure 16F] See legend to Figure 16A. [Figure 16G] See legend to Figure 16A. [Figure 16H] See legend to Figure 16A. [Figure 17-1] A table showing the CDR sequences of representative anti-FRα antibodies as defined by the IMGT, Chothia, Kabat, Contact and AbM definitions is provided. [Figure 17-2] See description of Figure 17-1. [Figure 17-3] See description of Figure 17-1. [Figure 17-4] See description of Figure 17-1. [Figure 17-5] See description of Figure 17-1. [Figure 17-6] See description of Figure 17-1. [Figure 17-7] See description of Figure 17-1. [Figure 18-1] A table showing the VH and VL sequences of representative anti-FRα antibodies is provided. [Figure 18-2]See description of Figure 18-1. [Figure 18-3] See description of Figure 18-1. [Figure 19-1] 19A shows the cell proliferation inhibition (cytotoxicity) ability of an ADC comprising humanized antibody v30384 conjugated to drug linker DL1, and an ADC comprising affinity matured variant v35356 conjugated to drug linker DL1 in cell lines: KB-HeLa (19A), IGROV-1 (19B), JEG-3 (19C), SKOV-3 (19D) and MDA-MB-468 (19E). [Figure 19-2] See description of Figure 19-1. [Figure 19-3] See description of Figure 19-1. [Figure 20] Figures 20A-D show the penetration of anti-FRα humanized antibody variant v36675 into JEG-3 cell spheroids compared to mirvetuximab and negative control palivizumab at 4 hours (20A), 24 hours (20B), 48 hours (20C), and 96 hours (20D). [Figure 21A] Figures 21A and B show fixed cell confirmation screening images from screening anti-FRα humanized antibody variant v36675 at 20 μg / mL (21A) and control antibody (rituximab biosimilar) at 1 μg / mL (21B) for specific off-target binding interactions using Retrogenix Cell Microarray Technology. [Figure 21B] See legend to Figure 21A. [Figure 22] 1 shows competitive binding between chimeric anti-FRα antibody v23294 and the anti-FRα antibodies mirvetuximab and farletuzumab assessed in H2110 cells. [Diagram 23] 1 shows the receptor-mediated internalization capacity of humanized variant v30384 compared to biparatopic anti-FRα antibody B5327A (v36264) and anti-FRα antibody mirvetuximab (v17716) in the FRα-expressing cell line IGROV-1 as determined by flow cytometry after a 5 hour incubation period. [Figure 24] 1 shows the penetration of anti-FRα humanized antibody variant v36675 in JEG-3 cell spheroids compared to biparatopic anti-FRα antibody B5327A (v36264) and anti-FRα antibody mirvetuximab (v17716) at 96 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description The present disclosure relates to antibody constructs that bind to human folate receptor alpha (FRα; also referred to herein as FOLR1), but do not exhibit significant binding to folate receptor beta (FOLR2), gamma (FOLR3), or delta (FOLR4). In certain embodiments, the anti-FRα antibody constructs of the present disclosure can bind to cynomolgus monkey FRα.

[0020] The present disclosure also relates to antibody-drug conjugates (ADCs) comprising the anti-FRα antibody constructs described herein conjugated to a drug, such as a cytotoxin or an immunomodulator. The anti-FRα antibody constructs and ADCs of the present disclosure can be used, for example, as therapeutic or diagnostic agents. Certain aspects of the present disclosure relate to therapeutic methods and uses of the anti-FRα antibody constructs and ADCs, for example, in the treatment of cancer. Some aspects relate to diagnostic methods and uses of the anti-FRα antibody constructs and ADCs, for example, in the diagnosis or analysis of cancer.

[0021] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0022] As used herein, the term "about" refers to approximately a ±10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0023] The use of the words "a" or "an" when used herein in conjunction with the term "comprising" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0024] As used herein, the terms "comprising," "having," "including," and "containing," as well as grammatical variations thereof, are inclusive, i.e., open ended, and do not exclude additional unrecited elements and / or method steps. The term "consisting essentially of," when used herein in connection with a composition, use, or method, indicates that additional elements and / or method steps may be present, but that these additions do not substantially affect the manner in which the recited composition, method, or use functions. The term "consisting of," when used herein in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and may consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically referred to.

[0025] "Complementarity determining regions" or "CDRs" are amino acid sequences that contribute to antigen binding specificity and affinity. "Framework" regions (FRs) can help maintain the proper conformation of CDRs to facilitate binding between the antigen binding region and the antigen. From the N-terminus to the C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for binding an antibody to an antigen or epitope. In many cases, three heavy chain CDRs and three light chain CDRs are required to bind an antigen. However, in some cases, even a single variable domain can confer binding specificity to an antigen. Furthermore, as is known in the art, in some cases, antigen binding may also occur through a combination of at least one or more CDRs, e.g., HCDR3s, selected from the VH and / or VL domains.

[0026] Several different definitions of CDR sequences are in common use, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and the definitions of IMGT, AbM (University of Bath) and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). By way of example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM and Contact are set forth in Table 1 below. Thus, as will be readily apparent to one of skill in the art, the exact numbering and arrangement of CDRs may vary based on the numbering system used. However, it should be understood that the disclosure of VH herein includes the disclosure of the associated (unique) heavy chain CDRs (HCDRs) defined by any of the known numbering systems. Similarly, disclosure herein of a VL includes disclosure of the associated (unique) light chain CDR (LCDR), as defined by any known numbering system.

[0027] [Table 1]

[0028] The term "identical" in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have the same percentage of amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95%, or about 98% identity over a designated region) when compared and aligned for maximum correspondence over a comparison window or over a designated region, as measured using one of the commonly used sequence comparison algorithms known to those skilled in the art, or by manual alignment and visual inspection. For sequence comparison, typically, a designated reference sequence and a test sequence are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence compared to the reference sequence based on the program parameters.

[0029] A "comparison window" refers to a segment of a sequence that includes consecutive amino acid or nucleotide positions, which may be, for example, about 10 to about 600 consecutive amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 consecutive amino acid or nucleotide positions, and a test sequence may be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are known to those of skill in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA or TFASTA, from the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0030] The term "subject" as used herein refers to an animal, in some embodiments a mammal, that is the object of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, non-human primate, etc.). In certain embodiments, the subject is a human.

[0031] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0032] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein can be combined in any suitable manner with the features, structures and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0033] It should also be understood that the positive recitation of a feature in one embodiment serves as a basis for excluding features in alternative embodiments. For example, when a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be deleted from the list and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically referenced.

[0034] Anti-FRα antibody construct The present disclosure relates to an antibody construct that specifically binds to human FRα (hFRα). In this context, the term "antibody construct" refers to a polypeptide or set of polypeptides that includes one or more antigen-binding domains, each of which specifically binds to an epitope or antigen. When an antibody construct includes two or more antigen-binding domains, each of the antigen-binding domains can bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they can bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct can further include a scaffold, and one or more antigen-binding domains can be fused or covalently linked to the scaffold, optionally via a linker, as described herein.

[0035] According to the present disclosure, the anti-FRα antibody construct comprises at least one antigen binding domain that specifically binds to hFRα. By "specifically binds" to hFRα, it is meant that the antibody construct binds to hFRα but does not show significant binding to any of the human folate receptors beta (FOLR2), gamma (FOLR3) or delta (FOLR4). In certain embodiments, the anti-FRα antibody construct of the present disclosure can bind to FRα from one or more non-human species. In certain embodiments, the anti-FRα antibody construct of the present disclosure can bind to cynomolgus monkey FRα.

[0036] Human FRα is also known as "human folate receptor 1" or "FOLR1". Protein sequences of hFRα from various sources are known in the art and are readily available from publicly accessible databases such as GenBank or UniProtKB. Examples of hFRα sequences include, for example, sequences provided under NCBI reference numbers P15328, AAX29268.1, AAX37119.1, NP_057937.1 and NP_057936.1. An exemplary hFRα protein sequence is provided in Table 2 as SEQ ID NO: 1 (NCBI Reference Sequence: NP_057936.1). An exemplary cynomolgus monkey FRα protein sequence is also provided in Table 2 (SEQ ID NO: 2; NCBI Reference Sequence: XP_005579002.2).

[0037] [Table 2]

[0038] Specific binding of the antigen-binding domain to a target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., using a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or traditional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as binding to a non-target protein (such as FOLR2, FOLR3 or FOLR4) being about less than about 10% of binding to hFRα, as measured, for example, by ELISA or flow cytometry. In certain embodiments, specific binding of an antibody construct to FRα has a dissociation constant (K ) of ≦1 μM, e.g., ≦500 nM, ≦250 nM, ≦100 nM, ≦50 nM, or ≦10 nM. D In certain embodiments, the specific binding of an antibody construct to a particular antigen or epitope may be defined by a specific binding affinity of 10 -6 M or less, e.g., 10 -7 M or less, or 10 -8The dissociation constant (K D In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope may be defined by a specific binding constant greater than 10 -6 M~10 -9 M, for example, 10 -7 M~10 -9 Dissociation constant of M (K D )

[0039] In certain embodiments, the anti-FRα antibody constructs of the present disclosure exhibit higher internalization into FRα-expressing cells than the reference antibodies mirvetuximab (huMov19 or huFR107) and farletuzumab (MORAb-003).

[0040] Antibody internalization can be measured using methods known in the art, for example, the direct internalization method following the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as pHAb dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dye (ThermoFisher Scientific Corporation, Waltham, MA) and Incucyte® Fabfluor-pH antibody labeling reagent (Sartorius AG, Göttingen, Germany), as well as analytical techniques such as microscopy, FACS, high content imaging or other plate-based assays.

[0041] In certain embodiments, an anti-FRα antibody construct is considered to exhibit higher internalization into FRα-expressing cells than the corresponding reference antibody (mirvetuximab or farletuzumab) if the amount of the anti-FRα antibody construct internalized into the FRα-expressing cells is at least 1.2 times higher than the amount of the reference antibody internalized into the same FRα-expressing cells under the same test conditions. In certain embodiments, the amount of internalized antibody is determined using an appropriate fluorescent dye and high content imaging. In some embodiments, the amount of internalized antibody is determined in cells expressing FRα at high levels. In some embodiments, the amount of internalized antibody is determined in IGROV-1 cells or cells expressing FRα at levels similar to IGROV-1 cells. In some embodiments, the amount of internalized antibody is determined after a 6 hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 24 hour incubation period.

[0042] In certain embodiments, an anti-FRα antibody construct is considered to exhibit higher internalization into FRα-expressing cells than the corresponding reference antibody (mirvetuximab or farletuzumab) if the amount of anti-FRα antibody construct internalized into FRα-expressing cells is at least 1.3 times greater, at least 1.4 times greater, at least 1.5 times greater, 1.6 times greater, 1.7 times greater, 1.8 times greater, 1.9 times greater, or 2.0 times greater than the amount of reference antibody internalized into the same FRα-expressing cells under the same test conditions. In certain embodiments, the amount of internalized antibody is determined using an appropriate fluorescent dye and high content imaging. In some embodiments, the amount of internalized antibody is determined in cells expressing FRα at high levels. In some embodiments, the amount of internalized antibody is determined in IGROV-1 cells or cells expressing FRα at a level similar to IGROV-1 cells. In some embodiments, the amount of internalized antibody is determined after a 6 hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 24 hour incubation period.

[0043] Antigen-binding domain The anti-FRα antibody construct of the present disclosure comprises at least one antigen-binding domain capable of binding to hFRα. The at least one antigen-binding domain capable of binding to hFRα is typically an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fab (scFab), single-chain Fv (scFv), and single-domain antibodies (sdAbs).

[0044] A "Fab fragment" comprises the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), together with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of a Fab light chain may be connected to the N-terminus of a Fab heavy chain in a single-chain Fab molecule.

[0045] An "scFv" comprises an antibody heavy chain variable domain (VH) and a light chain variable domain (VL) in a single polypeptide chain. An scFv may optionally further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For example, an scFv may comprise a VL connected from its C-terminus to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv may comprise a VH connected via its C-terminus to the N-terminus of the VL by a polypeptide linker (see Pluckthun's review, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0046] The "sdAb" format refers to a single immunoglobulin domain. sdAbs may be, for example, of Camelidae origin. Camelidae antibodies lack light chains and their antigen binding site consists of a single domain, called "VHH". sdAbs contain three CDRs / hypervariable loops that form the antigen binding site: CDR1, CDR2 and CDR3. sdAbs are fairly amenable to stable expression, for example as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0047] In those embodiments in which the anti-FRα antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain can independently be an immunoglobulin-based domain, e.g., an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, e.g., a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, e.g., a natural or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, finomers, affimers, alphabodies, DARPins, and avimers.

[0048] The present disclosure describes herein an antibody that specifically binds to hFRα (variant v23924), as well as the identification of representative humanized versions of this antibody (variants v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425 and v31426) and representative affinity matured versions of this antibody (variants v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168 and v36675) (see Examples and Sequence Listing). Epitope mapping using the hFRα sequence shown in Figure 7 (sequence number 15) determined that the epitope within the hFRα protein bound by variant v23924 includes amino acid residues E120, D121, R123, T124, S125, and Y126 of sequence number 15 (see Example 13).

[0049] In certain embodiments, at least one antigen-binding domain that binds hFRα contained in an anti-FRα antibody construct of the present disclosure binds to an epitope in the hFRα protein that includes amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15. In some embodiments, the hFRα epitope bound by the anti-FRα antibody construct is a non-linear (or discontinuous) epitope that includes amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15. In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain that competes for binding to hFRα with an antibody that binds to an epitope in the hFRα protein that includes amino acid residues E120, D121, R123, T124, S125, and Y126. In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain that competes for binding to hFRα with antibody v23924 described herein.

[0050] Using competitive assays known in the art, it can be determined whether an antibody construct competes with an antibody that binds to an epitope in the hFRα protein that includes amino acid residues E120, D121, R123, T124, S125, and Y126, or antibody v23924 (reference antibody) for binding to hFRα. For example, an antibody that binds to an epitope in the hFRα protein that includes amino acid residues E120, D121, R123, T124, S125, and Y126, or antibody v23924 (reference antibody), is first allowed to bind to hFRα under saturating conditions, and then the ability of the test antibody construct to bind to hFRα is measured. If the test antibody construct can bind to hFRα simultaneously with the reference antibody, the test antibody construct is considered to bind to a different epitope from the reference antibody. Conversely, if the test antibody construct is unable to bind to hFRα simultaneously with the reference antibody, it is considered to bind to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the reference antibody. Competitive assays may also be performed by reversing the binding order of the reference and test antibodies, i.e., allowing the test antibody to bind to hFRα first under saturating conditions, and then measuring the ability of the reference antibody construct to bind to hFRα.

[0051] Such competitive assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interferometry, flow cytometry, etc. An "antibody that competes with" a reference antibody refers to an antibody that blocks binding of the reference antibody to its epitope by 50% or more in a competitive assay.

[0052] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises at least one antigen binding domain that specifically binds to hFRα, and the antigen binding domain comprises a set of CDRs based on the CDRs of antibody variant v23924 described herein. The CDR sequences of antibody v23924 and representative humanized or affinity matured versions of this antibody are shown in FIG. 17. Analysis of the CDR sequences from the parent and affinity matured anti-FRα antibodies identified the minimal amino acid sequences present in each CDR as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. These amino acid sequences are represented by the minimal consensus CDR sequences provided in Table 3. Expanded versions of these CDR consensus sequences based on the CDR sequences defined by the AbM numbering system are shown in Table 4.

[0053] [Table 3]

[0054] [Table 4]

[0055] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8.

[0056] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (i) the HCDR1 amino acid sequence set forth in SEQ ID NO: 3, the HCDR2 amino acid sequence set forth in SEQ ID NO: 4, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 5 (X 2 is L and X 3 is A or X 2 is H and X 3is P), and (ii) the LCDR1 amino acid sequence set forth in SEQ ID NO:6 (X 4 is G and X 5 is D or X 4 is W and X 5 is Y), the LCDR2 amino acid sequence set forth in SEQ ID NO: 7, and the LCDR3 amino acid sequence set forth in SEQ ID NO: 8 (X 6 is S and X 7 is N and X 8 is V and X 9 is D or X 6 is W and X 7 is H and X 8 is I and X 9 is L).

[0057] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 9, 10, and 11, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 12, 13, and 14.

[0058] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (i) the HCDR1 amino acid sequence set forth in SEQ ID NO: 9, the HCDR2 amino acid sequence set forth in SEQ ID NO: 10 (X 11 is S or A, and X 12 is V or X 11 is S and X 12 is L), and the HCDR3 amino acid sequence set forth in SEQ ID NO: 11 (X 13 is L and X 14 is A or X 13 is H and X 14 is P), and (ii) the LCDR1 amino acid sequence set forth in SEQ ID NO: 12 (X 15 is R or Q, and X 16 is G and X 17 is D or X 15 is R and X16 is W and X 17 is Y), the LCDR2 amino acid sequence set forth in SEQ ID NO: 13, and the LCDR3 amino acid sequence set forth in SEQ ID NO: 14 (X 18 is S and X 19 is N and X 20 is V and X 21 is D or X 18 is W and X 19 is H and X 20 is I and X 21 is L).

[0059] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (i) Variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v3616 8, or v36675, variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v3534 HCDR2 amino acid sequences selected from any one of the HCDR2 amino acid sequences of v23924, v30618, v30384, v30389, v30394, v30399, v31 HCDR3 amino acid sequence selected from any one of the HCDR3 amino acid sequences: v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675; and (ii) Variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v3616 8, or v36675, variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347 , v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, and variants v23924, v30618, v30384, v30389, v30394, v30399, v3142 v35354, v35356, v35358, v36167, v36168, or v36675; The CDR amino acid sequences are as defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Figure 17).

[0060] In certain embodiments, the anti-FRα antibody construct of the disclosure comprises a heavy chain CDR amino acid sequence (HCDR1, HCDR2 and HCDR3) selected from the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. R3), and any one of the light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0061] In certain embodiments, the anti-FRα antibody constructs of the disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v31427, v31429, v31430, v31431, v31432, v31433, v31434, v31435, v31436, v31437, v31438, v31439, v31439, v31440, v31441, v31442, v31443, v31444, v31445, v31446, v31447, v31448, v31449, v31450, v31451, v31452, v31453, v31454, v31455, v31456, v31457, v31458, v31459, v31460, v31461, v31462, v31463, v31464, v31465, v31466, v31467, v31468, v31469, v31470, v31471, v31472, v31473, v31474, v31475, v31476, v31477, v31478, v31479, v31480, v31481, v31482, v31483, v31484, v31485, v31 and a light chain CDR amino acid sequence (LCDR1, LCDR2, and LCDR3) of any one of v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0062] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising the CDR sequences of the VH domain of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising the CDR sequences of the VL domain of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. The VH and VL sequences of v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, and v36675 are provided in Figure 18.

[0063] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequence of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequence of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0064] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence and a VL amino acid sequence selected from the VH amino acid sequence and the VL amino acid sequence of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675.

[0065] Those skilled in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR sequence or VH or VL sequence of a known antibody without losing the ability of the antibody to bind to its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art, such as alanine scanning, and the resulting variants are tested for binding activity by standard techniques. Thus, in certain embodiments, the anti-FRα antibody constructs of the disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. and an antigen-binding domain comprising a set of CDRs (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3) that have 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to any one of the sets of CDRs, where the % sequence identity is calculated across all six CDRs, and wherein the antigen-binding domain retains the ability to bind to hFRα.

[0066] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a variant of the set of CDR sequences of any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, wherein the variant comprises 1-10 amino acid substitutions across the set of CDRs (i.e., the CDRs may be modified by up to 10 amino acid substitutions by modifying any combination of the six CDRs), and wherein the antigen-binding domain retains the ability to bind to hFRα. In some embodiments, the anti-FRα antibody constructs of the disclosure comprise variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v361 68, or v36675, wherein the variant comprises 1-7 amino acid substitutions, 1-5 amino acid substitutions, 1-4 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution across the set of CDRs, and wherein the antigen-binding domain retains the ability to bind to hFRα.

[0067] In certain embodiments, the anti-FRα antibody construct of the disclosure comprises variant v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v366 75, wherein the antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any one of the VH sequences ... In certain embodiments, the anti-FRα antibody construct of the disclosure comprises variant v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v366 75, wherein the antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any one of the VL sequences ...

[0068] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138, or 139; and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, 30, 107, 108, or 110; (ii) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, 45, 65, 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42, 47, 120, or 121.

[0069] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen binding domain comprising the CDR sequences of a VH domain having a sequence set forth in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136. In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen binding domain comprising the CDR sequences of a VL domain having a sequence set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0070] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (a) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, or 45, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; and an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31, an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 27, 29, or 32, and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; (b) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; and an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; (c) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47, and (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (ii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 58; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (iii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (d) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121, and (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (ii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; or (iii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (iv) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; or (v) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (vi) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 137, 138, or 139; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; or (e) an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65, an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46, and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; and (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; or (ii) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; and an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110.

[0071] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises the following antigen-binding domain: (a) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 27, 29, or 32; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 43, or 45; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (b) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (c) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 58; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (d) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (e) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (f) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 42 or 47; or (g) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (h) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (i) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (j) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 40, 45, or 65; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (k) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 107, 108, or 110; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (l) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121; or (m) an HCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 21, 24, 137, 138, or 139; an HCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 22, 25, or 30; an LCDR1 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 125, 126, or 127; an LCDR2 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from the amino acid sequences set forth in any one of SEQ ID NOs: 120 or 121.

[0072] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences set forth in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136. In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0073] In certain embodiments, an anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences set forth in any one of SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136, and a VL amino acid sequence selected from the VL amino acid sequences set forth in any one of SEQ ID NOs: 39, 64, 119, 124, or 130.

[0074] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising: (a) the VL amino acid sequence set forth in SEQ ID NO: 39 and the VH amino acid sequence set forth in SEQ ID NO: 19, or (b) a VL amino acid sequence set forth in SEQ ID NO: 124 and a VH amino acid sequence set forth in SEQ ID NO: 91; or (c) the VL amino acid sequence set forth in SEQ ID NO: 64, and (i) the VH amino acid sequence set forth in SEQ ID NO: 50, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 54, or (iii) the VH amino acid sequence set forth in SEQ ID NO: 57, or (iv) the VH amino acid sequence set forth in SEQ ID NO: 61, or (v) the VH amino acid sequence set forth in SEQ ID NO: 76, or (vi) the VH amino acid sequence set forth in SEQ ID NO: 79, or (vii) the VH amino acid sequence set forth in SEQ ID NO: 82; or (viii) the VH amino acid sequence set forth in SEQ ID NO: 85, or (ix) the VH amino acid sequence set forth in SEQ ID NO: 88, or (x) the VH amino acid sequence set forth in SEQ ID NO: 106, or (d) the VL amino acid sequence set forth in SEQ ID NO: 130, and (i) the VH amino acid sequence set forth in SEQ ID NO: 99, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 106, or (iii) the VH amino acid sequence set forth in SEQ ID NO: 113, or (iv) the VH amino acid sequence set forth in SEQ ID NO: 116, or (v) the VH amino acid sequence set forth in SEQ ID NO: 133, or (vi) the VH amino acid sequence set forth in SEQ ID NO: 136, or (e) the VL amino acid sequence set forth in SEQ ID NO: 119, and (i) the VH amino acid sequence set forth in SEQ ID NO: 106, or (ii) the VH amino acid sequence set forth in SEQ ID NO: 116.

[0075] In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises an antigen-binding domain comprising: (i) a VH amino acid sequence set forth in SEQ ID NO: 19 and a VL amino acid sequence set forth in SEQ ID NO: 39, or (ii) a VH amino acid sequence set forth in SEQ ID NO: 50 and a VL amino acid sequence set forth in SEQ ID NO: 64; or (iii) a VH amino acid sequence set forth in SEQ ID NO: 54 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (iv) a VH amino acid sequence set forth in SEQ ID NO: 57 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (v) a VH amino acid sequence set forth in SEQ ID NO: 61 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (vi) a VH amino acid sequence set forth in SEQ ID NO: 76 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (vii) a VH amino acid sequence set forth in SEQ ID NO: 79 and a VL amino acid sequence set forth in SEQ ID NO: 64; or (viii) a VH amino acid sequence set forth in SEQ ID NO: 82 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (ix) a VH amino acid sequence set forth in SEQ ID NO: 85 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (x) a VH amino acid sequence set forth in SEQ ID NO: 88 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (xi) a VH amino acid sequence set forth in SEQ ID NO: 91 and a VL amino acid sequence set forth in SEQ ID NO: 124, or (xii) a VH amino acid sequence set forth in SEQ ID NO: 99 and a VL amino acid sequence set forth in SEQ ID NO: 130, or (xiii) a VH amino acid sequence set forth in SEQ ID NO: 106 and a VL amino acid sequence set forth in SEQ ID NO: 64, or (xiv) a VH amino acid sequence set forth in SEQ ID NO: 106 and a VL amino acid sequence set forth in SEQ ID NO: 119, or (xv) a VH amino acid sequence set forth in SEQ ID NO: 106 and a VL amino acid sequence set forth in SEQ ID NO: 130, or (xvi) the VH amino acid sequence set forth in SEQ ID NO: 113 and the VL amino acid sequence set forth in SEQ ID NO: 130, or (xvii) the VH amino acid sequence set forth in SEQ ID NO: 116 and the VL amino acid sequence set forth in SEQ ID NO: 119, or (xviii) a VH amino acid sequence set forth in SEQ ID NO: 116 and a VL amino acid sequence set forth in SEQ ID NO: 130, or (xix) a VH amino acid sequence set forth in SEQ ID NO: 133 and a VL amino acid sequence set forth in SEQ ID NO: 130, or (xx) A VH amino acid sequence set forth in SEQ ID NO: 136, and a VL amino acid sequence set forth in SEQ ID NO: 130.

[0076] format The anti-FRα antibody construct of the present disclosure may have various formats. The minimum component of the anti-FRα antibody construct is an antigen binding domain that binds to hFRα. The anti-FRα antibody construct may further optionally comprise one or more additional antigen binding domains and / or scaffolds. In those embodiments in which the anti-FRα antibody construct comprises two or more antigen binding domains, each additional antigen binding domain may bind to the same epitope in hFRα, may bind to a different epitope in hFRα, or may bind to a different antigen. Thus, the anti-FRα antibody construct may be, for example, monospecific, biparatopic, bispecific, or multispecific.

[0077] In certain embodiments, the anti-FRα antibody construct comprises at least one antigen-binding domain that binds to hFRα and a scaffold, and the antigen-binding domain is operably linked to the scaffold. The term "operably linked" as used herein means that the described components are in a relationship that allows them to function in their intended manner. Examples of suitable scaffolds are described below.

[0078] In certain embodiments, the anti-FRα antibody construct comprises two antigen binding domains operably linked, optionally to a scaffold. In some embodiments, the anti-FRα antibody construct may comprise three or four antigen binding domains and optionally a scaffold. In these formats, when a scaffold is included, at least the first antigen binding domain is operably linked to the scaffold, and the remaining antigen binding domain(s) may each be independently operably linked to the scaffold or to the first antigen binding domain, or may be linked to another antigen binding domain if more than two antigen binding domains are present.

[0079] Anti-FRα antibody constructs lacking scaffolds may comprise a single antigen-binding domain in a suitable format, such as sdAb, or they may comprise two or more antigen-binding domains, optionally operably linked by one or more linkers. In such anti-FRα antibody constructs, the antigen-binding domain may be in the form of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as antigen-binding domain, formats such as tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may be used to construct diabody formats that include two scFvs connected by a short linker (usually about 5 amino acids in length). The limited length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFv may be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond may be introduced between the VL and VH by introducing an additional cysteine ​​residue in each chain (e.g., at position 44 in the VH and position 100 in the VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct with a DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0080] Similarly, in some embodiments, a format may be used that includes two sdAbs, such as VH or VHH, connected together via a suitable linker. Other examples of anti-FRα antibody construct formats that lack a scaffold include those based on Fab fragments, such as Fab2 and F(ab')2 formats, where the Fab fragments are connected via a linker or IgG hinge region.

[0081] Combinations of different forms of antigen-binding domains can also be used to create alternative scaffold-less formats, for example, scFvs or sdAbs can be fused to the C-terminus of either or both the light and heavy chains of a Fab fragment, resulting in a bivalent (Fab-scFv / sdAb) construct.

[0082] In certain embodiments, the anti-FRα antibody construct may be an immunoglobulin (Ig)-based antibody format. In certain embodiments, the anti-FRα antibody construct may be based on an IgG class immunoglobulin, such as an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-FRα antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when the anti-FRα antibody construct is based on a particular immunoglobulin isotype, it means that the anti-FRα antibody construct comprises all or a portion of the constant region of the particular immunoglobulin isotype. For example, an anti-FRα antibody construct based on a given Ig isotype may comprise at least one antigen binding domain operably linked to an Ig scaffold, the scaffold comprising an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It should also be understood that in some embodiments, the anti-FRα antibody construct may also comprise a hybrid of isotypes and / or subclasses. It is also understood that the Fc region and / or hinge region may optionally be modified to impart one or more desired functional properties as known in the art.

[0083] In some embodiments, an anti-FRα antibody construct may be derived from two or more immunoglobulins from different species, for example, an anti-FRα antibody construct may be a chimeric or humanized antibody. The terms "chimeric antibody" and "humanized antibody" both generally refer to antibodies that combine immunoglobulin regions or domains from two or more species.

[0084] A "chimeric antibody" typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., mouse) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain that it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Patent No. 4,816,567.

[0085] A "humanized antibody" is a type of chimeric antibody that contains minimal sequences derived from a non-human antibody. In general, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region (CDR) of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that has the desired specificity and affinity for the target antigen. This technique for producing humanized antibodies is often called "CDR grafting".

[0086] In some cases, additional modifications are made to the humanized antibody to further improve antibody performance. For example, framework region (FR) residues of the human immunoglobulin may be replaced with corresponding non-human residues, or the humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. Generally, the variable domains of a humanized antibody contain all or substantially all of the hypervariable regions from the non-human immunoglobulin and all or substantially all of the FRs from the human immunoglobulin sequence. Humanized antibodies are described in detail, for example, by Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596.

[0087] Numerous approaches are known in the art for selecting the optimal human framework for grafting non-human CDRs. Early approaches used a limited subset of well-characterized human antibodies, regardless of their sequence identity with the non-human antibody providing the CDRs ("fixed framework" approach). More recent approaches employ variable regions that have high amino acid sequence identity with the variable regions of the non-human antibody providing the CDRs ("homology matching" or "best-fit" approach). Another approach is to select fragments of framework sequences in the light or heavy chain variable regions from several different human antibodies. CDR grafting may in some cases result in partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some of the human-derived residues to the corresponding non-human-derived residues. Methods for preparing humanized antibodies by these approaches are well known in the art (see, e.g., Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0088] Alternatively or in addition to such traditional approaches, more recent techniques may be employed to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, a framework based on human germline sequence or consensus sequence may be employed as the acceptor human framework, rather than a human framework with somatic mutation(s). Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only specificity determining residues (SDRs). In this approach, only the minimum CDR residues ("SDRs") required for antigen binding activity are grafted onto a human germline framework. This method may help improve the "humanity" of humanized antibodies (i.e., similarity to human germline sequences), thus reducing the risk of immunogenicity of the variable region. These techniques are described in various publications (see, e.g., Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872; and Kashmiri, et al., 2005, Methods, 36:25-34).

[0089] In certain embodiments, the anti-FRα antibody constructs of the present disclosure comprise a humanized antibody sequence, e.g., one or more humanized variable domains. In some embodiments, the anti-FRα antibody constructs can be humanized antibodies. Non-limiting examples of humanized antibodies based on the anti-FRα antibody v23924 are described herein (v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, and v31426; see the Examples and Sequence Listing).

[0090] scaffold In certain embodiments, the anti-FRα antibody construct of the present disclosure comprises one or more antigen binding domains operably linked to a scaffold. The antigen binding domain(s) may be one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (e.g., leucine zipper, heterodimer-forming "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase barstar toxin), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0091] Scaffold can be peptide, polypeptide, polymer, nanoparticle or other chemical.When scaffold is polypeptide, each antigen binding domain of anti-FRα antibody construct can be linked to either N-terminus or C-terminus of polypeptide scaffold.Anti-FRα antibody construct comprising polypeptide scaffold in which one or more antigen binding domains are linked to other regions than N-terminus or C-terminus, for example, via amino acid side chain with or without linker, is also contemplated in certain embodiments.

[0092] In embodiments in which the anti-FRα antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen binding domain(s) can be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen binding domain(s) is linked to the scaffold by genetic fusion. In some embodiments in which the scaffold is a polymer or nanoparticle, the antigen binding domain(s) can be linked to the scaffold by chemical conjugation.

[0093] Several protein domains that comprise selective pairs of two different polypeptides are known in the art and can be used to form scaffolds. One example is the leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny, et al., J Immunol, 148:1547-53 (1992); Wranik, et al., J. Biol. Chem., 287:43331-43339 (2012)). Other selective pairing molecular pairs include, for example, the barnase barster pair (Deyev, et al. Nat Biotechnol, 21:1486-1492 (2003)), the DNA strand pair (Chaudri, et al., FEBS Letters, 450 (1-2): 23-26 (1999)), and the split fluorescent protein pair (International Patent Application Publication No. WO2011 / 135040).

[0094] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, e.g., Muller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U.S. Patent Application Publication No. 2009 / 0285816).

[0095] For example, fusing an antigen-binding moiety, such as an scFv, diabody, or single-chain diabody, to albumin has been shown to improve the serum half-life of the antigen-binding moiety (Muller et al., ibid.). The antigen-binding moiety can be fused, optionally via a linker, at the N-terminus and / or C-terminus of albumin.

[0096] Derivatives of albumin in the form of heteromultimers have been described that contain two transporter polypeptides obtained by segmentation of the albumin protein, such that the transporter polypeptides self-assemble to form quasi-native albumin (see International Patent Applications WO2012 / 116453 and WO2014 / 012082). As a result of the segmentation of albumin, the heteromultimer contains four ends and can therefore be fused, optionally via linkers, to up to four different antigen-binding moieties.

[0097] In certain embodiments, the anti-FRα antibody construct may comprise a protein scaffold. In some embodiments, the anti-FRα antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, albumin, or an albumin analog or derivative. In some embodiments, the anti-FRα antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, for example, an IgG Fc region.

[0098] Fc area The term "Fc region," "Fc," or "Fc domain," as used herein, refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues within an Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0099] In certain embodiments, the anti-FRα antibody constructs of the present disclosure may comprise a scaffold based on an immunoglobulin Fc region. The Fc region may be dimeric and composed of two Fc polypeptides, or alternatively, the Fc region may be composed of a single polypeptide.

[0100] "Fc polypeptide" in the context of a dimeric Fc refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that comprises one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to polypeptides that form a dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" may be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0101] The Fc region may comprise a CH3 domain, or may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0102] In some embodiments, the anti-FRα antibody construct may comprise a scaffold based on an IgG Fc region. In some embodiments, the anti-FRα antibody construct may comprise a scaffold based on a human IgG Fc region. In some embodiments, the anti-FRα antibody construct may comprise a scaffold based on an IgG1 Fc region. In some embodiments, the anti-FRα antibody construct may comprise a scaffold based on a human IgG1 Fc region.

[0103] In certain embodiments, the anti-FRα antibody construct may comprise an IgG Fc region-based scaffold that is a homodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each of which comprises a CH3 sequence and, optionally, a CH2 sequence, and the amino acid sequences of the first and second Fc polypeptides are identical.

[0104] In certain embodiments, the anti-FRα antibody construct may comprise an IgG Fc region-based scaffold that is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each of which comprises a CH3 sequence and, optionally, a CH2 sequence, and the amino acid sequences of the first and second Fc polypeptides are different. In some embodiments, the anti-FRα antibody construct may comprise an Fc region-based scaffold that comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-FRα antibody construct may comprise an Fc region-based scaffold that comprises two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications.

[0105] In some embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, the modified CH3 domain being an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modifications" refers to modifications, such as substitutions or insertions, in which an amino acid at a particular position on a first CH3 or CH2 sequence is different from an amino acid at the same position on a second CH3 or CH2 sequence. These asymmetric amino acid modifications may be the result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or of different modifications of both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may comprise one or more asymmetric amino acid modifications.

[0106] In some embodiments, the anti-FRα antibody construct can include a heterodimeric Fc that includes a modified CH3 domain, the modified CH3 domain includes one or more amino acid modifications that promote the formation of a heterodimeric Fc over the formation of a homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0107] Amino acid modifications that may be made to the CH3 domain of an Fc to promote the formation of a heterodimeric Fc are known in the art and include, for example, those described in WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include the combined positive and negative design strategies described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702 that result in asymmetrically modified stable Fc regions. In certain embodiments, the anti-FRα antibody construct may include a scaffold based on a modified Fc region as described in International Publication Nos. WO2012 / 058768 or WO2013 / 063702.

[0108] Table 5 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full-length human IgG1 heavy chain. Table 5 also lists the CH3 domain amino acid modifications that promote the formation of a heterodimeric Fc, as described in International Patent Applications WO2012 / 058768 and WO2013 / 063702.

[0109] In certain embodiments, the anti-FRα antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising any one of variant 1, variant 2, variant 3, variant 4 or variant 5 modifications as shown in Table 5.

[0110] [Table 5]

[0111] In some embodiments, the anti-FRα antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications. Modifications of the CH2 domain can affect Fc receptor (FcR) binding to Fc, such as receptors of the FcγRI, FcγRII and FcγRIII subclasses.

[0112] In some embodiments, the anti-FRα antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, where modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII and FcγRIII receptors.

[0113] Several amino acid modifications to the CH2 domain that selectively alter the affinity of Fc to different Fcγ receptors are known in the art. Amino acid modifications that result in increased binding and decreased binding, respectively, may be useful in certain applications. For example, increased binding affinity of Fc to FcγRIIIa (an activating receptor) may result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of target cells. Similarly, decreased binding to FcγRIIb (an inhibitory receptor) may be beneficial in some circumstances. In certain applications, reduced or eliminated ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb, or amino acid modifications that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) may be useful.

[0114] Examples of amino acid modifications to the CH2 domain that alter Fc binding by Fcγ receptors include, but are not limited to, S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al., 2007, J Immunol Methods, 365(1-2):132-41), al.,2011,Breast Cancer Res,13(6):R123), F243L (increased affinity for FcγRIIIa) (Stewart,et al.,2011,Protein Eng Des Sel.,24(9):671-8), S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields,et al.,2001,J Biol Chem,276(9):6591-604), S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar,et al.,2006,Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter Fc binding by FcγRIIb are described in International Publication No. WO2021 / 232162. Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012, page 283).

[0115] In certain embodiments, the anti-FRα antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain that contains one or more amino acid modifications that result in reduced or eliminated binding of the entire Fc region to Fcγ receptors (i.e., a "knockout" variant).

[0116] Various publications describe strategies used to engineer antibodies to generate "knockout" variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp225-249). These strategies include modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domains of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0117] Examples of mutations that can be introduced into the hinge or CH2 domain to generate "knock-out" variants include the amino acid modifications L234A / L235A, and L234A / L235A / D265S.

[0118] In certain embodiments, the anti-FRα antibody construct described herein may comprise a scaffold based on IgG Fc with modified native glycosylation.As known in the art, glycosylation of Fc may be modified to increase or decrease effector function.For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine (i.e., N297A, Q, K or H) results in non-glycosylated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao&Morrison, 1989, J. Immunol., 143:2595-2601).

[0119] Conversely, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC based on improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such reduced fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 that has a reduced ability to attach fucose to the N297-linked carbohydrate (International Publication No. WO 03 / 035835), or in other cells that produce non-fucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid, and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). In addition, WO 2009 / 135181 describes the addition of fucose analogues to the culture medium during antibody production to inhibit the incorporation of fucose into the carbohydrate on the antibody.

[0120] Other methods for producing antibodies with little or no fucose on the Fc glycosylation site (N297) are known in the art, e.g., GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0121] Other glycosylation variants include those that contain bisected oligosaccharides, for example, variants in which the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, for example, International Publication No. WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include variants that have at least one galactose residue on the oligosaccharide attached to the Fc region, which may have improved CDC function (see, for example, International Publication Nos. WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0122] In certain embodiments, the anti-FRα antibody construct has a format of a full size antibody (FSA). In some embodiments, the anti-FRα antibody construct has a format of an IgG FSA, e.g., an IgG1 FSA. In some embodiments, the anti-FRα antibody construct is an FSA comprising a first heavy chain sequence (H1), a second heavy chain sequence (H2), a first light chain sequence (L1) and a second light chain sequence (L2). In some embodiments, the anti-FRα antibody construct is a monospecific FSA with a homodimeric Fc, comprising H1, H2, L1 and L2 sequences, H1 and H2 having the same amino acid sequence, and L1 and L2 having the same amino acid sequence. In some embodiments, the anti-FRα antibody construct is a monospecific FSA with a heterodimeric Fc, comprising H1, H2, L1 and L2 sequences, H1 and H2 having different amino acid sequences, and L1 and L2 having the same amino acid sequence. In some embodiments, the anti-FRα antibody construct is a bispecific or biparatopic FSA having a heterodimeric Fc and comprises H1, H2, L1 and L2 sequences, wherein H1 and H2 have different amino acid sequences and L1 and L2 have different amino acid sequences.

[0123] In certain embodiments, the anti-FRα antibody construct is a FSA having a set of H1, H2, L1, and L2 sequences that include the H1, H2, L1, and L2 amino acid sequences shown in Tables A and B for any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675. As is known in the art, expression of antibody heavy chain sequences from particular cell lines or particular expression vectors can result in one or both of the heavy chains containing a C-terminal lysine residue. Thus, certain embodiments of the present disclosure relate to anti-FRα antibody constructs that are FSAs having a set of H1, H2, L1, and L2 sequences that include the H1, H2, L1, and L2 amino acid sequences set forth in Tables A and B for any one of variants v23924, v30618, v30384, v30389, v30394, v30399, v31422, v31423, v31424, v31425, v31426, v35305, v35342, v35347, v35348, v35350, v35354, v35356, v35358, v36167, v36168, or v36675, wherein one or both of the H1 and H2 sequences include a C-terminal lysine (see, e.g., SEQ ID NO: 157).

[0124] Preparation of anti-FRα antibody constructs The anti-FRα antibody constructs described herein can be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2 nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0125] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-FRα antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. The polynucleotide(s) encoding the anti-FRα antibody construct can be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and "Antibodies: A Laboratory Manual," 2001). nd (See, for example, The 1990s American Journal of Clinical Chemistry, Vol. 13, No. 1, pp. 111-114, 2012.) As will be understood by those skilled in the art, the number of polynucleotides required for the expression of anti-FRα antibody construct will depend on the format of the construct, including whether the antibody construct includes a scaffold. For example, if the anti-FRα antibody construct is a mAb format with homodimeric Fc, two polynucleotides each encoding one polypeptide chain will be required, whereas if the anti-FRα antibody construct is a mAb format with heterodimeric Fc, three polynucleotides each encoding one polypeptide chain will be required. If multiple polynucleotides are required, they can be incorporated into one vector or into two or more vectors.

[0126] Generally, for expression, a polynucleotide or set of polynucleotides is incorporated into an expression vector(s) together with one or more regulatory elements, e.g., transcription elements, necessary for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the choice of regulatory elements will depend on the host cell selected for expression of the antibody construct, and such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal vector or an integrating vector.

[0127] Suitable host cells for cloning or expressing anti-FRα antibody constructs include various prokaryotic or eukaryotic cells, as known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, E. coli cells, A. salmonicida cells, or B. subtilis cells.

[0128] In certain embodiments, anti-FRα antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed. Humana Press, Totowa, NJ, 2003.

[0129] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, particularly fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0130] The host cell suitable for expressing glycosylated anti-FRα antibody construct is usually eukaryotic cell.For example, US Patent No. 5,959,177, US Patent No. 6,040,498, US Patent No. 6,420,548, US Patent No. 7,125,978 and US Patent No. 6,417,429 describe the PLANTIBODIES™ technology for producing antigen-binding construct in transgenic plants.The mammalian cell line adapted to grow in suspension can be particularly useful for expressing antibody construct. Examples include SV40 (COS-7) transformed monkey kidney CV1 line, human embryonic kidney (HEK) line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad Sci. 2010, 14:131-132), and the like. Sci, 383: 44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR -Exemplary mammalian host cell lines suitable for the production of antibody constructs include, but are not limited to, CHO cells, see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216, and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines suitable for the production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed. Humana Press, Totowa, NJ, 2003).

[0131] In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NS0 or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows mature glycosylation of the antibody construct.

[0132] Host cells containing an expression vector(s) encoding an anti-FRα antibody construct may be cultured using conventional methods to produce the anti-FRα antibody construct. Alternatively, in some embodiments, host cells containing an expression vector(s) encoding an anti-FRα antibody construct may be used therapeutically or prophylactically to deliver the anti-FRα antibody construct to a subject, or a polynucleotide or expression vector may be administered ex vivo to cells from a subject, which may then be returned to the subject's body.

[0133] Typically, the anti-FRα antibody construct is purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3). rd(See Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques (including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration and reversed phase chromatography) performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods also include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration are also useful. As is well known in the art, various natural proteins bind Fc and antibodies, and these proteins can be used to purify specific antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can also be enabled by specific fusion partners. For example, antibodies can be bound to glutathione resins when GST fusions are used, Ni when His tags are used, or 100% glycerol when His tags are used. +2 It may be purified by affinity chromatography or, if a flag tag is used, by using immobilized anti-flag antibody. The degree of purification required varies depending on the use of the anti-FRα antibody construct. In some cases, purification may not be necessary.

[0134] In certain embodiments, the anti-FRα antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used in reference to the anti-FRα antibody constructs described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, e.g., in a natural cell, or in the case of a recombinantly produced construct, in a host cell. In certain embodiments, a substantially pure anti-FRα antibody construct is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating proteins.

[0135] Certain embodiments of the present disclosure relate to methods of producing an anti-FRα antibody construct, comprising culturing a host cell into which one or more polynucleotides encoding the anti-FRα antibody construct, or one or more expression vectors encoding the anti-FRα antibody construct, have been introduced under conditions suitable for expression of the anti-FRα antibody construct, and, optionally, recovering the anti-FRα antibody construct from the host cell (or host cell culture medium).

[0136] Post-translational modifications In certain embodiments, the anti-FRα antibody constructs described herein may include one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the anti-FRα antibody construct from a host cell.

[0137] Post-translational modifications include various modifications known in the art (see, for example, Proteins-Structure and Molecular Properties, 2nd Ed., TECreighton, WH Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, pgs.1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. NY Acad. Sci., 663:48-62). In embodiments in which the anti-FRα antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or more sites, or may comprise different modifications at different sites.

[0138] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage (with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4).

[0139] Other examples of post-translational modifications include, for example, the addition or removal of N-linked or O-linked glycans, chemical modification of N-linked or O-linked glycans, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, and addition or deletion of N-terminal methionine residues resulting from expression in a prokaryotic host cell. Post-translational modifications can also include modification with a detectable label, such as an enzyme label, a fluorescent label, a luminescent label, an isotopic label, or an affinity label, to allow for detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin, and aecolin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0140] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0141] Polynucleotides, Vectors and Host Cells Certain embodiments of the present disclosure relate to an isolated polynucleotide or set of polynucleotides that encode the anti-FRα antibody constructs described herein. A polynucleotide in this context may encode all or part of an anti-FRα antibody construct.

[0142] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0143] A polynucleotide that "encodes" a given polypeptide is one that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0144] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) that contain one or more polynucleotides encoding the anti-FRα antibody constructs described herein. The polynucleotide(s) can be comprised by a single vector or by more than one vector. In some embodiments, the polynucleotide can be comprised by a multicistronic vector.

[0145] Certain embodiments of the present disclosure relate to host cells that contain a polynucleotide(s) encoding one or more vectors that contain the anti-FRα antibody constructs or polynucleotide(s) described herein. In some embodiments, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0146] Antibody-drug conjugates Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising the anti-FRα antibody constructs described herein conjugated to one or more drug moieties, such as a cytotoxin or an immunomodulator.

[0147] Typically, in ADCs, the anti-FRα antibody construct is conjugated to a drug moiety via a linker, which may be a cleavable or non-cleavable linker. The anti-FRα antibody construct may be conjugated to a single drug molecule or may be conjugated to multiple drug molecules. The number of drug molecules conjugated to a single anti-FRα antibody construct is defined by the drug-antibody ratio (DAR). In certain embodiments, in the ADCs of the present disclosure, the DAR is within the range of about 1 to about 12, or about 2 to about 12, or about 2 to about 8.

[0148] In certain embodiments, the ADC comprising the anti-FRα antibody construct has the general formula I: A-(L-(D) m ) n (I) having wherein A is an anti-FRα antibody construct described herein, L is a linker, D is a drug moiety, m is 1 to about 8, and n is 1 to about 12.

[0149] In certain embodiments of Formula I, m is 1 to 6. In some embodiments, m is 1 or 2. In some embodiments, n is from about 1 to about 8, e.g., from about 2 to about 8.

[0150] Various compounds known to be useful as cytotoxic or immunomodulatory ADC payloads can be used as drug moieties in ADCs, including anti-FRα antibody constructs. Examples include, but are not limited to, maytansinoids and maytansinoid analogs, benzodiazepines and pyrrolobenzodiazepines, duocarmycins and their analogs, such as CC-1065, calicheamicins and calicheamicin analogs, auristatins and auristatin analogs, hemiasterlins and hemiasterlin analogs, tubulysins and tubulysin analogs, amatoxins and amatoxin analogs, camptothecins and camptothecin analogs, eribulin, TLR agonists (such as agonists of TLR7 and / or TLR8), and STING agonists.

[0151] In certain embodiments, the drug moiety comprised by an ADC of the disclosure is an auristatin or an auristatin analog, hemiasterlin or a hemiasterlin analog, camptothecin or a camptothecin analog, or eribulin.

[0152] Typically, in the ADCs of the present disclosure, the drug moiety is attached to the anti-FRα antibody construct by a linker. The linker is a bifunctional or multifunctional moiety that can attach one or more drug molecules to the antibody construct. In some embodiments, the linker can be bifunctional (or monovalent) to allow a single drug molecule to be attached to a single site on the antibody construct. In some embodiments, the linker can be multifunctional (or multivalent) to allow two or more drug molecules to be attached to a single site on the antibody construct. In some embodiments, a multifunctional linker may be used to attach one drug molecule to two or more sites on the antibody construct.

[0153] Attachment of the linker to the anti-FRα antibody construct can be achieved in a variety of ways, such as through surface lysines, by reductive coupling to oxidized carbohydrates, or through cysteine ​​residues liberated by reducing interchain disulfide bonds. Alternatively, attachment of a linker to the anti-FRα antibody construct can be achieved by modification of the antibody construct to include additional cysteine ​​residues (see, e.g., U.S. Pat. Nos. 7,521,541, 8,455,622, and 9,000,130), or by non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine, to allow site-specific conjugation (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2009, PNAS, 106:3000-3005). (See, e.g., E. et al., 2014, Bioconj. Chem., 25:351-361). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of antibody glycans to allow for the attachment of linkers via metal-free click chemistry (see, e.g., European Patent No. EP2911699).

[0154] The linker typically comprises a functional group capable of reacting with the targeting group(s) on the antigen-binding construct and one or more functional groups capable of reacting with the targeting group on the drug moiety. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (GT Hermanson, 2013, Academic Press). Non-limiting examples of functional groups for reacting with free cysteine ​​or thiols include maleimides, haloacetamides, haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. In this context, "self-stabilizing" maleimides are also useful, such as those described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062. Non-limiting examples of functional groups for reacting with surface lysines and amines include activated esters (e.g., N-hydroxysuccinamide (NHS) esters, sulfo-NHS esters), imidoesters (e.g., Traut's reagent), isothiocyanates, aldehydes, and acid anhydrides (e.g., diethylenetriaminepentaacetic anhydride (DTPA)). As another example, succinimide-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU) or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) may be used to convert carboxylic acids to activated esters, which are then reacted with amines. Non-limiting examples of functional groups that can react with electrophilic groups on antibody constructs or drug moieties (such as aldehyde or ketone carbonyl groups) include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0155] In certain embodiments, linkers containing functional groups that allow cross-linking of two interchain cysteines on the antibody-binding construct may be used, such as the ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem., 25:1124-1136), dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm., 12:3986-3998), dithioaryl (TCEP) pyridazinedione-based linkers (Lee et al., 2016, Chem. Sci., 7:799-802) or dibromopyridazinedione-based linkers (Maruani et al., 2015, Nat. Commun., 6:6645).

[0156] A variety of linkers are known in the art for linking drugs to antibodies, including hydrazone, disulfide and peptide-based linkers. Linkers may be cleavable or non-cleavable. Cleavable linkers are typically susceptible to cleavage under intracellular conditions, for example, via lysosomal processes. Examples include protease-sensitive, acid-sensitive or reduction-sensitive linkers. In contrast, non-cleavable linkers generally rely on the degradation of antibody in cells, which results in the release of amino acid-linker-drug moieties.

[0157] Examples of cleavable linkers that may be useful in certain embodiments are peptide-containing linkers that are cleavable by intracellular proteases, such as lysosomal or endosomal proteases. Examples include dipeptide-containing linkers, such as the dipeptides Val-Cit, Phe-Lys, Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro , linkers comprising phenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, or Met-(D)Lys, tripeptide-containing linkers, such as linkers comprising the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, or (D)Ala-Phe-Lys, and tetrapeptide-containing linkers, such as linkers comprising the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, or Ala-Leu-Ala-Leu.

[0158] Additional useful cleavable linkers include linkers that are hydrolyzable at a particular pH or within a pH range, such as disulfide-containing linkers and hydrazone linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimidyl-4-(2-pyridyldithio)butanoic acid (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoic acid (sulfo-SPDB). Optionally, disulfide-containing linkers may contain additional groups that provide steric hindrance adjacent to the disulfide bond, such as, for example, geminal dimethyl groups, to improve the extracellular stability of the linker. Linkers that contain a combination of these functional groups may also be useful, for example, linkers that contain both hydrazones and disulfides are known in the art.

[0159] A further example of a cleavable linker is a linker that contains a β-glucuronide that is cleavable by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (e.g., De Graaf et al., 2002, Curr. Pharm. Des., 8:1391-1403).

[0160] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative / self-leaving group, a stretcher, or a hydrophilic moiety.

[0161] Self-immolative and self-leaving groups that find use as linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED) and hemiaminal groups. Other examples of self-immolative groups include, but are not limited to, heterocyclic derivatives, such as aromatic compounds that are electronically similar to PABC or PABE groups, such as the 2-aminoimidazole-5-methanol derivatives described in U.S. Pat. No. 7,375,078. Other examples include groups that cyclize upon hydrolysis of the amide bond, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology, 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem., 55:5867-5877). Self-immolative / self-leaving groups, alone or in combination, are often included in peptide-based linkers, but may also be included in other types of linkers. In some embodiments, a linker may include one or more self-immolative / self-leaving groups, such as a PABC group, a PABE group, or a combination of a PABC or PABE group with MED.

[0162] Stretchers used in linkers for ADCs include, for example, alkylene groups and fatty acid, diacid, amine or diamine based stretchers, such as diglycolate, malonate, capronate and caproamide. Other stretchers include, for example, glycine based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers can also function as hydrophilic moieties and be particularly useful with hydrophobic drugs, although their use in linkers with other drugs is also contemplated in some embodiments.

[0163] In certain embodiments, the stretcher has the following structure: TIFF2025513721000006.tif39152, where: R is H or C1-C6 alkyl; t is an integer from 2 to 10; u is an integer from 1 to 10.

[0164] In some embodiments, in the ADC of formula I, the linker L is of formula II: TIFF2025513721000007.tif19128, wherein Z is a linking group that attaches the linker to a targeting group on the anti-FRα antibody construct A; Str is the stretcher AA1 and AA2 are each independently an amino acid, and AA1-[AA2] r forms a protease cleavage site, X is a self-immolative group, q is 0 or 1, r is 1, 2, or 3; s is 1 or 2, # is the point of attachment to anti-FRα antibody construct A, % is the point of attachment to the drug moiety D.

[0165] In some embodiments, in the linker of formula (II): Z is TIFF2025513721000008.tif22128, where # is the attachment point to A and * is the attachment point to the rest of the linker.

[0166] In some embodiments, in the linker of formula (II), Str is TIFF2025513721000009.tif39152, wherein: R is H or C1-C6 alkyl; t is an integer from 2 to 10; u is an integer from 1 to 10.

[0167] In some embodiments, the ADC of formula I may include a disulfide-containing linker. In some embodiments, in the ADC of formula I, the linker L is of formula III: TIFF2025513721000010.tif20128, wherein Z is a linking group that attaches the linker to a targeting group on the anti-FRα antibody construct A; Q is -(CH2) p -or- (CH2CH2O) q -, and p and q are each independently an integer of 1 to 10; each R is independently H or C1-C6 alkyl; n is 1, 2 or 3; # is the point of attachment to anti-FRα antibody construct A, % is the point of attachment to the drug moiety D.

[0168] In some embodiments, the ADC of Formula I may include a β-glucuronide-containing linker.

[0169] A variety of non-cleavable linkers for linking drugs to antibodies are known in the art and may be useful in certain embodiments for the ADCs of the present disclosure. Examples of non-cleavable linkers include linkers that have N-succinimidyl ester or N-sulfosuccinimidyl ester moieties for reaction with antibodies, or maleimide or haloacetyl-based moieties for reaction with drugs, or vice versa. An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs through a maleimide group that reacts with sulfhydryls (thiols, -SH), while sulfo-NHS esters are reactive to primary amines. Other non-limiting examples of such linkers include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) ("long chain" SMCC or LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-succinimidyl ester (GMC), Examples of linkers based on N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimide-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI). Other examples include those containing haloacetyl-based functional groups such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0170] ADCs comprising the anti-FRα antibody constructs described herein can be prepared by one of several routes known in the art using standard organic chemistry reactions, conditions, and reagents (see, for example, Bioconjugate Techniques (GTHermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) reacting a functional group of an antibody construct with a bivalent linker reagent to form an antibody-linker intermediate AL via a covalent bond, followed by reaction with an activated drug moiety D, or (2) reacting a functional group of a drug moiety with a linker reagent to form a drug-linker intermediate DL via a covalent bond, followed by reaction with a functional group of an antibody construct. Conjugation methods (1) and (2) can be used with a variety of antibody constructs, drug moieties, and linkers to prepare the ADCs described herein.

[0171] Various prepared linkers, linker components, and drugs are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, e.g., March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., 2002, J. Org. 67:1866-1872; Frisch et al., 1997, Bioconj. 7:180-186; Bioconjugate Techniques (GT Hermanson, 2013, Academic Press), and Antibody-Drug Conjugates: Methods in Molecular Biology (Ducry (Ed.), 2013, Springer)). In addition, several preformed drug linkers suitable for reaction with a selected antibody construct are also commercially available, for example, drug linkers including DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative BioLabs (Shirley, NY). In addition, various antibody-drug conjugation services are commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramal Pharma Solutions (Grangemouth, UK).

[0172] Once prepared, the ADCs can be purified by standard techniques, such as chromatography (e.g., HPLC, size exclusion, adsorption, ion exchange and / or affinity capture), dialysis and / or tangential flow filtration.

[0173] How to use Certain aspects of the disclosure relate to therapeutic or diagnostic uses of anti-FRα antibody constructs and ADCs. FRα is overexpressed in a wide variety of cancers, and therefore, certain embodiments of the disclosure relate to methods of using anti-FRα antibody constructs and ADCs in the treatment or diagnosis of FRα-positive cancers.

[0174] Certain embodiments relate to methods of inhibiting the growth of FRα-positive tumor cells, comprising contacting the cells with an anti-FRα antibody construct or ADC described herein. The cells may be in vitro or in vivo. In certain embodiments, the anti-FRα antibody constructs and ADCs may be used in methods of treating FRα-positive cancers or tumors in a subject.

[0175] Cancers that overexpress FRα are typically solid tumors. Examples include, but are not limited to, ovarian cancer, endometrial cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer (including triple-negative breast cancer (TNBC)), colorectal cancer, biliary tract cancer, pancreatic cancer, and esophageal cancer. Certain embodiments of the present disclosure relate to a method of treating FRα-positive cancer with the anti-FRα antibody constructs or ADCs described herein, wherein the cancer is ovarian cancer, endometrial cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer, colorectal cancer, biliary tract cancer, pancreatic cancer, or esophageal cancer. In some embodiments, the anti-FRα antibody constructs or ADCs described herein may be useful for treating triple-negative breast cancer (TNBC).

[0176] Treating an FRα-positive cancer may result in one or more of the following: alleviation of symptoms, reduction in tumor size, inhibition of tumor growth, reduction in one or more direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or palliation of the disease state, improved survival, progression-free survival, remission, and / or improved prognosis.

[0177] In certain embodiments, when used to treat cancer, the anti-FRα antibody construct or ADC can be administered systemically to the subject to be treated, for example, by bolus injection or continuous infusion into the subject's bloodstream. In certain embodiments, when used to treat cancer, the anti-FRα antibody construct or ADC can be administered locally to the subject at the site to be treated.

[0178] It is contemplated that anti-FRα antibody constructs or ADCs can be used alone or in combination with one or more known chemotherapeutic or immunotherapeutic agents that are typically used in the treatment of cancer. The combination of anti-FRα antibody constructs or ADCs with standard chemotherapeutic or immunotherapeutic agents can act to improve the efficacy of chemotherapeutic or immunotherapeutic agents, and thus improve standard cancer therapy. This application can be important in the treatment of drug-resistant cancer that does not respond to standard therapy. When used in conjunction with one or more known chemotherapeutic or immunotherapeutic agents, anti-FRα antibody constructs or ADCs can be administered before or after the administration of chemotherapeutic or immunotherapeutic agents, or they can be administered simultaneously.

[0179] The dosage of the anti-FRα antibody construct or ADC administered will be a therapeutically effective amount, without being subject to defined limitations. "Therapeutically effective amount" refers to an amount of the anti-FRα antibody construct or ADC described herein that is sufficient to achieve treatment of a particular indication when administered to a subject. A therapeutically effective amount of the anti-FRα antibody construct or ADC for cancer treatment can, for example, have one or more of the following effects: reducing the number of cancer cells, reducing tumor size, inhibiting the invasion of cancer cells into peripheral organs, inhibiting tumor metastasis, inhibiting tumor growth, increasing survival time, and / or relieving to some extent one or more of the symptoms associated with cancer. For cancer treatment, efficacy can alternatively be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0180] Certain embodiments relate to methods of detecting the presence of FRα in a biological sample, such as a sample containing cells or tissue, using the anti-FRα antibody constructs described herein. In some embodiments, the biological sample may be taken from a patient, for example, a patient known or suspected to have cancer. Some embodiments relate to methods of detecting the presence of FRα in a biological sample, comprising contacting the sample with an anti-FRα antibody construct described herein.

[0181] Certain embodiments relate to a method of diagnosing disorders associated with increased expression of FRα, such as cancer, using the anti-FRα antibody constructs described herein. The diagnostic method may be an in vivo method in which the anti-FRα antibody construct is administered to a subject, or an in vitro method in which a sample taken from a subject is contacted with the anti-FRα antibody construct. For in vivo methods, administration may be systemic or local.

[0182] In methods for detecting the presence of FRα or diagnosing a disorder associated with increased expression of FRα, the anti-FRα antibody construct may be labeled with a detectable label, such as a fluorescent, luminescent, colorimetric, chemiluminescent, radioactive, or enzymatic label known in the art.

[0183] Pharmaceutical Compositions For therapeutic use, the anti-FRα antibody constructs and ADCs can be provided in the form of a pharmaceutical composition comprising the anti-FRα antibody construct or ADC and a pharma- ceutically acceptable carrier or diluent. The compositions can be prepared by known procedures using well-known and readily available ingredients.

[0184] The pharmaceutical composition may be formulated for administration to a subject, for example, parenterally, orally (e.g., buccal or sublingual), topically, rectally or vaginally, or by inhalation or spray. "Parenteral" administration may be subcutaneous injection, or intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion. The pharmaceutical composition will generally be formulated in a manner suitable for administration to a subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard capsule, soft capsule, pill, suppository, oily suspension, aqueous suspension, dispersible powder, dispersible granule, emulsion, injection or solution. The pharmaceutical composition may be provided as a unit dosage formulation.

[0185] In certain embodiments, a pharmaceutical composition comprising an anti-FRα antibody construct or ADC can be formulated for parenteral administration by injection, for example, as a lyophilized preparation or an aqueous solution, or in a unit dose injectable form.

[0186] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin or or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0187] In certain embodiments, pharmaceutical compositions containing anti-FRα antibody constructs or ADCs may be in the form of sterile injectable aqueous or oily solutions or suspensions. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. Sterile injectable solutions or suspensions may contain anti-FRα antibody constructs or ADCs in non-toxic parenterally acceptable diluents or solvents. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution. In addition, sterile fixed oils may be employed as a solvent or suspending medium. For this purpose, various non-irritating fixed oils may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used in the preparation of injectables. Adjuvants such as local anesthetics, preservatives and / or buffers may also be included in the injectable solutions or suspensions.

[0188] In certain embodiments, pharmaceutical compositions comprising anti-FRα antibody constructs or ADCs can be formulated for intravenous administration to subjects, e.g., humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the compositions can also include a solubilizing agent and / or a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by injection, it can be dispensed using an infusion bottle containing sterile water or saline of pharmaceutical grade. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to allow the ingredients to be mixed prior to administration.

[0189] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"), Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0190] Medicine Kit Certain embodiments relate to pharmaceutical kits comprising the anti-FRα antibody constructs or ADCs described herein.

[0191] The kit will typically include a container that holds the anti-FRα antibody construct or ADC, and a label and / or package insert on or associated with the container. The label or package insert includes instructions that are customarily included in commercial packages of therapeutic products, and includes information or instructions about indications, use, dosage, administration, contraindications, and / or warnings for the use of the therapeutic product. The label or package insert may further include instructions in a format prescribed by a government agency that has jurisdiction over the manufacture, use, or sale of pharmaceuticals or biological products, and the instructions incorporate the approval by the government agency of manufacture, use, or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

[0192] In addition to the container holding the anti-FRα antibody construct or ADC, the kit may optionally include one or more additional containers containing other components of the kit, such as a pharma- ceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), other buffers, or diluents.

[0193] Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. The containers can be made of various materials, such as glass or plastic. If appropriate, one or more components of the kit can also be lyophilized or provided in a dry form, such as a powder or granule, and the kit can further include a suitable solvent for reconstituting the lyophilized or dried component(s).

[0194] The kit may also contain other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0195] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present invention in any way. EXAMPLES

[0196] General Provisions Biological Assays: Expression levels of FRα in cell lines and CDX models were assessed in-house using a laboratory-grade IHC assay and assigned relative expression levels (high / medium / low or strong / medium / weak). PDX models were similarly assessed using archival tumor samples.

[0197] Example 1: Preparation of anti-folate receptor alpha antibodies Antibodies that specifically bind to folate receptor alpha (FRα) were generated by immunizing rabbits with human FRα antigen, and isolated and sequenced as described below.

[0198] 1.1 Immunization Antibodies against FRα were developed in rabbits immunized with soluble HIS-tagged human folate receptor 1 antigen (FRα-HIS) (ACROBiosystems, Newark, DE; Cat#FO1-H82E2). Briefly, two New Zealand White rabbits were immunized with a primary boost consisting of 200 μg of FRα-HIS antigen mixed with Alum (5 mg / injection) / CpG (10 μg / injection) administered subcutaneously at three sites (0.333 mL / site) along the dorsal body of the rabbit. These were followed by four immunizations with 100 μg of FRα-HIS antigen mixed with Alum (5 mg / injection) / CpG (10 μg / injection). Each immunization was separated by 14 days. Animals were bled prior to the fourth immunization for serological testing.

[0199] 1.2 Selection of animals to sample The titer of anti-human FRα antibody was determined by flow cytometry using CHO cells expressing human FRα. Briefly, CHO cells were transiently transfected with a pTT5-based expression plasmid (National Research Council of Canada) encoding human FRα according to the manufacturer's instructions for Lipofectamine™ 2000 (Thermo Fisher Scientific Corp., Waltham, MA). Starting from 1:400, the dilutions of immunized rabbit serum, serially diluted 1:2 over 11 points, were incubated with 50,000 CHO cells transiently expressing human FRα for 30 minutes. The samples were then washed, and antibody binding was detected by flow cytometry with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor-647 (Jackson Immuno Research Labs, West Grove, PA). The titer was determined by identifying the highest dilution sample that showed at least 2-fold fluorescence signal above background.

[0200] 1.3 Recovery of B cells and discovery of anti-human FRα antibodies Immunized rabbits with desired titers of over 100,000 were sacrificed and spleens were harvested. Lymphocytes were dissociated by trituration in FACS buffer (PBS, 2% v / v FBS) to release cells from the tissue. Cells were pelleted and then suspended in 5 mL of Pharm Lyse™ (Becton, Dickinson & Co., Franklin Lakes, NJ) for 1 minute to lyse red blood cells. An equal volume of FACS buffer was added to neutralize the Pharm Lyse™, and the resulting lymphocyte sample was pelleted and resuspended in FACS buffer.

[0201] The lymphocyte suspension was then stained with goat anti-rabbit IgG Alexa Fluor-647 (Jackson Immuno Research Labs, West Grove, PA) to identify IgG+ B cells. After 30 minutes of staining, IgG+ B cells were sorted and counted on a FACSAria™ (Becton, Dickinson & Co., Franklin Lakes, NJ). Using the Selected Lymphocyte Antibody Method (SLAM) (Babcook et al., 1996, Proc Natl Acad Sci USA, 93(15):7843-7848), B cells were seeded at different densities ranging from a single cell up to 50 cells in 384-well plates, expanded in culture for 7 days, and the supernatant was harvested for detection of anti-human FRα antibodies. The 384-well plates were stored at -80°C.

[0202] The supernatants were screened for human FRα-specific monoclonal antibodies by ELISA. 384-well ELISA plates were coated with 25 μL / well of human FRα-HIS (2 μg / mL) in PBS and then incubated overnight at 4° C. After incubation, the plates were washed twice with water, 90 μL / well of blocking buffer (2% nonfat milk, PBS) was added, and the plates were incubated at room temperature for 1 hour. After incubation, the plates were washed with 12.5 μL / well of antibody-containing supernatants + 12.5 μL of blocking buffer, positive and negative controls were added, and the plates were incubated at room temperature for 2 hours.

[0203] After incubation, the plate was washed and 25 μL of 0.4 μg / mL goat anti-rabbit IgG Fc-HRP detection antibody (Jackson Immuno Research Labs, West Grove, PA) was added to each well and the plate was incubated at room temperature for 1 hour. After incubation, the plate was washed and 25 μL of tetramethylbenzidine (TMB) was added and the plate was developed for about 10 minutes (until the negative control wells began to show signal). Then, 25 μL of stop solution (1N HCL) was added to each well and the plate was read at a wavelength of 450 nm on a Synergy™ H1 microplate (BioTek Instruments, Winooski, VT).

[0204] 1.4 Sequencing of anti-human FRα antibodies Total RNA was extracted from wells containing antibodies with the desired characteristics using RNeasy (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The resulting total RNA was used as a template with SuperScript™ III (Thermo Fisher Scientific Corp., Waltham, MA) and oligo-dT20 (Integrated DNA Technologies, Inc., Coralville, IA) to transcribe cDNA from the mRNA. The cDNA was subsequently treated with RNase H (New England Biolabs, Ipswich, MA). Initial PCR of heavy and light chain antibody coding sequences was performed with cDNA as the nucleic acid template using primers and methods modified from Babcook et al., 1996, Proc Natl Acad Sci USA, 93(15):7843-7848 and von Boehmer et al., 2016, Nat Protoc., 11(10):1908. PCR products were cloned into pCRTOPO4 using the Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher Scientific Corp., Waltham, MA) and transformed into E. cloni® cells (Lucigen Corporation, Middleton, WI). Antibiotic-resistant clones were Sanger sequenced and analyzed for unique antibody coding sequences.

[0205] Subsequent PCR reactions were then performed on these unique sequences using V-segment family and J-segment family specific primers. The resulting amplicons were cloned into pTT5-based expression plasmids (National Research Council of Canada). The unique heavy and light chain sequences emerging from single well samples were co-expressed in HEK293-6E cells (National Research Council of Canada) in all possible combinations to determine the correct heavy and light chain pairing. The produced antibodies were assayed for binding to antigens transiently expressed on HEK293 cells.

[0206] 1.5 Generation of chimeric antibodies The coding sequences of the antibody variable regions were cloned in frame into huIgG1 and huCK expression vectors (based on the pTT5 vector). The huIgG1 constant region starts with alanine Kabat-118 and the huCK constant region starts with arginine Kabat-108. The activity of the resulting recombinant chimeric antibodies was confirmed in specificity binding assays and found to be comparable to the parent antibodies.

[0207] Example 2: Humanization of anti-FRα antibodies One of the chimeric anti-human FRα (anti-hFRα) variants v23924, generated as described in Example 1, was selected for humanization. The CDR sequences of v23924 are provided in Table 2.1, and the VH and VL sequences are provided in Table 2.2. Humanization was performed as described below.

[0208] [Table 2.1]

[0209] [Table 2.2]

[0210] 2.1 Humanization Sequence alignment of the rabbit VH and VL sequences of v23924 to their respective human germline sequences identified IGHV3-23*01 and IGKVI-39*01 as the closest and most frequent human germline sequences. As shown in Figure 1, AbM-defined CDR sequences (see Table 2.1) were grafted onto the frameworks of these selected human germline sequences. Backmutations to rabbit residues in the resulting sequences at positions deemed likely to be important for retaining binding affinity to the antigen (hFRα) were included to create several humanized sequences, most of which were generated sequences built on previous sequences, with the first humanized sequence containing the least number of backmutations. None of the variants modified the CDRs of the parent antibody as defined by the AbM method.

[0211] This process was carried out in two cycles, with the first cycle ("Cycle 1") resulting in six variable heavy chain humanized sequences and five variable light chain humanized sequences. The second cycle ("Cycle 2") was expanded to an additional five variable heavy chain humanized sequences in pursuit of closer parent-like affinity of the humanized antibody to hFRα. Complete heavy chain sequences containing a humanized heavy chain variable domain (VH) and a hIgG1 heavy chain constant domain (CH1, hinge, CH2, CH3), and complete light chain sequences containing a humanized light chain variable domain (VL) and a human kappa light chain constant domain (kappa CL) were assembled. Monoclonal antibody (mAb) variants were then assembled in cycle 1 by pairing each of the humanized heavy chains with each of the humanized light chains to provide 30 humanized variants, and in cycle 2 by pairing an additional humanized heavy chain with the selected humanized light chain to provide an additional 15 humanized variants, for a total of 45 humanized variants to be experimentally evaluated.

[0212] 2.2 Production of humanized antibodies Each of the 45 humanized mAb constructs, as well as the parental v23924 mAb construct, were produced in a full-size antibody (FSA) format containing either two identical full-length heavy chains (parental v23924 and 15 humanized variants), resulting in a homodimeric Fc region (HomoFc), or a heterodimeric full-length heavy chain containing complementary mutations in the CH3 region to drive exclusive heavy chain pairing (30 humanized variants), resulting in a heterodimeric Fc region (HetFc). A version of v23924 containing HetFc instead of HomoFc was also generated (variant v30618). All constructs contain two identical kappa light chains.

[0213] The two identical full-length heavy chains composed of HomoFc regions contained the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 (SEQ ID NO: 146, see Table 2.3). The heterodimeric full-length heavy chains composed of HetFc regions (HetFc-A and HetFc-B) contained the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 with the following mutations in the Fc region: HetFc-A:T350V_L351Y_F405A_Y407V HetFc-B:T350V_T366L_K392L_T394W

[0214] The sequences of HetFc-A (SEQ ID NO: 148) and HetFc-B (SEQ ID NO: 149) are shown in Table 2.3. The human kappa CL sequence of IGKC*01 (SEQ ID NO: 147, see Table 2.3) was used for all constructs.

[0215] [Table 2.3]

[0216] Each of the humanized VH domain sequences from cycle 1 was added to the human CH1-hinge-CH2-CH3 (HetFc-A and HetFc-B) domain sequence of IGHG1*01 to obtain 12 humanized complete heavy chain sequences (6 humanized VH x 2). Each of the rabbit VH and five additional humanized VH domain sequences from cycle 2 were added to the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 to provide the parent rabbit-human chimeric complete heavy chain sequence and five additional humanized complete heavy chain sequences. Each of the VL domain sequences was added to the human kappa CL sequence of IGKC*01 to provide five humanized light chain sequences. All sequences were reverse translated into DNA, codon optimized for mammalian expression, and genes were synthesized.

[0217] The heavy chain vector insert containing the signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 150) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842)) and the heavy chain clone ending at residue G446 (EU numbering) of the CH3 domain was ligated into the pTT5 vector to generate the heavy chain expression vector. The light chain vector insert containing the same signal peptide was ligated into the pTT5 vector to generate the light chain expression vector. The resulting heavy and light chain expression vectors were sequenced to confirm the correct reading frame and sequence of the coding DNA.

[0218] The heavy and light chains of each of the humanized antibody variants were expressed in 200 mL cultures of CHO-3E7 cells. Briefly, 1.7–2 × 10 6CHO-3E7 cells at a density of cells / ml with viability >95% were grown in FreeStyle™ F17 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (GE Life Sciences, Marlborough, MA) and 0.1% Pluronic® F-68 (Gibco / Thermo Fisher Scientific, Waltham, MA) at 37° C. A total of 200 ml of CHO-3E7 cells + 1× antibiotic / antimycotic (GE Life Sciences, Marlborough, MA) were transfected with a total of 200 ug of DNA (100 ug of antibody DNA and 100 ug of GFP / AKT / stuffer DNA) at a DNA:PEI ratio of 1:4 (w / w) using PEI-MAX® (Polyscience, Inc., Philadelphia, PA). 24 hours after addition of the DNA-PEI mixture, 0.5 mM valproic acid (final concentration) + 1% w / v tryptone (final concentration) was added to the cells, which were then transferred to 32°C and incubated for a further 6 days before harvesting.

[0219] Protein A purification was performed in batch mode or using 1 mL HiTrap™ MabSelect™ SuRe™ columns (Cytiva, Marlborough, MA). In batch mode, clarified supernatant samples were incubated in batch with NaOH-cleaned-in-place (CIP'd) mAb Select SuRe™ resin (GE Healthcare, Chicago, IL) and equilibrated in Dulbecco's PBS (DPBS). The resin was poured into the CIP'd column and the column was washed with DPBS. In both purification modes, protein was eluted with 100 mM sodium citrate buffer pH 3.0. The eluted fractions were pH adjusted by adding 10% (v / v) 1 M HEPES (pH approx. 10.6-10.7) to a final pH of 6-7. Samples were buffer exchanged into DPBS. Protein was quantified based on absorbance at 280 nm (A280nm). The parental rabbit-human antibody chimeric variants (v23924 and v30618) were further purified by preparative SEC chromatography on a Superdex200 Increase 10 / 30 column (GE Healthcare, Chicago, IL) in DPBS mobile phase after Protein A purification.

[0220] After purification, sample purity was assessed by electrophoresis under non-reducing and reducing conditions using the High Throughput Protein Express assay and Caliper LabChip® GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). The procedure was performed according to the HT Protein Express LabChip® User Guide version 2 with the following modifications: Either 2 μl or 5 μl (concentration range 5-2000 ng / μl) of antibody sample was added to separate wells in a 96-well plate (BioRad, Hercules, CA) along with 7 μl of HT Protein Express Sample Buffer (Perkin Elmer, Cat. No. 760328). The antibody samples were then denatured at 70° C. for 15 min. The LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and Ab-200 assay settings.

[0221] The overall yields (after Protein-A purification) of the 45 humanized antibody variants and the parental chimeric antibody v23924 ranged from approximately 9-17 mg (or 45-85 mg / L). Figures 2A and 2C show the Caliper electrophoresis results of the parental chimeric antibody v23924 and a representative humanized variant v30384. As can be seen in Figure 2, for the representative humanized antibody sample, the non-reduced (NR) and reduced (R) Calipers reflected a single species corresponding to the full size antibody and intact heavy and light chains. This was also true for the other humanized variants.

[0222] 2.3 Quality assessment of humanized antibodies The species homogeneity of the humanized antibody variants was assessed by UPLC-SEC after Protein A purification and after preparative SEC purification of the parental chimeric antibody, v23924.

[0223] UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 × 150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) set at 30 °C and installed in a Waters Acquity UPLC™ H-class biosystem equipped with a photodiode array (PDA) detector. The mobile phase was Dulbecco's phosphate buffered saline (DPBS) with 0.02% Tween 20 at pH 7.4, and the flow rate was 0.4 mL / min. The total run time of each injection was 7 min, and the total mobile phase volume was 2.8 mL. Elution was monitored by UV absorbance in the range of 210-500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower® 3 software employing Apex Track™ and detecting shoulder features.

[0224] Figures 2B and 2D show the UPLC-SEC profiles of the parental chimeric antibody v23924 (after SEC purification) and the representative humanized antibody v30384 (after Protein A purification), respectively. The UPLC-SEC profile of the representative humanized antibody sample reflected high species homogeneity comparable to the parental chimeric antibody sample. Samples from the remaining humanized antibody variants had similar profiles to those shown for the representative humanized antibody sample.

[0225] Example 3: Binding of humanized antibodies to hFRα 3.1 Affinity evaluation of humanized antibodies against hFRα To determine whether the humanization process affected the affinity of the humanized variants for their target, the ability of the 45 purified humanized antibody variants to bind to the hFRα antigen was assessed by biolayer interferometry (BLI) as follows.

[0226] hFRα antigen binding was assessed using an Octet® RED96 system (ForteBio, Fremont, CA) by cycling the following steps: antibody (0.9 μg / mL) loading onto an anti-human IgG Fc capture (AHC) biosensor for 200 s; baseline stabilization for 60 s; expected K for 400–500 s. D association to recombinant His-tagged human FRα (ACROBiosystems, Newark, DE) at multiple relevant concentrations spanning 100-200 s; recording of dissociation for 500-1000 s; and regeneration performed by cycling three times between 10 mM glycine pH 1.5 (15 s) and assay buffer (1 s) before proceeding to the next antibody. The assay buffer used was KB buffer (kinetics buffer consisting of PBS pH 7.4, 0.1% BSA, 0.02% Tween 20, 0.05% sodium azide) supplemented with 0.06% Tween 20 and in some instances also 1% BSA. Experiments were performed at 30 °C and with a shaking speed of 1000 rpm.

[0227] Data analysis was performed using Data Analysis Software 9.0 (ForteBio, Fremont Calif.). Reference-subtracted binding curves were globally fitted to a 1:1 interaction model to derive the binding kinetics parameter k on , k off , and the dissociation constant K D was generated.

[0228] Nine of the 45 humanized antibody variants have K values ​​ranging from approximately 63 nM to 210 nM. D The parent chimeric antibody (v23924) was found to bind to hFRα with K values ​​(see Table 3.1). Dwas determined to be 27 nM. The humanized antibody variants that showed binding to hFRα were characterized by an affinity that was reduced by about 2-fold to about 8-fold compared to that of the parent chimeric antibody. As can be seen from Table 3.1, all successful humanized variants shared the same variable light chain (4L) but differed in variable heavy chain composition. The light and heavy chain sequences of the successful humanized variants are provided in Table 3.2. Figure 3 shows the BLI sensorgrams of the parent chimeric antibody v23924 and a representative humanized antibody v30384.

[0229] [Table 3.1]

[0230] [Table 3.2]

[0231] 3.2 Evaluation of binding activity of humanized antibodies against hFRα Antigen binding avidity for the parental and selected humanized variants in the FSA format was assessed by surface plasmon resonance (SPR) as described below.

[0232] SPR assays to determine the hFRα affinity and binding activity of the parental chimeric antibody (v23924) and humanized variants were performed on a Biacore™ T200SPR system at a temperature of 25° C. using PBS-T (PBS + 0.05% (v / v) Tween 20) running buffer (0.5 M EDTA stock solution added to a final concentration of 3.4 mM). CM5 series S sensor chips, Biacore™ amine coupling kit (NHS, EDC and 1 M ethanolamine), and 10 mM sodium acetate buffer were purchased from GE Healthcare Life Science (Mississauga, ON, Canada). PBS running buffer with 0.05% Tween 20 (PBST) was purchased from Teknova Inc. (Hollister, CA). Recombinant human FRα was purchased from ACRobiosystems (Newark, DE).

[0233] Screening of variants for binding to the hFRα antigen was performed in two steps: capture of hFRα on the surface followed by injection of three to five concentrations of variants. hFRα surfaces were prepared on CM5 series S sensor chips by standard amine coupling method as described by the manufacturer (GE Healthcare Life Science, Mississauga, ON, Canada). Immobilization of hFRα was performed using the Biacore™ T200 immobilization wizard using the amine coupling method aiming for resonance units (RU) in the range of 5-200 RU. Using multi-cycle kinetics, three to five concentrations of a two-fold dilution series of samples starting at 300 nM with a blank buffer control were injected at 50 uL / min for 180 s with a dissociation step of 600 s to obtain a set of sensorgrams with a buffer blank reference. The hFRα surface was regenerated and the next injection cycle was prepared by one pulse of 10 mM glycine / HCl pH 1.5 for 30 s at 30 uL / min. The blank-subtracted sensorgrams were analyzed using Biacore™ T200 Evaluation Software v3.0. The blank-subtracted sensorgrams were then fitted to a 1:1 Langmuir binding model.

[0234] The results are shown in Table 3.3. Both the parent antibody (v23924) and the two humanized variants (v30384 and v30399) in the normal bivalent antibody format (FSA) showed avidity in binding to the hFRα antigen, i.e., approximately 17-fold lower K D values, and approximately 27-39 times lower K D Values ​​were obtained with FSA compared to the one-arm antibody (OAA) format at low and medium antigen densities, which were further reduced to approximately 108-fold and approximately 116-181-fold, respectively, at high antigen density.

[0235] [Table 3.3]

[0236] Example 4: Purity of humanized anti-FRα antibodies The apparent purity of the humanized antibody variants from Example 3 was assessed using mass spectrometry after Protein A purification (Example 2) and native deglycosylation.

[0237] Since the antibody variant samples contained only Fc N-linked glycans, the samples were treated with N-glycosidase F (PNGase-F) only. Purified samples were deglycosylated with PNGaseF as follows: 0.1 U PNGaseF / μg antibody in 50 mM Tris-HCl pH 7.0, overnight incubation at 37° C., final protein concentration of 0.48 mg / mL. After deglycosylation, samples were stored at 4° C. prior to LC-MS analysis.

[0238] Deglycosylated protein samples were analyzed by intact LC-MS using an Agilent 1100 HPLC system coupled to an LTQ-Orbitrap™ XL mass spectrometer (ThermoFisher, Waltham, MA) with an Ion Max electrospray source (tuned for optimal detection of larger proteins (>50 kDa)). Samples were injected onto a 2.1 × 30 mm Poros R2 reversed-phase column (Applied Biosystems Corp., Waltham, MA) and resolved using a linear gradient consisting of 0.1% aqueous formic acid / acetonitrile (degassed), increasing concentrations of acetonitrile (20-90%). The column was heated to 82.5 °C and the solvent was heated to 80 °C with a precolumn to improve protein peak shape. The cone voltage (source fragmentation setting) was approximately 40 V, the FT resolution setting was 7,500, and the scan range was m / z 400-4,000. The LC-MS system was evaluated for IgG sample analysis using a deglycosylated IgG standard (Waters IgG standard) and a deglycosylated mAb standard mixture (25:75 half:full size antibody). For each LC-MS analysis, mass spectra acquired over the antibody peak (typically 3.6-4.1 min) were summed and the entire multiply charged ion envelope (m / z 1,200-4,000) was deconvoluted into a molecular weight profile using the MaxEnt1 module of MassLynx™ data analysis software (Waters, Milford, MA). The apparent amount of each antibody species in each sample was determined from the peak heights of the resulting molecular weight profile.

[0239] The results are shown in Table 4.1. Almost all humanized variants were highly pure with a range of about 89-100% of the desired species, with only v30389 showing a lower purity (82.6%). The slightly lower purity of four variants, v30384, v30389, v30394 and v30399, compared to that of the remaining variants, is due to these variants containing a heterodimeric CH3 region (see Example 2) resulting in the presence of some half antibodies. Figure 4 shows the LC / MS profiles for two representative humanized variants, v30384 and v31422. In the LC / MS profiles of all samples, a side peak at about +266 Da was observed, which is likely an artifact of the analysis.

[0240] [Table 4.1]

[0241] Example 5: Thermal stability of humanized anti-FRα antibodies The thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.

[0242] 400 μL of purified samples, mainly at a concentration of 0.4 mg / mL in PBS, were used for DSC analysis using a VP-Capillary DSC (GE Healthcare, Chicago, IL). At the beginning of each DSC run, five buffer blank injections were performed to stabilize the baseline, and a buffer injection was installed before each sample injection for reference. Each sample was scanned from 20 °C to 100 °C at a rate of 60 °C / h using low feedback, an 8-second filter, a 3-minute prescan thermostat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine the melting temperature (Tm) as an indicator of thermal stability.

[0243] The Fab Tm values ​​determined for the humanized variants are shown in Table 5.1. All humanized variants showed increased thermal stability compared to the parent antibody v23924 (Fab Tm of approximately 72° C.), with Fab Tm values ​​ranging from approximately 81 to 84° C. Among the humanized variants, v30394 showed the highest thermal stability.

[0244] [Table 5.1]

[0245] Example 6: Determination of the isoelectric point of humanized anti-FRα antibodies The isoelectric points of the humanized antibody variants were determined by capillary isoelectric focusing (cIEF) as described below.

[0246] cIEF was performed using a CE-UV Agilent 7100 capillary electrophoresis (CE) system. 5ug (or up to 2.5uL) of sample was applied to the capillary (ampholyte range 3.0-10.0). pI marker mixes, 4.1, 4.22, 5.5, 7.0 and 10.0 for system suitability testing, and 4.1 and 10.0 for sample analysis were used. An Agilent 7100 CE system with an external water bath set at 6°C, detector filter assembly (280nm), and 9 bar external pressure was used for all CE runs. A neutral coated capillary (fluorocarbon) was cut at both ends at a distance of 8.5cm and 24.5cm, respectively, from the detection window, equipped with a green alignment interface and mounted in an Agilent capillary cassette. Once a day the capillary was conditioned as follows: high pressure flush with 350 mM acetic acid at 3.5 bar for 5 min, water for 2 min, and cIEF gel for 5 min. Before each run the capillary was conditioned as follows: 4.3 M urea solution for 3 min, water for 2 min, high pressure flush at 3.5 bar. Samples were injected by applying a high pressure of 2 bar for 100 s, followed by water immersion of both the inlet and outlet electrodes. Focusing was performed for 10 min at 25 kV with 200 mM phosphoric acid as anolyte and 300 mM NaOH as catholyte. Using chemical mobilization the outlet vial was exchanged for 350 mM acetic acid and 30 kV was applied for 30 min. After each run a high pressure flush was performed with water for 2 min at 3.5 bar. Peak RTs and manual integration of electropherograms were obtained using Agilent OpenLAB Intelligent Reporting A.01.06.111 software. Raw data (signal vs. retention time) were exported to a CVS file and the major isoform pI, pI range and pI at the center of mass (based on internal pI markers) were calculated in Microsoft Excel.

[0247] The results are shown in Table 6.1. The pI values ​​determined for the major isoforms for the majority of the humanized variants range from 7.78 to 7.97, with one variant having a pI of 8.25 (v31426), all within the typical range for therapeutic antibodies and relatively similar to that of the parental chimeric antibody v23924 (pI of 7.65).

[0248] [Table 6.1]

[0249] Example 7: Chromatographic analysis of anti-FRα antibodies The parental and humanized variants were analyzed by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as shown below.

[0250] 7.1 HIC analysis Antibody hydrophobicity / hydrophilicity was evaluated by HIC as described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, vol. 1045, pp. 275-284. L. Ducry, Ed. Experiments were performed on an Agilent Infinity II 1290 HPLC using a TSKgel® Butyl-NPR column (2.5 μm, 4.6 × 35 mm, TOSOH Bioscience GmbH, Griesheim, Germany) pre-equilibrated with 5 column volumes of buffer A (1.5 M (NH4)2SO4, 25 mM NaH2PO4, pH = 6.95) at room temperature. Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was diluted with 95% buffer A and 5% buffer B (75% 25 mM NaH2PO4). 3- Plus 25% isopropanol, pH 6.95) was loaded onto the column and run for 15 minutes at 0.5 mL / min using the gradient shown in Table 7.1. The HIC chromatograms were integrated using appropriate parameters that provided complete baseline-to-baseline integration.

[0251] [Table 7.1]

[0252] 7.2 SEC analysis Analytical SEC was performed using an Agilent Infinity II 1260 HPLC equipped with an Advance Bio SEC column (300 Å, 2.7 μm, 7.8 × 150 mm) equilibrated with 5 column volumes of mobile phase A (150 mM Na2PO4, pH 6.95) at room temperature. Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was isostatically eluted at 1 mL / min for 7 min while monitoring absorbance at A280. Chromatograms were integrated to obtain full baseline-to-baseline integrals of each peak, with moderate resolution between partially resolved peaks. The peak corresponding to the major component of IgG (approximate retention time 3.3 min) was reported as monomeric based on the SEC profile of the control, trastuzumab. Any peak occurring before 3.3 min was designated as a high molecular weight species (HMWS) and any peak occurring after 3.3 min, except for the solvent peak (>5.2 min), was designated as a low molecular weight species (LMWS).

[0253] 7.3 Results A summary of HIC retention times (HIC-RT) and %SEC monomer for the parental and humanized variants is provided in Table 7.2. Overall, HIC and SEC showed favorable biophysical behavior of all humanized variants. The parental chimeric antibody v23924 eluted at 6.5 min in the HIC gradient, while all humanized variants eluted between 6.0-6.7 min. The SEC profiles showed >90% monomer for all humanized variants, with variant v30389 having the lowest % monomer (94%). All of these variants had HMWS <5% and LMWS <5%.

[0254] [Table 7.2]

[0255] Example 8: Additional Stability Studies To further investigate the stability of the humanized antibody variants, 40° C. stability and acid stability studies were performed in which samples were characterized at specific time points by Caliper and UPLC-SEC as described in Example 2, and in the case of 40° C. stability studies by cIEF and octet antigen binding as described in Examples 6 and 3, respectively. Trastuzumab was used as a control in these studies.

[0256] Selected humanized variants (v30389, v30394, v30399, v31423, and v31424) were subjected to stability testing at 40° C. for 14 days at a sample concentration of approximately 1 mg / ml in PBS pH 7.4 buffer. Samples were characterized at 0, 5, 7, 11, and 14 days. Acid stability testing was performed upon buffer exchange of samples into acetate buffer pH 3.6 at various sample concentrations at 25° C. for 1 hour and characterized at 0, 15, 30, and 60 minutes. Additionally, freeze-thaw (from −80° C. to room temperature) consisting of 3 cycles of 30 minutes per cycle was performed at a sample concentration of approximately 1 mg / ml in PBS pH 7.4.

[0257] The results are shown in Table 8.1. No significant issues regarding the stability of the humanized variants were identified in the study. Freeze-thaw and acid stability studies revealed no change in sample composition over the course of the study as determined by Caliper and UPLC-SEC. The 40°C stability study showed slight changes in the Caliper and UPLC-SEC profiles as the study progressed (specifically, the appearance of some amounts of lower molecular weight species ranging from approximately 10-17%). No changes in antigen binding affinity were observed. cIEF revealed the expected slight increase in more acidic species compared to the main isoform.

[0258] [Table 8.1]

[0259] Example 9: Functional characterization of anti-FRα antibodies - FRα specificity The binding cross-reactivity of the parental chimeric antibody v23924 to FRα (FOLR1), FOLR2, FOLR3 and FOLR4 was assessed by flow cytometry and ELISA. Binding of FRα, FOLR2 and FOLR4 was assessed by flow cytometry using HEK293 transfected cells. FOLR3 binding was assessed by ELISA since FOLR3 is a soluble protein. Control anti-FOLR2 (mouse anti-human FOLR2; Nordic BioSite AB, Taby, Sweden; Catalog No. AFC-4544-2), anti-FOLR3 (mouse anti-human FOLR3; LS Bio, Seattle, WA; Catalog No. LS-C125621) and anti-FOLR4 (mouse anti-His DyLight™ 650; Novus Biologicals, Littleton, CO; Catalog No. NBP2-31055C) antibodies were included in these experiments.

[0260] FRα, FOLR2 and FOLR4 binding: Briefly, HEK293-6e cells were transfected for approximately 24 hours to transiently express (1ug of DNA per million cells) human FRα (Cat. No. 13420), FOLR2 (Cat. No. 13481) and FOLR4 (Cat. No. 13483) (all from GenScript Biotech, Piscataway, NJ). After transfection, 50,000 cells were seeded into V-bottom 96-well plates and incubated with 50nM primary antibody for 45 minutes under standard culture conditions. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; catalog number 109-605-098) for 45 minutes at room temperature. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) and a minimum of 1,000 events were collected per well.

[0261] FOLR3 Binding: ELISA 96-well plates were coated with commercially available purified FOLR3 protein (R&D Systems, Inc., Minneapolis, MN; Catalog No. 5319-FR) for 1 hour at 37°C. Plates were blocked with 1% milk in PBS, pH 7.4, for 1 hour at room temperature. After blocking, primary antibody was added at 7 nM for 1 hour at room temperature. HRP-conjugated secondary antibody (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 109-035-098) was then added at 0.4 μg / ml for 1 hour at room temperature. Plates were developed using tetramethylbenzidine (TMB) and the reaction was stopped using HCl. Absorbance was read at 450 nm using a Synergy™ H1 microplate reader (BioTek Instruments, Winooski, VT).

[0262] result The results are shown in Tables 9.1 and 9.2.

[0263] Anti-FRα, anti-FOLR2, anti-FOLR3 and anti-FOLR4 control antibodies showed expected binding to their respective target proteins by flow cytometry or ELISA. By flow cytometry, FlowJo™ v8 software (BD Biosciences, Franklin Lake, NJ) was used to gate on the live singlet cell population and determine AF647GeoMean and % positive binding in this population for each antibody. For ELISA, raw absorbance values ​​were used to determine % positive binding of anti-FOLR3 and v23924 antibodies compared to the negative control absorbance signal. v23924 showed expected binding to human FRα and did not show binding cross-reactivity to FOLR2, FOLR3 or FOLR4 indicating FRα specificity.

[0264] [Table 9.1]

[0265] [Table 9.2]

[0266] Example 10: Functional characterization of anti-FRα antibodies - Binding to cynomolgus monkey FRα The cross-reactivity of the parental chimeric antibody v23924 against human and cynomolgus monkey FRα was assessed by flow cytometry using transfected CHO-S cells as described below. Palivizumab (anti-RSV) (v22277) was used as a negative control.

[0267] Briefly, CHO-S cells were transfected for approximately 24 hours to transiently express human or cynomolgus monkey FRα, 1 ug DNA per million cells. After transfection, cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibodies for 24 hours at 4°C to prevent internalization. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, Catalog No. 109-605-098) for 30 minutes at 4°C. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ).

[0268] The results are shown in Table 10.1. v23924 showed comparable binding to human and cynomolgus FRα on CHO-S transfected cells, with apparent Kd values ​​of 83.89 pM and 121.60 pM on human FRα and cynomolgus FRα transfected cells, respectively. As expected, no binding was observed with the control v22277.

[0269] [Table 10.1]

[0270] Example 11: Functional characterization of anti-FRα antibodies - cell binding The on-cell binding capacity of the parent chimeric antibody v23924 and a representative humanized variant v30384 was assessed by flow cytometry on JEG-3 and HEC-1-A endogenous FRα-expressing cell lines as described below.

[0271] Briefly, cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibodies for 24 hours at 4°C to prevent internalization. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 109-605-098) for 30 minutes at 4°C. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) and a minimum of 1,000 events were collected per well. AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) in the live cell population was plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0272] The results are shown in Table 11.1. Both the chimeric (v23924) and humanized (v30384) antibodies yielded comparable apparent Kd and Bmax values ​​in both JEG-3 and HEC-1-A cell lines (high and moderate endogenous FRα expression, respectively).

[0273] [Table 11.1]

[0274] Example 12: Functional characterization of anti-FRα antibodies - internalization The receptor-mediated internalization capacity of the parental chimeric antibody v23924 and a representative humanized variant v30384 in FRα-expressing cell lines (IGROV-1 and OVCAR-3) was determined by high-content imaging as described below. The FRα-targeting antibodies mirvetuximab and farletuzumab were used as positive controls and palivizumab (anti-RSV) (v22277) was used as a negative control.

[0275] Briefly, antibodies were fluorescently labeled by coupling to anti-human IgG Fc Fab fragment pHAb dye conjugate (Promega Corporation, Madison, WI; Catalog No. G9841) (approximately 3 dye molecules per Fab fragment) at a molar excess of 5:1 for 24 hours at 4°C. Cells were seeded and incubated overnight at 37°C in 5% CO2 in 96-well plates. The coupled antibody was added to the cells the next day and incubated at 37°C for 6-24 hours to allow for internalization. After incubation, cells were stained with Dye Cycle Violet (ThermoFisher Scientific Corporation, Waltham, MA; Catalog No. V35003) for live cell identification and internalization fluorescence in live cells and analyzed by high content imaging using the CellInsight™ CX5 High Content Screening (HCS) Platform (ThermoFisher Scientific Corporation, Waltham, MA). The fold fluorescence of chimeric antibody v23924 was plotted using GraphPad Prism, Version 9 (GraphPad Software, San Diego, Calif.).

[0276] result Results are shown in Figures 5A and 5B (IGROV-1 cells) and Figures 6A and 6B (OVCAR-3 cells). Chimeric antibody v23924 and humanized variant v30384 showed comparable levels of internalization in both IGROV-1 cells (high FRα) and OVCAR-3 cells (moderate FRα). In both IGROV-1 and OVCAR cells, chimeric antibody v23924 and humanized variant v30384 showed increased internalization compared to mirvetuximab and farletuzumab positive controls across all tested concentrations (25-1 nM) and time points (6 and 24 hours). For example, after 6 hours of incubation in IGROV-1 cells, humanized variant v30384 showed a 3.3-fold and 6.1-fold increase in internalization fluorescence at 25 nM compared to mirvetuximab and farletuzumab, respectively, and a 2.6-fold and 19.1-fold increase in internalization fluorescence at 5 nM compared to mirvetuximab and farletuzumab, respectively (Figure 5A). Similarly, after 24 hours of incubation in IGROV-1 cells, humanized variant v30384 showed a 2.1-fold and 3.9-fold increase in internalization fluorescence at 25 nM compared to mirvetuximab and farletuzumab, respectively, and a 1.9-fold and 4.7-fold increase in internalization fluorescence at 5 nM compared to mirvetuximab and farletuzumab, respectively (Figure 5B).

[0277] Example 13: Epitope Mapping High-resolution epitope mapping of the parental chimeric antibody v23924 on the human FRα antigen (hFRα) was performed by hydrogen / deuterium exchange mass spectrometry (HDX-MS) at NovoAb Bioanalytics Inc. (Victoria, BC, Canada) as described below.

[0278] 13.1 Sample preparation for HDX-MS Lyophilized hFRα was purchased from ACROBiosystems (Newark, DE; Catalog No.: FO1-H5229) and dissolved at a concentration of 2.5 mg / ml. Antigen-antibody complexes were prepared by mixing hFRα with parental chimeric antibody v23924 at a molar ratio of 2:1. All samples were pH 7.4 and clear (no precipitate was observed). For peptide identification, hFRα at a concentration of 10 μM was reduced with 100 mM tris-(2-carboxyethyl)phosphine (TCEP) in the presence of 2 M guanidine at pH 2.4, and then digested with pepsin at an enzyme-protein molar ratio of 1:1. HDX was initiated by mixing the protein sample with D2O buffer at a ratio of 2:8 (v / v). The resulting solution was incubated at 26° C. and aliquots were taken at 20 s, 7 min, 1 h, and 4 h and immediately quenched by the addition of 200 mM TCEP solution containing 4 M guanidine. These samples were flash frozen in liquid nitrogen and stored at −80° C. During the LC-MS experiments, protein aliquots were quickly thawed and kept on ice to allow reduction to proceed for 2 min, then digested with pepsin at 0° C. for 2 min.

[0279] 13.2 LC-MS and LC-MS / MS For LC-MS experiments, 20 μL aliquots of each sample were immediately injected onto a C18 analytical column and separated by reversed-phase liquid chromatography at a flow rate of 100 μL / min using a Dionex UHPLC system (Thermo Fisher Scientific, Bremen, Germany). The UHPLC system was coupled to a Thermo Scientific Orbitrap Fusion™ mass spectrometer equipped with a heated electrospray ionization (HESI) II source. The column, accessories, injector and solvent delivery lines were embedded in an ice bucket to minimize H / D back exchange. Syringes used for injections were cooled on ice. The mobile phases were 0.1% formic acid (A) and 100% acetonitrile / 0.1% formic acid (B), and peptides were separated with a 13 min gradient. MS survey scans were performed in the range m / z 300–1600 with a mass resolution of 120,000 FWHM. The Orbitrap detector was calibrated to an error of <3 ppm using Calibration Mix (Calmix; ThermoFisher Scientific Corporation, Waltham, MA). For electron transfer dissociation (ETD) experiments, fluoranthene radical anions were introduced into the ion trap for over 50 ms. Collision-induced dissociation (CID) and ETD fragment ions were detected in the Orbitrap using a scan range of 150–2000 m / z.

[0280] For data analysis, raw bottom-up LC-MS / MS data were processed using the Proteome Discoverer™ software suite (Thermo Fisher Scientific). The generated peak lists were submitted to an in-house Mascot 2.2 server and searched against the sequence of hFRα. Peptides thus identified were used for HDX data analysis. ETD data were processed using Xcalibur™ software (ThermoFisher Scientific Corporation, Waltham, MA), and the generated ETD peak lists were searched against the sequence of hFRα using Protein Prospector (available online at the University of California, San Francisco website; http: / / prospector.ucsf.edu). Matched ions were also checked and confirmed by manual inspection. Mass transfers of peptides and deuteration states of individual amides were determined based on their centroid m / z values ​​before and after H / D exchange. All HDX data were normalized to 100% D2O content (80% D exchange in buffer at all time points). The percentage of deuterium incorporation was obtained by comparing the number of deuterium obtained with the total number of amide hydrogens contained in each peptide. The deuteration information at the amide level was calculated based on the deuterium incorporation of the ETD fragments.

[0281] 13.3 Results Protein sequence coverage and peptide identification The presence of protein disulfide bonds has a significant effect on the pepsin digestion pattern and efficiency in peptide-based HDX-MS analysis. Because both hFRα and antibody v23924 contain multiple disulfide bridges, an optimized protocol for rapid protein disulfide reduction and pepsin digestion was first developed. The reduction time and digestion time were optimized to 2 min, respectively, and these conditions were applicable to both hFRα and hFRα-v23924 complex. The peptides identified in this way covered 100% of the antigen sequence (see Figure 7).

[0282] Peptide level HDX comparison The deuterium uptake levels of peptides derived from hFRα and v23924 complexes were plotted against HDX time (20 s, 7 min, 1 h, and 4 h). The results are summarized and shown in Figure 8. Most peptides have the same deuterium uptake behavior before and after antibody binding (Figure 8A). This suggests that the binding site (epitope) of the v23924 antibody is highly localized. In the difference plot shown in Figure 8B, three peptides (numbers 14, 15, and 16) showed a significant decrease in deuterium uptake after v23924 binding, indicating that they are in the epitope region. The sequences of these peptides are WWEDCRTSY(118-126) (SEQ ID NO: 151), WEDCRTSY(119-126) (SEQ ID NO: 152), and WEDCRTSYTCKSNWHKGWNWTSGF(119-142) (SEQ ID NO: 153), respectively.

[0283] Epitope determination at the amino acid level Although the three epitope peptides differ in sequence, their HDX differences are the same (Figure 8), and they all contain the sequence WEDCRTSY(119-126) (SEQ ID NO: 152). This indicates that all epitope residues are contained in the shortest peptide 119-126. The observation of multiple difference peptides in the same region provides further confirmation that this region of the protein is the binding site for the v23924 antibody. To further identify the HDX differences and pinpoint the epitope down to individual amino acids, MSMS was performed on peptide 119-126 using ETD. The 1 hour HDX time point was chosen as this yielded the greatest differences. The ETD fragment provided single residue resolution. The deuteration levels of each amino acid were calculated and compared between the hFRα and v23924 complexes (Figure 9). Based on the difference plot results, the epitope residues were determined to be E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15 (i.e., the epitope sequence is EDRTSY; SEQ ID NO: 154).

[0284] Example 14: Affinity maturation of anti-FRα antibodies Humanized antibody v30384 (see Example 3) was affinity matured using the HuTarg™ system (Innovative Targeting Solutions, Vancouver, BC, Canada). Genetic engineering was applied to the variable regions of humanized variant v30384, and high affinity mutants were identified using next generation sequencing (NGS).

[0285] 14.1 Design of library plasmid pools The CDR loops of the humanized variant v30384 variable domains were interspersed with RAG1 / 2 recombination signal sequences (RSSs). These variable domains were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) and cloned into plasmid E951 (Innovative Targeting Solutions, Vancouver, BC, Canada).

[0286] 14.2 HuTarg™ Library Generation The following steps were carried out according to the protocol of Innovative Targeting System. The E951-based plasmid pool was integrated into HuTarg™ cells, and RAG1 / 2 expression was induced for 48 hours. The HuTarg™ cells that successfully showed recombinant antibodies, as shown after staining with PE-conjugated goat anti-human kappa light chain antibody (Bio-Rad Laboratories, Hercules, CA; Cat. No. 206009), were then selected for further investigation.

[0287] 14.3 FACS-based selection of affinity mutants HuTarg™ cells were subjected to multiple rounds of FACS-based sorting on a BD FACSAria™ flow cytometer (BD Biosciences, Franklin Lakes, NJ), each round using decreasing amounts of biotinylated soluble HIS-tagged FRα antigen (FRα-HIS; ACROBiosystems Newark, DE; Catalog No. FO1-H82E2) and detection with streptavidin conjugated to AlexaFluor-647 (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. S11223). HuTarg™ cells that showed increased binding to biotinylated FRα-HIS were sorted directly into RNAzol RT (Sigma-Aldrich, St. Louis, MI; Catalog No. R4533) in preparation for next-generation sequencing.

[0288] 14.4 Next-generation sequencing of affinity mutants Total RNA from cells lysed in RNAzol was isolated according to the manufacturer's instructions. RNA was digested with ezDNase™ (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. 11766051) and cDNA was transcribed using Superscript™ IV (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. 18090010) and gene-specific primers. VH and VK domains were targeted for PCR amplification and molecular barcoded with the NEBNext® Ultra™ DNA Library Preparation Kit (New England Biolabs, Ipswich, MA; Catalog No. E7370L). Samples were pooled and run on an Illumina MiSeq™ sequencer using a 500 cycle kit using v2 chemistry (Illumina, San Diego, CA; Catalog No. MS-102-2003). Sequence analysis was performed to identify mutations within the VH and VK sequences that were shown to likely confer increased affinity.

[0289] 14.5 Recombinant Expression of Affinity Mutants DNA sequences encoding the mutated VH and VK domains were synthesized as "MiniGenes" (Integrated Technologies, Inc., Coralville, IA) and cloned into an expression vector to provide expression plasmids encoding fully human IgG1 heavy chains and human kappa light chains, respectively. The expression plasmids were matrixed together such that every heavy chain plasmid was paired with every light chain plasmid. This matrix was recombinantly expressed in Expi293™ cells (Thermo Fisher Scientific Corp., Waltham, MA; Catalog No. A14635) according to the manufacturer's instructions to generate 64 samples.

[0290] 14.6 Evaluation of affinity mutants Protein G particles (Spherotech Inc., Lake Forest, IL) were coated with humanized variant v30384 or affinity matured antibody at normalized concentrations. Soluble human FRα was diluted to limiting antigen concentration and incubated with antibody-coated beads. FRα antigen binding and antibody capture were detected using AlexaFluor-647 conjugated streptavidin and AlexaFluor-488 conjugated goat anti-human IgG Fcγ (both from Jackson Laboratories, Bar Harbor, ME), respectively. Samples were analyzed by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). The geometric mean of FRα binding and antibody capture was analyzed for each sample. Antibody capture was normalized to FRα binding to affinity-ranked humanized variant v30384 for affinity matured antibody.

[0291] Single point affinity ranking was performed by measuring the ratio of antibody captured on beads to the amount of antigen captured by the antibody. Binding of variant v30384 was minimal (3-fold above background) using a human FRα concentration of 1.9 nM, but the majority of mature variants showed higher binding ratios. Of the 64 mutated variants, 3 variants showed a 4-fold increase in binding ratio and 10 showed comparable binding ratios.

[0292] Example 15: Evaluation of affinity of affinity matured antibodies to FRα Ten of the affinity matured variants described in Example 14 were produced in full size antibody (FSA) format at WuXi Biologics (Hong Kong) Limited, China, via transient transfection in CHO-K1 cells and affinity capture purification with subsequent polishing steps, mainly including preparative SEC or CEX chromatography (if necessary), to obtain sample purity of more than 97% by HPLC-SEC. The FSA format was similar to that of the parent humanized variant v30384, except that these variants contained HomoFc instead of HetFc. The ten affinity matured variants were characterized for binding to hFRα using the Octet® RED96 system, as described in Example 3.

[0293] result The results are shown in Table 15.1. Affinity maturation was performed on the parent humanized antibody v30384 (K D. We have succeeded in obtaining humanized variants with substantially higher affinity for hFRα than the 1.27E-07M. D was observed for variant v35348. The improved affinity was determined to be achieved primarily through a decrease in the dissociation constant.

[0294] [Table 15.1]

[0295] Example 16: Chromatographic analysis of affinity matured antibodies The ten affinity matured variants from Example 15 were analyzed by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) as described in Example 7. The results are shown in Table 16.1. Affinity maturation of the antibodies resulted in changes in hydrophobicity / hydrophilicity as demonstrated by variable HIC-RT. Antibody monomer content was above 97% in all cases and did not correlate with HIC-RT.

[0296] [Table 16.1]

[0297] Example 17: Functional characterization of affinity matured antibodies - cell binding The cellular binding capacity of a representative affinity matured variant v35356 was evaluated on IGROV-1 and JEG-3 endogenous FRα expressing cell lines by flow cytometry as described in Example 11.

[0298] result The results are shown in Table 17.1. The parent humanized variant v30384 and the affinity matured variant v35356 yielded comparable apparent Kd and Bmax values ​​in both IGROV-1 and JEG-3 cell lines (high and moderate endogenous FRα expression, respectively).

[0299] [Table 17.1]

[0300] Example 18: Functional characterization of affinity matured antibodies - internalization The receptor-mediated internalization capacity of the parent humanized variant, v30384, and a representative affinity matured variant, v35356, in FRα-expressing cell lines (IGROV-1 and JEG-3) was determined by flow cytometry as described below. Palivizumab (anti-RSV) (v22277) was used as a negative control.

[0301] Briefly, antibodies were fluorescently labeled by coupling to Fab-AF488 anti-human IgG Fc labeling reagent (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 109-547-008) at a 1:1 molar ratio for 24 hours at 4°C. Cells were seeded and incubated overnight at 37°C in 5% CO2 in 48-well plates. The coupled antibody was added to the cells the next day and incubated at 37°C for 24 hours to allow internalization. After incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using 100 nM anti-488 antibody incubated at 4°C for 45 minutes. Quenched AF488 fluorescence (internalized fluorescence) was analyzed by flow cytometry for all samples on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) and a minimum of 1,000 events were collected per well. AF488 / FITC-A GeoMean in live cell populations was plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0302] result The results are shown in Figure 10. The parent humanized variant v30384 and the affinity matured variant v35356 showed comparable internalization in IGROV-1 (Figure 10(A)) and JEG-3 (Figure 10(B)) cells when administered at 20 nM for both 5 and 24 hour exposures.

[0303] Example 19: Preparation of antibody-drug conjugates - cysteine ​​conjugation Antibody-drug conjugates (ADCs) containing chimeric parent antibody v23924, humanized antibody variants or affinity matured antibody variants conjugated to maleimide-containing drug linkers were prepared at various drug-to-antibody ratios (DARs). Exemplary protocols are provided below. The drug linkers used are listed in Table 19.1. The ADCs prepared and the conjugation conditions used are summarized in Table 19.2.

[0304] 19.1 Conjugation of v23924 by partial reduction of interchain disulfide bonds (DAR4) A solution (5.14 mL) of chimeric antibody v23924 (25 mg) was diluted to 3.57 mg / mL with a 5 mM solution of DTPA (diethylenetriaminepentaacetic acid) in PBS (pH 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) (1.78 equiv., 31 μL) was added to this solution. After 2 h incubation in a 37 °C water bath, drug-linker DL1 (10 equiv., 87 μL) as a 20 mM DMSO stock was added. The conjugation reaction was mixed thoroughly by pipetting and the reaction was allowed to proceed on ice for up to 1 h, followed by quenching the excess drug linker with 20 mM N-acetylcysteine ​​aqueous stock (9 equiv., 78 μL) for 30 min before purifying the ADC from the small molecule. v23924 was also conjugated to drug linkers DL2 and DL3 at DAR4 using the same conjugation procedure.

[0305] 19.2 Conjugation of v23924 after complete reduction of interchain disulfide bonds (DAR10) A solution (3.1 mL) of chimeric antibody v23924 (15 mg) was diluted to 3.57 mg / mL with a 5 mM solution of DTPA in PBS, pH 7.4. 10 mM TCEP (18 equivalents, 188 μL) was added and the reaction mixture was incubated in a 37° C. water bath for 3 h, after which excess TCEP was removed using a 10 mL 40 kD Zeba™ Spin Desalting Column (Thermo Fisher Scientific, Waltham, MA) pre-equilibrated with PBS, pH 7.4. The fully reduced antibody was conjugated to drug linker DL1 (18 equivalents, 94 μL) from a 20 mM DMSO stock on ice for up to 1 h, after which the excess drug linker was quenched with a 20 mM aqueous stock of N-acetylcysteine ​​(12 equivalents, 63 μL) for 30 min before the ADC was purified from the small molecule.

[0306] 19.3 Conjugation of v30384 by partial reduction of interchain disulfide bonds (DAR4) A solution (2.1 mL) of humanized antibody variant v30384 (10 mg) was diluted to 3.52 mg / mL in a 5 mM solution of DTPA in PBS (pH 7.4). 1 mM TCEP (2.37 eq., 164.5 μL) was added and the reaction mixture was incubated in a 37 °C water bath for 2 h before conjugation to a 20 mM DMSO stock of drug linker DL1 (10 eq., 34.7 μL) on ice for up to 1 h, followed by quenching the excess drug linker with a 20 mM aqueous stock of N-acetylcysteine ​​(9 eq., 31.2 μL) for 30 min before purifying the ADC from the small molecule.

[0307] 19.4 Conjugation of v30384 after complete reduction of interchain disulfide bonds (DAR8) A solution (958.3 μL) of humanized antibody variant v30384 (20 mg) was diluted to 5 mg / mL with 5 mM DTPA solution in PBS (pH 7.4). 10 mM TCEP (12 eq, 167 μL) was added and the reaction mixture was incubated in a 37° C. water bath for 3 hours, after which excess TCEP was removed using a 10 mL 40 kD Zeba™ Spin Desalting Column (Thermo Fisher Scientific, Waltham, MA) pre-equilibrated with 10 mM Na-acetate, pH 5.5. Fully reduced antibody was conjugated to a 10 mM DMSO stock of drug linker DL5 (15 equiv., 208 μL) in the presence of 10% DMSO (vol / vol) for up to 2 h in the dark at room temperature with continuous stirring, followed by quenching excess drug linker with a 10 mM aqueous stock of N-acetylcysteine ​​(20 equiv., 274 μL) for 30 min prior to purification of the ADC from the small molecule.

[0308] [Table 19.1] MTvc: TIFF2025513721000031.tif32128MCvcPABC: TIFF2025513721000032.tif40128MC-GGFG: TIFF2025513721000033.tif25128MTvk: TIFF2025513721000034.tif28128 Compound 1: TIFF2025513721000035.tif30128 (see International Patent Application Publication No. WO2016 / 041082) Compound 2: TIFF2025513721000036.tif30128 (see International Patent Application Publication No. WO2014 / 144871)

[0309] [Table 19.2] TIFF2025513721000038.tif219161TIFF2025513721000039.tif215161TIFF2025513721000040.tif16161

[0310] Example 20: Preparation of antibody-drug conjugate-lysine conjugation Antibody-drug conjugates (ADCs) comprising humanized antibody variant v30384 (see Table 20.1) conjugated to drug linkers DL6 or DL8 at target DARs of 3.3 or 4, respectively, were prepared as described below and summarized in Table 20.2.

[0311] DL6: A solution (207 μL) of humanized antibody v30384 (1 mg) was reacted with a 10 mM DMSO stock of drug linker DL6 (14 equiv, 9.7 μL) in PBS, pH 7.4. The conjugation reaction was mixed thoroughly by pipetting and the reaction was allowed to proceed at room temperature for up to 17 hours.

[0312] DL8: A solution (5.9 mL) of humanized antibody v30384 (30 mg) was reacted with a 20 mM DMSO stock of drug linker DL8 (9.5 equiv, 99 μL) in PBS, pH 7.4. The conjugation reaction was mixed thoroughly by pipetting and the reaction was allowed to proceed at room temperature for up to 18 hours.

[0313] [Table 20.2] sSPDB: TIFF2025513721000042.tif24128NHS-ADvc: TIFF2025513721000043.tif30128

[0314] [Table 20.2]

[0315] Example 21: Purification and characterization of ADCs ADCs prepared as described in Examples 19 and 20 were purified using appropriately sized 40 kD Zeba™ Spin Desalting Columns (Thermo Fisher Scientific, Waltham, Mass.) pre-equilibrated with PBS, pH 7.4, or 10 mM Na-acetate, pH 5.5. ADCs produced at >1 mg scale were sterile filtered (0.22 μm).

[0316] The purified ADCs were stored at 4°C and analyzed for total protein content using absorbance at 280 nm or using a bicinchoninic acid (BCA) assay referenced to a standard curve generated from 1 mg / mL trastuzumab. The ADCs were also characterized by HPLC-HIC, SEC, CE-SDS and RP-HPLC-MS as described below. The mean DAR and DAR distribution of the ADCs were obtained from the HIC and LC-MS data. Endotoxin levels were assessed using the ToxinSensor™ Single Test Kit (Genescript BioTech, Piscataway, NJ; Catalog No. L00450) with the threshold set at 0.5EU / mg. Residual free drug and drug-linker levels (%FD) were assessed by RP-HPLC-MS and calculated based on the following equation with the threshold set at 1 mol% DAR: TIFF2025513721000045.tif10128

[0317] The biophysical properties determined for the ADCs are summarized in Table 21.4.

[0318] 21.1 DAR Measurement of Cysteine-Conjugated ADCs by HIC The average DAR by HIC was evaluated as described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, vol.1045, pp.275-284. L. Ducry, Ed. Experiments were performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a TSKgel® Butyl-NPR column (2.5 μm, 4.6×35 mm, TOSOH Bioscience GmbH, Griesheim, Germany) pre-equilibrated with 5 column volumes of buffer A (1.5 M (NH4)2SO4, 25 mM NaH2PO4, pH=6.95) at room temperature. Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was loaded onto the column with 95% Buffer A and 5% Buffer B (75% 25 mM NaH2PO4 plus 25% isopropanol, pH 6.95) and run for 15 minutes at 0.5 mL / min using the gradient shown in Table 21.1. HIC chromatograms were integrated using appropriate parameters that resulted in full baseline-to-baseline integration of each peak, followed by integration of each peak to show reasonable separation. As a reference, unconjugated naked antibody was run with the same gradient to give a HIC retention time of the DAR=0 species.

[0319] [Table 21.1]

[0320] 21.2 DAR Determination of Lysine-Conjugated ADCs by RP-HPLC-MS ADC samples were deglycosylated with EndoS for 1 hour at room temperature and injected into an Agilent 1290 Infinity II LC coupled to an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA). Protein species were separated using a PLRP-S column (1000 Å, 8 uM, 50×2.1 mm) at a flow rate of 0.3 mL / min using the gradient shown in Table 21.2. Buffer A: 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA), and 10% isopropyl alcohol (IPA) in water. Buffer B: 0.1% FA and 10% IPA in acetonitrile (ACN).

[0321] [Table 21.2]

[0322] The MS source conditions are shown in Table 21.3 and the acquisition parameters were as follows:

[0323] Mode: MS; mass range: 500-7000 m / z; acquisition speed: 1 spectrum / sec, and 1000 ms / spectrum, 3354 transients / spectrum.

[0324] [Table 21.3]

[0325] Quantitative analysis using MassHunter software (Agilent Technologies, Santa Clara, Calif.) was used for deconvolution and data analysis. Deconvolution parameters were as follows:

[0326] Deconvolution algorithm: maximum entropy; mass range: 70000-160000; mass step: 1.0; used limit m / z range: 1000-7000; subtract baseline: 7.0; adducts: protons; isotope width: automatic; height filter: peak signal to noise >= 30.0. Maximum number of peaks: limited to height 100.

[0327] The average DAR was calculated from the deconvoluted spectra using the following formula: TIFF2025513721000049.tif9128

[0328] 21.3 SEC-HPLC Analysis of ADCs Analytical SEC was performed using an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) equipped with an Advance Bio SEC column (300 Å, 2.7 μm, 7.8 × 150 mm) equilibrated with 5 column volumes of buffer (150 mM Na2PO4, pH 6.95) at room temperature. Typically, 20-30 μg of sample at a concentration of 2-3 mg / mL was isostatically eluted at 1 mL / min for 7 min and absorbance was monitored at A280. Chromatograms were integrated to obtain full baseline-to-baseline integrals of each peak, with moderate resolution between partially resolved peaks. The peak corresponding to the major component of IgG (approximate retention time 3.3 min) was reported as monomeric based on the SEC profile of the unmodified chimeric antibody v23924. Any peak occurring before 3.3 min was designated as a high molecular weight species (HMWS) and any peak occurring after 3.3 min, except for the solvent peak (>5.2 min), was designated as a low molecular weight species (LMWS).

[0329] 21.4 CE-SDS Analysis of ADCs All samples were first diluted to 1 mg / mL and then prepared in a 96-well PCR plate (Protein Express Assay LabChip™; PerkinElmer, Inc., Waltham, MA) according to the manufacturer's procedure. Briefly, 2 μg of ADC was mixed with 7 uL of Protein Express buffer in the presence (reducing) or absence (non-reducing) of 400 mM dithiothreitol (DTT), followed by heat denaturation at 95° C. for 5 minutes. Samples were then diluted in a 1:2 ratio in dH2O before data acquisition. After each CE-SDS run, the gels and corresponding electropherograms were analyzed using LabChip™ Reviewer (PerkinElmer, Inc., Waltham, MA).

[0330] [Table 21.4] TIFF2025513721000051.tif61162

[0331] Example 22: In vitro cytotoxicity of ADCs The cell proliferation inhibition (cytotoxicity) potency of ADCs containing the humanized variant v30384 or the affinity matured variant v35356 (see Example 19), each conjugated to the drug linker DL1, was determined in a panel of FRα-expressing cell lines as described below.

[0332] Briefly, cells were seeded at 1000 cells / well in 384-well plates and treated with titrations of test articles produced in complete cell growth medium. Treated cells were incubated for 4 days under standard culture conditions (37°C / 5% CO2). After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI; Cat. No. G7570) was spiked into all wells and luminescence corresponding to ATP present in each well was measured using a Synergy™ H1 plate reader (BioTek Instruments, Winooski, VT). % cytotoxicity values ​​were calculated (relative light units) using ATP measured RLU values ​​based on blank wells (medium only, no test article added) and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, San Diego, CA).

[0333] result The results are shown in Tables 22.1 and 19. Both the ADCs containing the humanized variant v30384 and the ADCs containing the affinity matured variant v35356 showed comparable in vitro cytotoxicity in FRα-expressing cells KB-Hela, IGROV-1, JEG-3 and SKOV-3. None of the ADCs showed non-specific cytotoxicity in the FRα-negative MDA-MB-468 cell line.

[0334] [Table 22.1]

[0335] Example 23: Release and quantification of intracellular payload The intracellular payload delivery capabilities of representative ADCs in cell lines JEG-3 (high FRα), Caov-3 (moderate FRα), HEC-1-A (moderate FRα), and H2110 (moderate / low FRα) were evaluated using mass spectrometry as described below. The ADCs tested contain the humanized variant v30384 or the affinity matured variant v35356, respectively, conjugated to the drug linker DL1 (see Example 19).

[0336] Briefly, cells were seeded at 80,000 cells / well in 12-well plates and treated with various concentrations of ADC for 24 hours under standard culture conditions. After incubation, cells were washed using PBS pH 7.4, harvested, counted, and frozen at -80°C. After thawing, cells were lysed using pure acetonitrile. Cell lysate supernatants were injected into an Agilent 1290 Infinity II LC coupled to an Agilent 6470 triple quadrupole (QQQ) mass spectrometer (Agilent Technologies, Santa Clara, CA), and free payload (compound 1) in cell lysate samples was quantified using free payload standards of known concentrations.

[0337] result The results are shown in Figure 11. In the high FRα expressing cell line JEG-3, both ADCs showed comparable intracellular payload (compound 1) delivery. In the lower FRα expressing cell lines Caov-3, H2110 and HEC-1-A, the ADC containing affinity matured variant v35356 showed higher payload delivery at 24 hours compared to the ADC containing parent humanized variant v30384.

[0338] Example 24: In vivo efficacy testing - chimeric anti-FRα antibody ADC The in vivo antitumor activity of parental chimeric antibody v23924 conjugated to drug linker DL1 or DL7 was evaluated in several xenograft models expressing various levels of FRα, as described below. The activity of a control ADC, including a known FRα-targeting antibody, mirvetuximab, was evaluated for comparison. The control ADCs were v17717 (mirvetuximab Fab with HetFc) conjugated to drug linker DL1, and v17717 conjugated to drug linker DL6. For statistical analysis, a linear mixed-effects model was fitted to the log-transformed tumor volumes, followed by F-tests of the null hypothesis that the mean growth rates are equal and post-hoc pairwise comparisons.

[0339] A summary of the ADCs utilized in each xenograft study, the xenograft model, dose, and study duration are shown in Table 24.1. In each xenograft study, tumor volumes and body weights of animals were measured twice weekly.

[0340] [Table 24.1]

[0341] For the CTG-0848 PDX model, tumor fragments were implanted subcutaneously into female nude mice. The mean tumor volume was approximately 100–250 mm. 3 Upon reaching tumor mass index (MMI), animals were matched by tumor size, assigned to treatment groups (n=5 per group) and treated with a single IV dose of ADC on day 0 as shown in Table 24.1.

[0342] For the OV90 model, tumor cell suspension (1×10 in 0.1 ml of 50% Matrigel®) was 7 The tumors were subcutaneously implanted into female CB.17 SCID mice. The mean tumor volume was 100–150 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=9 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 1.

[0343] In the case of the OVCAR3 model, tumor fragments (approximately 1 mm 3) were implanted subcutaneously into female CB.17 SCID mice. The average tumor volume was approximately 100–150 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=8 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 1.

[0344] For the LXFA737 PDX model, tumor fragments (edge ​​length of 3–4 mm) were implanted subcutaneously into female nude mice. The mean tumor volume was approximately 80–200 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=6 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 0.

[0345] For the JEG3 CDX model, tumor cell suspension (1×10 in 0.1 ml of 50% Matrigel®) was 7 The tumors were subcutaneously transplanted into NOD / SCID mice. The average tumor volume was approximately 100 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=5 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 0.

[0346] For the HCC1954 CDX model, tumor cell suspension (5×10 in 0.1 ml of 50% Matrigel®) was 6 The average tumor volume was approximately 150 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=5 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 0.

[0347] For the SKOV3 CDX model, tumor cell suspension (1×10 in 0.1 ml of 50% Matrigel®) was 7 The average tumor volume was approximately 170 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=5 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 0.

[0348] For the KB CDX model, tumor cell suspension (3 × 10 in 0.1 ml of PBS) was6 The average tumor volume was approximately 100-150 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=5 per group) and treated with a single IV dose of the ADC shown in Table 24.1 on Study Day 1.

[0349] For all models except the HCC1954 and KB CDX models, serum was collected at several time points for pharmacokinetic analysis, as described in Example 28.

[0350] result The results are shown in Figure 12.

[0351] In the CTG-0848 PDX model, v23924-DL1 caused sustained regression of tumor growth when dosed at 5 and 10 mg / kg (Figure 12A). In the OV90 model, v23924-DL1 caused modest inhibition of tumor growth rate when dosed at 9 and 18 mg / kg (Figure 12B), which was not statistically significantly different from vehicle control (mixed effects model). In the OVCAR3 model, both v23924-DL1 and v17717-DL6 caused statistically significant inhibition of tumor growth rate when dosed at 9 mg / kg (p<0.001 mixed effects model). v23924-DL1 caused sustained tumor regression and caused significantly greater inhibition of tumor growth rate than v17717-DL6 (p<0.001) (Figure 12C).

[0352] In the LXFA737 PDX model, v23924-DL1, v17717-DL6, and v17717-DL1, when dosed at 5 mg / kg, caused tumor growth rate inhibition of 198%, 144%, and 165%, respectively (each tumor growth rate significantly different from control, p<0.001) (Figure 12D). In the JEG3 CDX model, v23924-DL1, v17717-DL6, and v17717-DL1, when dosed at 3 mg / kg, caused transient tumor regression with tumor growth rate inhibition of 132%, 148%, and 139%, respectively (each tumor growth rate significantly different from control, p<0.001) (Figure 12E). In the HCC1954 CDX model, when administered at 10 mg / kg, v23924-DL1, v17717-DL6, and v17717-DL1 caused transient tumor regression with tumor growth rate inhibition of 287%, 278%, and 242%, respectively (each tumor growth rate significantly different from control, p<0.01) (FIG. 12F).

[0353] In the SKOV3 CDX model, v23924-DL1, v17716-DL6, and v17717-DL1, when administered at 10 mg / kg, caused transient tumor regression with tumor growth rate inhibition of 287%, 243%, and 278%, respectively (each tumor growth rate significantly different from control, p<0.01) (Figure 12G). In the KB CDX model, v23924-DL1, v23924-DL7, and v17717-DL6, when administered at 5 mg / kg, caused tumor growth rate inhibition of 289%, 171%, and -1%, respectively, with both v23924 ADCs being significantly superior to the vehicle control and v17717-DL6 (p<0.01). When administered at 10 mg / kg, all ADCs caused sustained tumor regression in this model (Figure 12H).

[0354] Example 25: In vivo efficacy studies - chimeric and humanized anti-FRα antibody ADCs The in vivo antitumor activity of the chimeric antibody v23924 and humanized variants v30384 and v30399 (see Example 3) conjugated to drug linker DL1 (see Example 19) was evaluated in the medium / high level FRα expressing OVCAR3 ovarian cancer model as described below. For statistical analysis, linear mixed-effect models were fitted to log-transformed tumor volumes followed by F-tests of the null hypothesis of equal mean growth rates and post-hoc pairwise comparisons.

[0355] Tumor fragments (approximately 1 mm 3 ) were implanted subcutaneously into female CB.17 SCID mice. The mean tumor volume was approximately 100–150 mm. 3 When tumor volume reached 100 mg / kg, animals were assigned to groups (n=8 per group) and treated with a single IV dose of either 4 mg / kg or 9 mg / kg ADC on Study Day 1. Tumor volumes and body weights were measured twice weekly for the 60-day study period.

[0356] result The results are shown in Figure 13. When administered at 4 mg / kg, v23924-DL1 and humanized v30384-DL1 and v30399-DL1 caused similar tumor regression followed by regrowth. When administered at 9 mg / kg, all ADCs caused sustained tumor regression.

[0357] Example 26: In vivo efficacy study - humanized anti-FRα antibody ADC#1 The in vivo antitumor activity of humanized variant v30384 (see Example 3) conjugated to drug linker DL1 or DL8 (see Examples 19 and 20) was evaluated in several xenograft models expressing various levels of FRα, as described below. The activity of a control ADC containing mirvetuximab, a known FRα-targeting antibody, was evaluated in selected models for comparison. The control ADCs were v17717 (mirvetuximab Fab with HetFc) conjugated to drug linker DL6 (v17717-DL6) and v17716 (mirvetuximab Fab with HomoFc) conjugated to drug linker DL6 (v17716-DL6). In the H2110 CDX model, v31629 (farletuzumab) conjugated to drug linker DL4 (v31629-DL4) was also included as a control. For statistical analysis, linear mixed-effects models were fitted to log-transformed tumor volumes followed by F-tests of the null hypothesis of equal mean growth rates and post-hoc pairwise comparisons.

[0358] A summary of the ADCs utilized in each xenograft study, the xenograft model, dose, and study duration are shown in Table 26.1. In each xenograft study, tumor volumes and body weights of animals were measured twice weekly.

[0359] [Table 26.1]

[0360] For the H2110 CDX model, tumor cell suspension (1 × 10 in 0.1 ml of 50% Matrigel®) was 7 The tumors were subcutaneously implanted into CB.17 SCID mice. The average tumor volume was approximately 155 mm. 3 When the IV dose reached 0, animals were assigned to groups (n=8 per group) and treated with a single IV dose of the ADCs shown in Table 26.1 on Study Day 0. Serum was collected at several time points for pharmacokinetic analysis, as described in Example 28.

[0361] For the SKOV3 CDX model, tumor cell suspension (1 × 10 in 0.1 ml of 50% Matrigel®) was 7 The average tumor volume was approximately 175 mm. 3 When the pharmacokinetic (pharmacokinetic) mean serum concentration (SSC) was reached, animals were assigned to groups (n=9 per group for the repeated dose groups, n=7 for the single dose groups) and treated with ADC on Study Day 0 according to the dose levels and schedules as shown in Table 26.1. Serum was collected at several time points for pharmacokinetic analysis as described in Example 28.

[0362] For the IGROV-1 model, tumor cell suspension (1 × 10 in 0.1 ml of 50% Matrigel®) was 7 The average tumor volume was approximately 100–150 mm. 3 When the NIH score was reached, animals were assigned to groups (n=12 per group) and treated with ADC according to the dose levels and schedules as shown in Table 26.1.

[0363] For the LXFA737 CDX model, tumor fragments (edge ​​length 3–4 mm) were implanted subcutaneously into nude mice. The average tumor volume was approximately 100 mm 3 When the IV dose of ADC was reached, animals were assigned to groups (n=10 per group) and treated with a single IV dose of the ADC shown in Table 26.1 on Study Day 0.

[0364] result The results are shown in Figure 14.

[0365] In the H2110 CDX model, both v30384-DL1 and v17716-DL6, when administered at 1.25, 2.5 and 5 mg / kg, produced mild to moderate tumor growth inhibition across a shallow dose response, which was not statistically significant (Figure 14A). v31629-DL4, when administered at 2.5 mg / kg, produced mild inhibition of tumor growth rate comparable to v17716-DL6 and v30384-DL1.

[0366] In the SKOV3 CDX model, single doses of 8 mg / kg v30384-DL1, v30384-DL8, and v17716-DL6 resulted in significant inhibition of tumor growth rate of 203%, 178%, and 232% (p<0.01), respectively, with a moderate but statistically significant tumor growth rate inhibition by v17716-DL6 (p<0.01) (Figure 14B). Repeated dosing (v30384-DL1 every 10 days for a total of four doses, v17716-DL6 every 15 days for a total of three doses) had little effect on tumor growth compared to single dose administration (Figure 14C).

[0367] In the IGROV-1 model, when administered at 8 mg / kg, both v30384-DL1 (every 10 days for a total of 4 doses) and v17716-DL6 (every 15 days for a total of 3 doses) resulted in significant inhibition of tumor growth compared to control (p<0.001). When administered at 4 mg / kg, only v30384-DL1 (every 10 days for a total of 4 doses) resulted in significant inhibition of tumor growth compared to control (p<0.001). v30384-DL1 resulted in significantly greater tumor growth rate inhibition than v17716-DL6 at both the 4 and 8 mg / kg dose levels (p<0.001 and p=0.014, respectively) (Figure 14D).

[0368] In the LXFA737 CDX model, when administered at 8 mg / kg, v30384-DL8 and v17716-DL6 produced significant inhibition of tumor growth rate of 154% and 313%, respectively (p<0.01). v17716-DL6 produced significantly greater inhibition of tumor growth rate compared to v30384-DL8 at both the 4 mg / kg (132% and 21%) and 8 mg / kg (313% and 154%) dose levels (p<0.01) (Figure 14E).

[0369] Example 27: In vivo efficacy study - humanized anti-FRα antibody ADC#2 The in vivo antitumor activity of humanized variant v30384 (see Example 3) conjugated to drug linker DL5 (see Example 19) was evaluated in several xenograft models expressing various levels of FRα, as described below. For statistical analysis, linear mixed-effect models were fitted to log-transformed tumor volumes, followed by F-tests of the null hypothesis of equal mean growth rates and post-hoc pairwise comparisons.

[0370] A summary of the ADCs utilized in each xenograft study, the xenograft model, dose, and study duration are shown in Table 27.1. In each xenograft study, tumor volumes and body weights of animals were measured twice weekly.

[0371] [Table 27.1]

[0372] For the OV90 CDX model, tumor cell suspension (1 × 10 in 0.1 ml of 50% Matrigel®) was 7 The tumors were subcutaneously transplanted into CB.17 SCID mice. The average tumor volume was approximately 100–150 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=6 per group) and treated with a single IV dose of the ADC shown in Table 27.1 on Study Day 1.

[0373] For the H2110 CDX model, tumor cell suspension (1 × 10 in 0.1 ml of 50% Matrigel®) was 7 The tumors were subcutaneously implanted into CB.17 SCID mice. The average tumor volume was approximately 150 mm. 3 When the IV dose of ADC was reached, animals were assigned to groups (n=5 per group) and treated with a single IV dose of the ADC shown in Table 27.1.

[0374] In the OVCAR3 CDX model, tumor fragments (approximately 1 mm 3 ) were subcutaneously implanted into CB.17 SCID mice. The average tumor volume was approximately 100–150 mm. 3When the IV dose of ADC was reached, animals were assigned to groups (n=5 per group) and treated with a single IV dose of the ADC shown in Table 27.1 on Study Day 1.

[0375] For each model, serum was collected at several time points for pharmacokinetic analysis, as described in Example 28.

[0376] result The results are shown in Figure 15. In all three models, v30384-DL5 produced a dose response across the doses tested.

[0377] In the OV90 CDX model, v30384-DL5 produced a dose response across 1, 3, and 10 mg / kg (25%, 47%, and 185% inhibition of tumor growth rate, respectively, significant at the 3 and 10 mg / kg dose levels (p<0.01)) (Figure 15A). In the H2110 CDX model, v30384-DL5 produced a dose response across 0.3, 1, 3, and 10 mg / kg dose levels (Figure 15B). In the OVCAR3 CDX model, v30384-DL5 produced a dose response across 0.25, 0.75, 1.5, and 3 mg / kg dose levels, with the higher dose groups producing sustained regressions with regrowth approximately 5 weeks after dosing (Figure 15C). In further studies in the OVCAR3 CDX model, v30384-DL5 was administered at 6 mg / kg and produced sustained regressions (Figure 15D).

[0378] Example 28: Pharmacokinetic study Serum was collected as described from a subset of the xenograft studies described in Examples 24, 26, and 27 and analyzed for pharmacokinetics (PK) of the ADC, with assessments described below. Overall, these studies in immunocompromised tumor-bearing mice demonstrate that ADCs based on v30384 have favorable PK properties.

[0379] Test article (total antibody and / or intact ADC) concentrations were measured from mouse serum by 384-well plate ELISA. For detection of total IgG in serum from mice administered chimeric v23924, humanized v30384, and comparator v17717 and v17716-based ADCs, ELISA plates were coated with goat anti-human IgG Fc capture antibody (Jackson Immuno Research Laboratories, West Grove, PA). For ELISA of DL1 intact ADCs, rabbit anti-compound 1 capture antibody was used. For ELISA of DL6 intact ADCs, mouse anti-DM1 / 4 capture antibody was used (Levena Biopharma, San Diego, CA). Serum samples were applied after blocking, followed by detection antibody: goat anti-human IgG F(ab')2 conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research Laboratories, West Grove, PA). Absorbance was measured at 450 nm after application of 3,3',5,5'-tetramethylbenzidine (TMB) and HCl quenching. Concentrations were determined by reference to standards (GraphPad Prism software (GraphPad Software, San Diego, Calif.)).

[0380] Results are shown in Figure 16. Analysis across multiple studies demonstrated comparable total IgG and ADC PK for v23924-DL1 and v17717-DL1, respectively, with typical antibody-like prolonged exposure (see Figures 16A-F). v17717-DL6 and v17716-DL6 ADC PK demonstrated consistently lower exposure and higher clearance compared to their respective total IgG PK.

[0381] v30384-DL5 demonstrated proportional PK across doses of 1, 3 and 10 mg / kg in the OV90 model (Figure 16G). In the H2110 model, v30384-DL5 demonstrated prolonged exposure at 10 mg / kg and greater clearance at lower doses (Figure 16H).

[0382] Example 29: Penetration of anti-FRα antibodies in multicellular tumor spheroids The ability of anti-FRα antibodies to penetrate FRα-expressing cell line spheroids was evaluated according to the methods described below. Spheroids provide a three-dimensional cellular organization with layers of different cell populations and different gradients from the outer to the inner regions. Cell signaling is more complex in spheroids than in two-dimensional cell cultures. As a result of these characteristics, spheroids may recapitulate drug resistance and metabolic adaptation.

[0383] The spheroid penetration ability of humanized variant v36675 was compared to mirvetuximab (v17716) and non-FRα-targeted control palivizumab (anti-RSV) (v22277). Variant v36675 is the same as variant v30384, but contains HomoFc instead of HetFc. The cell line used was high FRα-expressing JEG-3 (placental choriocarcinoma).

[0384] The antibodies were fluorescently labeled by coupling to anti-human IgG Fc-targeting Fab fragment AF488 conjugate (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Catalog No. 10010-023) for 24 hours at 4°C.

[0385] JEG-3 cells were detached from the culture vessel with TrypLE™ Express Enzyme (1X) (Thermo Fisher Scientific, Waltham, MA) and counted using a Cellaca® MX high-throughput automated cell counter (Nexcelom Bioscience LLC, Lawrence, MA). Cells were diluted in complete growth medium (minimum essential medium supplemented with 10% fetal bovine serum (both from Thermo Fisher Scientific, Waltham, MA)) and seeded at 3,000 cells / well into 96-well CellCarrier Spheroid ultra-low attachment plates (Perkin Elmer, Waltham, MA), centrifuged, and incubated under standard culture conditions for 3 days to allow spheroid formation and growth.

[0386] After spheroid formation, Fab-AF488 coupled antibody was added to the spheroids at a final concentration of 25 nM and incubated for 4–96 h under standard culture conditions. After incubation, excess antibody was removed by adding 100 μL of complete growth medium, removing 100 μL of medium from the wells, and performing a total of three washes. Spheroids were treated with a solution of 1 μM Hoechst33342 (Thermo Fisher Scientific, Waltham, MA) and 100 nM anti-Alexa Fluor488 antibody (Thermo Fisher Scientific, Waltham, MA; catalog no. A-11094) and incubated for 2 h at 37 °C / 5% CO2.

[0387] Imaging was performed using an Operetta CLS™ High Content Analysis System (Perkin Elmer, Waltham, MA) with confocal acquisition and a 10x magnification air objective. Z-stacks of 15 planes separated by 15 μm were acquired, and the largest diameter slice representing the central slice of the spheroid was selected for 2D analysis. Image analysis was performed using Harmony® 4.5 software (Perkin Elmer, Waltham, MA). Briefly, spheroid identification was performed by applying a mask around Hoechst33342 positive objects, one spheroid per well. The spheroid area was divided into concentric band subregions, each representing 10% area of ​​the spheroid area. The mean AF488 fluorescence within each subregion band was quantified, corrected by subtracting the inner 10% mean AF488 fluorescence, and plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0388] result The results are summarized in Table 29.1 and Figure 20. Humanized antibody variant v36675 demonstrated a greater degree of penetration into JEG-3 spheroids than mirvetuximab, as measured by AF488 intensity in each subregion band, as measured by the distance from the spheroid edge where AF488 fluorescence was detectable, at all time points evaluated. The non-binding control palivizumab demonstrated less AF488 signal throughout the spheroid compared to both anti-FRα antibodies at all time points evaluated.

[0389] [Table 29.1]

[0390] Example 30: Evaluation of specificity of anti-FRα antibody Retrogenix Cell Microarray Technology (Charles River Laboratories, Wilmington, Mass.) was used to screen the following anti-FRα antibodies for any specific off-target binding interactions: humanized variant v36675 and affinity matured variant v35356.

[0391] Retrogenix Cell Microarray Technology identifies interactions with both cell surface receptors and secreted proteins by screening test ligands for binding against a library of cDNA clones representing over 6,300 human proteins. These proteins include plasma membrane monomers, heterodimers (formed by co-expression of separate subunits), and secreted proteins (expressed with inert plasma membrane tethers). Each cDNA is spotted in duplicate onto specialized slides and overlaid with HEK293 cells. These cells are reverse transfected, resulting in clusters of cells each overexpressing a different individual protein (or heterodimeric complex).

[0392] The study was conducted at Charles River Laboratories and consisted of three phases: pre-screening, library screening, and confirmatory screening. Pre-screening was performed first to determine the appropriate concentration of test antibodies for library screening (i.e., the concentration at which low levels of background binding to fixed, untransfected HEK293 cells and strong binding to cells overexpressing FRα were observed). In the library screening phase, test antibodies were screened as pools against fixed HEK293 cells expressing approximately 6,300 human proteins individually. In the confirmatory screening phase, each library hit was re-expressed and each test antibody was re-tested individually using both fixed and live HEK293 cells.

[0393] In all three stages, slides were individually spotted with expression vectors encoding both ZsGreen1 (to assess transfection efficiency) and either (1) in the pre-screening stage: human FRα or control receptors (EGFR and CD20), (2) in the library screening stage: the above protein libraries arrayed individually and in duplicate across multiple microarray slides ("slide sets"), with two replicate slides screened against each slide set, or (3) in the confirmation screening stage: protein hits identified in the library screening or control receptors arrayed in duplicate (EGFR and CD20).

[0394] In the pre-screening stage, 2, 5 or 20 μg / mL of each of the selected antibodies, 1 μg / mL of a control antibody (rituximab biosimilar binding to CD20), or PBS were added to the slides after cell fixation. In the library screening stage, a pool of two antibodies (variant v36675 at 20 μg / mL and variant v35356 at 5 μg / mL) was added to each slide after cell fixation. In the confirmatory screening stage, slides were treated with individual antibodies: variant v36675 at 20 μg / mL, variant v35356 at 5 μg / mL, or control antibody (rituximab biosimilar) at 1 μg / mL, or without test article in the absence of fixation (live cells; n=1 slide per treatment) and after cell fixation (n=2 slides per treatment).

[0395] Binding was detected using AlexaFluor® 647-labeled anti-human IgG H+L (AF647 anti-hIgG H+L) followed by fluorescence imaging. Fluorescence images were analyzed and quantified (for transfections) using ImageQuant™ software (version 8.2; GE Healthcare, Chicago, IL). Protein hits were defined as overlapping spots showing increased signal compared to background levels and were identified by visual inspection using images gridded on the ImageQuant™ software. Based on the intensity of overlapping spots, hits were classified as strong, medium, weak, or very weak by visual inspection by two experienced scientists.

[0396] result The majority of the initial hits identified in the library screening had spot intensities ranging from very weak to strong and were confirmed to be hits in the confirmatory screening of both variant v36675 and variant v35356. However, aside from the hits reflecting the expected strong interaction with FRα, other hits were considered nonspecific because these hits either 1) involved the FcgR receptor (signaling via Fc domain-mediated interaction), as was also observed for the control antibody (rituximab biosimilar), or 2) involved various immunoglobulins recognized by the detection antibody.

[0397] No other interactions were identified for humanized variant v36675 (see Figure 21), indicating high specificity of variant v36675 for its primary target, FRα. The weak interaction observed for this variant with the heterodimer FCGR3A+CD247 was not observed in the control (compare Figures 21A and B), but was not considered strong enough to be a hit.

[0398] For affinity matured variant v35356, we detected a weakly intense signal for the protein COL6A2 isoform 2C2A in the library screen. This "hit" was confirmed in the fixed cell screen but not in the live cell screen, indicating that the observed discrepancy is likely due to a fixation artifact.

[0399] Example 31: Competitive Assay Competitive binding to the FRα target between the parental chimeric antibody v23924 and the anti-FRα antibodies mirvetuximab (v17716) and farletuzumab (v31629) was assessed by flow cytometry in the moderately FRα-expressing tumor cell line H2110 as follows.

[0400] Briefly, antibodies were labeled with Alexa Fluor647 (AF647; ThermoFisher Scientific, Waltham, MA; Catalog No. A20006) according to the manufacturer's specifications prior to cell processing. Tumor cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with unlabeled antibodies for 1 hour at 4°C to prevent internalization. After incubation, cells were washed and fluorescently labeled antibodies were added for 1 hour at 4°C. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) and a minimum of 1,000 events were collected per well. AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) in the live cell population was used to calculate % competitive binding compared to untreated controls, and data were plotted using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0401] result The results are shown in Figure 22 and Table 31.1. Chimeric antibody v23924 was found to compete with farletuzumab for FRα binding in H2110 cell line, resulting in nearly 100% competitive binding in both binding orientations (primary antibody farletuzumab and secondary fluorescently labeled antibody variant v23924, and primary antibody variant v23924 and secondary fluorescently labeled antibody farletuzumab). Chimeric antibody v23924 was found not to compete with mirvetuximab for FRα binding in H2110 cell line. These antibodies showed low levels of % competition in both binding orientations. In contrast, farletuzumab showed competitive binding with mirvetuximab. Thus, chimeric antibody v23924 shows a binding profile that is distinct from both mirvetuximab and farletuzumab.

[0402] [Table 31.1]

[0403] Example 32: Further functional characterization of anti-FRα antibodies The functional activity of humanized antibody variants v30384 (HetFc) or v36675 (HomoFc) was assessed in comparison to the biparatopic anti-FRα antibody B5327A (IMGN151; v36264) and the anti-FRα antibody mirvetuximab (v17716) using FRα-expressing tumor cell lines by cell binding, antigen-mediated antibody internalization, antibody penetration into tumor cell spheroids, and 3D cell growth inhibition in spheroids.

[0404] 32.1 Cell binding The ability of humanized antibody variants v30384 (HetFc), v36264 (B5327A) and v17716 (mirvetuximab) to bind to FRα-expressing tumor cell lines was evaluated on the endogenous FRα-expressing tumor cell line IGROV-1 (ovarian adenocarcinoma; FRα-high) by flow cytometry following the same general procedure as described in Example 11.

[0405] The results are shown in Table 32.1. Humanized variants v30384 and mirvetuximab (v17716) showed similar on-cell binding capabilities with comparable Bmax values ​​(16,599 and 19,505 intensity geometric mean, respectively) and apparent Kd values ​​(0.43 and 0.69 nM, respectively). The biparatopic antibody B5327A (v36264) showed an increased Bmax compared to the other two antibodies (26,267 intensity geometric mean), as expected for a biparatopic antibody.

[0406] [Table 32.1]

[0407] 32.2 Internalization The receptor-mediated internalization capacity of humanized antibody variants v30384 (HetFc), v36264 (B5327A) and v17716 (mirvetuximab) was assessed by uptake into the endogenous FRα-expressing tumor cell line IGROV-1 (ovarian adenocarcinoma) by flow cytometry as follows.

[0408] Antibodies were fluorescently labeled by coupling to anti-human IgG Fc Fab fragment AF488 conjugate (Jackson Immuno Research Labs, West Grove, PA; Catalog No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Catalog No. 10010-023) for 24 hours at 4°C. Cells were seeded at 50,000 cells / well in 48-well plates and incubated overnight under standard culture conditions (37°C / 5% CO2) to allow binding. The coupled antibody was added to the cells the next day at 25 nM and incubated under standard culture conditions for 5 hours to allow internalization. After incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched with 100 nM anti-AF488 antibody (Life Technologies, Carlsbad, CA; Catalog No. A-11094) for 30 min at 4°C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with a minimum of 1,000 events collected per well. AF488 / FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human FabAF488 labeling) was calculated for the biological single cell population using FlowJo™ version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0409] The results are shown in Figure 23. After 5 hours of incubation of IGROV-1 cells with 25 nM of test antibody, the humanized antibody variant v30384 exhibited comparable internalization fluorescence to the biparatopic antibody v36264 (1.1 fold vs. v30384), whereas mirvetuximab (v17716) exhibited a significantly lower degree of internalization fluorescence (0.5 fold vs. v30384).

[0410] 32.3 Antibody penetration into 3D spheroids The ability of humanized antibody variants v36675 (HomoFc), v36264 (B5327A) and v17716 (mirvetuximab) to penetrate FRα-expressing cell line spheroids was assessed by treating spheroids formed from the highly FRα-expressing cell line JEG-3 (placental choriocarcinoma) with fluorescently labeled antibodies following the same general procedure as described in Example 29.

[0411] The results are shown in Figure 24. After 96 hours of incubation of JEG-3 spheroids with 25 nM fluorescently labeled antibodies, the humanized antibody variant v36675 showed greater fluorescence intensity from the second layer inwards than the biparatopic antibodies B5327A (v36264) or mirvetuximab (v17716), indicating a greater degree of cellular uptake of v36675 in all layers and, overall, greater penetration distances into the spheroids. In contrast, the biparatopic antibody B5327A (v36264) showed greater fluorescence intensity than v36675 or v17716 in the outermost layers, indicating accumulation of the antibody on the spheroid surface.

[0412] 32.4 3D spheroid growth inhibition The 3D cytotoxic potential of humanized antibody variants v30384 (HetFc), v36264 (B5327A), and v17716 (mirvetuximab), each conjugated to drug linker DL1, was evaluated in FRα-expressing IGROV-1 (ovarian adenocarcinoma) and JEG-3 (placental choriocarcinoma) cell line spheroids as follows.

[0413] Briefly, cells were seeded at 3,000 cells / well in ultra-low attachment 384-well plates (Corning, New York, NY), centrifuged, and incubated under standard culture conditions for 3 days to allow spheroid formation and growth. Spheroids generated in cell growth medium were then treated with titrations of test articles. Spheroids were incubated for 6 days under standard culture conditions. After incubation, CellTiter-Glo® 3D Reagent (Promega Corporation, Madison, WI) was added to all wells. Plates were incubated in the dark at room temperature for 1 hour, and luminescence was quantified using a BioTek Cytation 5 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA). Percent cytotoxicity values ​​were calculated based on blank wells (without test article added) and plotted against test article concentration using GraphPad Prism9 software (GraphPad Software, San Diego, CA). EC 50 Values ​​were calculated by GraphPad Prism9 based on nonlinear regression log(agonist) vs. response, variable slope (four parameters).

[0414] The results are shown in Table 32.2. For IGROV-1 spheroids, all three antibody-drug conjugates (ADCs) showed comparable cell growth inhibition abilities. For JEG-3 spheroids, v30384-DL1 was significantly more potent than v17716-DL1 (EC 50 The two ADCs showed similar potencies (0.18 and 0.17 nM), whereas the biparatopic ADC, v36264-DL1, showed slightly lower potency (0.35 nM).

[0415] [Table 32.4]

[0416] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are specifically incorporated by reference in their entireties into this specification to the same extent as if each such individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0417] Modifications of the particular embodiments described herein that will be obvious to those of skill in the art are intended to be included within the scope of the following claims.

[0418] Array Table [Table A]

[0419] [Table B] TIFF2025513721000062.tif163168TIFF2025513721000063.tif226168TIFF2025513721000064.tif229168TIFF2025513721000065.tif225168TIFF2025513721000066.tif197168TIFF2025513721000067.tif226168TIFF2025513721000068.tif229168TIFF2025513721000069.tif196168TIFF2025513721000070.tif226168TIFF2025513721000071.tif229168TIFF2025513721000072.tif225168TIFF2025513721000073.tif197168TIFF2025513721000074.tif226168TIFF2025513721000075.tif229168TIFF2025513721000076.tif194168TIFF2025513721000077.tif226168TIFF2025513721000078.tif229168TIFF2025513721000079.tif194168TIFF2025513721000080.tif226168TIFF2025513721000081.tif229168TIFF2025513721000082.tif196168TIFF2025513721000083.tif226168TIFF2025513721000084.tif229168TIFF2025513721000085.tif194168TIFF2025513721000086.tif226168TIFF2025513721000087.tif229168TIFF2025513721000088.tif194168TIFF2025513721000089.tif226168TIFF2025513721000090.tif229168TIFF2025513721000091.tif194168TIFF2025513721000092.tif226168TIFF2025513721000093.tif229168TIFF2025513721000094.tif225168TIFF2025513721000095.tif228168TIFF2025513721000096.tif228168TIFF2025513721000097.tif197168TIFF2025513721000098.tif226168TIFF2025513721000099.tif229168TIFF2025513721000100.tif225168TIFF2025513721000101.tif221163TIFF2025513721000102.tif225168TIFF2025513721000103.tif228168TIFF2025513721000104.tif66168.

Claims

1. Epitope within human folate receptor alpha (hFRα) containing amino acid residues E120, D121, R123, T124, S125, and Y126 of SEQ ID NO: 15 An antibody construct comprising an antigen-binding domain that specifically binds to an epitope, wherein the epitope is a discontinuous epitope.

2. An antibody construct comprising an antigen-binding domain that specifically binds to human folate receptor alpha (hFRα), wherein the antigen-binding domain comprises heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) containing the sequences described in SEQ ID NOs: 3, 4, and 5, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) containing the sequences described in SEQ ID NOs: 6, 7, and 8.

3. The antigen-binding domain, (a) A CDR sequence of a VH domain having the sequence described in any one of sequence numbers 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136, and (b) CDR sequence of a VL domain having the sequence described in any one of sequence numbers 39, 64, 119, 124, or 130 The antibody construct according to claim 2, comprising:

4. The antigen-binding domain, (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 20, 23, 26, 28, 31, 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 21, 24, 27, 29, 32, 51, 58, 100, 101, 102, 103, 109, 137, 138, or 139; and an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 22, 25, 30, 107, 108, or 110; (ii) An LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 40, 43, 45, 65, 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NOs: 42, 47, 120, or 121. The antibody construct according to claim 2, comprising:

5. The antigen-binding domain, (a) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 27, 29, or 32; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 43, or 45; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (b) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47, or (c) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 58; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47, or (d) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 51; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (e) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 24, 100, 101, 102, or 103; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (f) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 42 or 47; or (g) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (h) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (i) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (j) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 40, 45, or 65; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (k) an HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 107, 108, or 110; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (l) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 20, 23, 26, 28, or 31; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 29, 32, or 109; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121; or (m) An HCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 92, 93, 94, 95, or 96; an HCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 21, 24, 137, 138, or 139; an HCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 22, 25, or 30; an LCDR1 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 125, 126, or 127; an LCDR2 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 41, 44, or 46; and an LCDR3 amino acid sequence selected from any one of the amino acid sequences described in SEQ ID NO: 120 or 121. The antibody construct according to claim 2, comprising:

6. The antigen-binding domain, (a) A VH amino acid sequence selected from any one of the VH amino acid sequences described in SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136, or (b) A VL amino acid sequence selected from any one of the VL amino acid sequences described in SEQ ID NOs: 39, 64, 119, 124, or 130. The antibody construct according to claim 2, comprising:

7. The antibody construct according to claim 2, wherein the antigen-binding domain comprises a VH amino acid sequence selected from any one of the VH amino acid sequences described in SEQ ID NOs: 19, 50, 54, 57, 61, 76, 79, 82, 85, 88, 91, 99, 106, 113, 116, 133, or 136, and a VL amino acid sequence selected from any one of the VL amino acid sequences described in SEQ ID NOs: 39, 64, 119, 124, or 130.

8. The antigen-binding domain, (i) The VH amino acid sequence described in SEQ ID NO: 19 and the VL amino acid sequence described in SEQ ID NO: 39, or (ii) The VH amino acid sequence described in SEQ ID NO: 50, and the VL amino acid sequence described in SEQ ID NO: 64, or (iii) The VH amino acid sequence described in SEQ ID NO: 54, and the VL amino acid sequence described in SEQ ID NO: 64, or (iv) The VH amino acid sequence described in SEQ ID NO: 57, and the VL amino acid sequence described in SEQ ID NO: 64, or (v) The VH amino acid sequence described in SEQ ID NO: 61, and the VL amino acid sequence described in SEQ ID NO: 64, or (vi) The VH amino acid sequence described in SEQ ID NO: 76, and the VL amino acid sequence described in SEQ ID NO: 64, or (vii) The VH amino acid sequence described in SEQ ID NO: 79, and the VL amino acid sequence described in SEQ ID NO: 64, or (viiii) The VH amino acid sequence described in SEQ ID NO: 82, and the VL amino acid sequence described in SEQ ID NO: 64, or (ix) The VH amino acid sequence described in SEQ ID NO: 85, and the VL amino acid sequence described in SEQ ID NO: 64, or (x) The VH amino acid sequence described in SEQ ID NO: 88, and the VL amino acid sequence described in SEQ ID NO: 64, or (xi) The VH amino acid sequence described in SEQ ID NO: 91, and the VL amino acid sequence described in SEQ ID NO: 124, or (xi) The VH amino acid sequence described in SEQ ID NO: 99, and the VL amino acid sequence described in SEQ ID NO: 130, or (xiii) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 64, or (xiv) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 119, or (xv) The VH amino acid sequence described in SEQ ID NO: 106, and the VL amino acid sequence described in SEQ ID NO: 130, or (xvi) The VH amino acid sequence described in SEQ ID NO: 113, and the VL amino acid sequence described in SEQ ID NO: 130, or (xvii) The VH amino acid sequence described in SEQ ID NO: 116, and the VL amino acid sequence described in SEQ ID NO: 119, or (xviiii) The VH amino acid sequence described in SEQ ID NO: 116, and the VL amino acid sequence described in SEQ ID NO: 130, or (xix) The VH amino acid sequence described in SEQ ID NO: 133, and the VL amino acid sequence described in SEQ ID NO: 130, or (xx) VH amino acid sequence described in SEQ ID NO: 136, and VL amino acid sequence described in SEQ ID NO: 130 The antibody construct according to claim 2, comprising:

9. The antibody construct according to claim 2, further comprising a scaffold, wherein the scaffold comprises an IgG Fc region, and the antigen-binding domain is functionally linked to the scaffold.

10. The antibody construct according to claim 5, further comprising a scaffold, wherein the scaffold comprises an IgG Fc region, and the antigen-binding domain is functionally linked to the scaffold.

11. The antibody construct according to claim 8, further comprising a scaffold, wherein the scaffold comprises an IgG Fc region, and the antigen-binding domain is functionally linked to the scaffold.

12. The antibody construct according to claim 2, wherein the antibody construct further comprises a second antigen-binding domain.

13. The antibody construct according to claim 12, wherein the second antigen-binding domain specifically binds to hFRα.

14. The antibody construct according to claim 12, wherein the second antigen-binding domain binds to an antigen other than hFRα.

15. The antibody construct according to claim 12, further comprising one or more additional antigen-binding domains.

16. The antibody construct according to claim 12, further comprising a scaffold, wherein the scaffold comprises an IgG Fc region, and at least one antigen-binding domain is functionally linked to the scaffold.

17. The antibody construct according to claim 10, wherein the antibody construct further comprises a second antigen-binding domain.

18. An antibody construct comprising two antigen-binding domains functionally linked to the IgG Fc region, wherein each of the antigen-binding domains specifically binds to human folate receptor alpha (hFRα), (a) The VL amino acid sequence described in SEQ ID NO: 39, and the VH amino acid sequence described in SEQ ID NO: 19, or (b) The VL amino acid sequence described in SEQ ID NO: 124, and the VH amino acid sequence described in SEQ ID NO: 91, or (c) The VL amino acid sequence described in Sequence ID No. 64, and (i) The VH amino acid sequence described in Sequence ID No. 50, or (ii) The VH amino acid sequence described in Sequence ID No. 54, or (iii) The VH amino acid sequence described in Sequence ID No. 57, or (iv) The VH amino acid sequence described in SEQ ID NO: 61, or (v) The VH amino acid sequence described in SEQ ID NO: 76, or (vi) The VH amino acid sequence described in Sequence ID No. 79, or (vii) The VH amino acid sequence described in Sequence ID No. 82, or (viiii) The VH amino acid sequence described in Sequence ID No. 85, or (ix) The VH amino acid sequence described in Sequence ID No. 88, or (x) The VH amino acid sequence described in Sequence ID No. 106, or (d) The VL amino acid sequence described in SEQ ID NO: 130, and (i) The VH amino acid sequence described in Sequence ID No. 99, or (ii) The VH amino acid sequence described in Sequence ID No. 106, or (iii) The VH amino acid sequence described in Sequence ID No. 113, or (iv) The VH amino acid sequence described in SEQ ID NO: 116, or (v) The VH amino acid sequence described in SEQ ID NO: 133, or (vi) The VH amino acid sequence described in SEQ ID NO: 136, or (e) The VL amino acid sequence described in Sequence ID No. 119, and (i) The VH amino acid sequence described in Sequence ID No. 106, or (ii) VH amino acid sequence described in Sequence ID No. 116 The antibody construct comprising the above.

19. A polynucleotide or set of polynucleotides encoding an antibody construct according to any one of claims 1 to 18.

20. An expression vector or a set of expression vectors comprising a polynucleotide or a set of polynucleotides as described in claim 19.

21. A host cell comprising the expression vector or set of expression vectors described in claim 20.

22. A method for producing an antibody construct according to any one of claims 1 to 18, comprising introducing an expression vector or set of expression vectors according to claim 20 into a host cell, and culturing the host cell under conditions suitable for the expression of the antibody construct.

23. An antibody construct according to any one of claims 1 to 18, conjugated with one or more drug portions Antibody-drug conjugates, including those mentioned above.

24. The antibody-drug conjugate according to claim 23, wherein the antibody conjugate is conjugated to 1 to about 8 drug portions.

25. General formula I: A-(L-(D) ｍ ) ｎ (I) An antibody-drug conjugate having, in the formula, A is an antibody construct according to any one of claims 1 to 18, L is the linker, D is the drug part, m is between 1 and approximately 8. n is between 1 and approximately 12. The aforementioned antibody-drug conjugate.

26. The antibody-drug conjugate according to claim 25, wherein m is 1 or 2 and n is about 2 to about 8.

27. The antibody-drug conjugate according to claim 25, wherein the drug portion is a meitansinoid, a meitansinoid analog, a benzodiazepine, a pyrrolobenzodiazepine, a duocalmycin, a calicheamicin, a calicheamicin analog, auristatin, auristatin analog, aemiasterin, aemiasterin analog, a tubulicin, a tubulicin analog, amatoxin, an amatoxin analog, a camptothecin, a camptothecin analog, an eribulin, a TLR agonist, or a STING agonist.

28. The antibody-drug conjugate according to claim 25, wherein the drug portion is auristatin, auristatin analog, hemiasterin, hemiasterin analog, camptothecin, camptothecin analog, or eribulin.

29. A pharmaceutical composition for the treatment of cancer, comprising an antibody construct according to any one of claims 1 to 18.

30. A pharmaceutical composition for the treatment of cancer, comprising the antibody-drug conjugate described in claim 25.

Images